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Scientia Iranica F (2013) 20 (3), 1003–1013

Sharif University of Technology Scientia Iranica Transactions F: Nanotechnology www.sciencedirect.com

Invited/review paper Nanocarriers, nanovaccines, and nanobacteria as nanobiotechnological concerns in modern vaccines

P. Gill ∗ Department of Medical Nanotechnology (Nanomedicine), Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences (GOUMS), Gorgan, Iran Nanotechnology Group, Institute for Advanced Medical Technologies (IAMT), Tehran University of Medical Sciences (TUMS), Tehran, Iran

Received 20 March 2012; revised 13 November 2012; accepted 24 December 2012

KEYWORDS Abstract This review briefly describes the concerns of nanobiotechnology in the design and development Nanocarrier; of novel vaccines using the most known nanocarriers, including nature-made nanocarriers (such as Nanovaccine; bacterial spores, -like particles, exosomes, and bacteriophages), man-made nanocarriers (such as Bacterial spore; Proteosomes, liposomes, virosomes, SuperFluids, and nanobeads), and their applications in therapeutic Proteosome; and protective immunization, as well as their advantages and disadvantages. Here, we focus on the Exosome; development of nano-based vaccines as ‘‘nanovaccines’’ for inducing immune systems, and the foreseeable Liposome; promises and problems when compared with existing vaccines. Also, we review a potential nano-hazard Virosome; for vaccines, so-called nanobacterial contamination. SuperFluid; © 2013 Sharif University of Technology. Production and hosting by Elsevier B.V. All rights reserved. Nanobead; Virus-like particle (VLP); Bacteriophage; Nanobacteria.

Contents

1. Introduction...... 1004 2. Nature-made nanocarriers...... 1004 2.1. Bacterial spores...... 1004 2.2. Virus-like particles...... 1004 2.3. Bacteriophages...... 1005 2.4. Exosomes...... 1005 3. Man-made nanocarriers...... 1006 3.1. Proteosomes...... 1006 3.2. SuperFluids...... 1006 3.3. Nanobeads...... 1006 3.4. Virosomes...... 1007 3.5. Liposomes...... 1007 4. Nanobacteria...... 1008 5. Conclusions...... 1008 Acknowledgments...... 1009 References...... 1009

Peer review under responsibility of Sharif University of Technology. ∗ Correspondence to: Department of Medical Nanotechnology (Nanomedicine), Faculty of Advanced Medical Technologies, Golestan Univer- sity of Medical Sciences (GOUMS), Gorgan, Iran. Tel.: +98 171 4430563; fax: +98 171 4430563. E-mail addresses: [email protected], [email protected].

1026-3098 © 2013 Sharif University of Technology. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scient.2013.05.012 1004 P. Gill / Scientia Iranica, Transactions F: Nanotechnology 20 (2013) 1003–1013

1. Introduction or man-made nanocarriers. Bacterial spores, Proteosomes, exo- somes, liposomes, virosomes, SuperFluids, nanoparticle-based Vaccination is the most effective strategy for reducing high nanobeads, virus-like particles and bacteriophages are some in- morbidity and mortality rates, as well as diminishing the enor- stances of nano-sized vehicles that are reviewed here as poten- mous social and economic impact associated with disorders. tial nanocarriers for vaccine delivery to immune systems [28]. Vaccines are utilized to stimulate the immune system to initi- Nanobacteria are another important subject for review here, be- ate an immune response against specific targets, such as cancer cause of their role in the contamination of vaccines and some cells and microbial agents [1,2]. Various types of vaccine may be complicated disorders [74]. Because nanobacteria have been used to treat cancers, such as whole tumor vaccines, den- found to be a contaminant in previously assumed-to-be-sterile dritic cell vaccines, idiotype vaccines, antigen or adjuvant vac- medical products, we also aim to discuss it as a nano-hazard in cines, viral vectors, and DNA vaccines [3–8]. Although the this review [75]. immunologic effect is not always sufficient to reverse the pro- gression of cancer, vaccines are generally well tolerated and 2. Nature-made nanocarriers provide useful anticancer effects in some situations [9]. Antigen cancer vaccines are typically composed of a cancer associated 2.1. Bacterial spores antigen, not normally present on healthy cells, along with other components (i.e. adjuvants) utilized to stimulate the immune Through evolution, nature has produced highly complex responses against the antigens, resulting in the destruction of nanoarchitectures using macromolecules, especially polysac- malignant cells, without harming normal cells, and preventing charides, nucleic acids, and proteins [76]. Understanding the recurrence [10]. principle of how these macromolecules interact to fabricate On the other hand, infection can protect against subsequent nano-scaled structures, it should be possible to exploit this sci- disease by induction of both humoral and cellular immunity, ence in the design and development of novel artificial struc- but, inert protein-based vaccines are not as effective [11]. CD8 tures and tools [77]. The advantage of this ‘‘learning from T cells play a vital role in protective immunity against many in- nature’’ approach is that it well defines the bionanofabrication tracellular pathogens and cancer, but are notoriously difficult to processes that already exist in [78]. activate by vaccination and immunotherapy [12]. Vaccines that have novel and interesting structures, such as bacterial spore require a specific immunization sequence with repeated doses coats [79], which have protective characteristics [80]. Bacterial (such as prime-boost) are very efficient, but complicated to ad- spores are dormant forms with formidable resistance prop- minister, where logistic considerations of vaccine distribution erties. These nanostructures attribute multiple layers of pro- are critical [13]. tein, which encase the spores in a protective and flexible shell As a novel approach, simple inert nanocarriers able to gen- [81–85]. The shield has features that are pertinent for merging erate strong protection after a single dose could be useful with nanobiotechnological fields, such as self-assembling pro- for a variety of applications [14]. These nanomaterials could tomers, and a capacity for engineering and delivering foreign be classified as nature-made and man-made nanocarriers/ molecules [86]. In fact, the spore coat could be employed not nanoparticles. In fact, nanobiotechnology is particularly used only as a delivery vehicle for various molecules, but also as a in developing a new generation of more effective vaccines source of novel self assembling biomolecules [87]. The coat can (i.e., nanovaccines) capable of overcoming the many biologi- act as a device for heterologous antigen presentation and pro- cal, biophysical, and biomedical barriers that the body builds phylactic immunization [29]. against standard intervention [15]. Nanoparticles show much There are a number of attributes that make bacillus spores promise in cancer therapy, by selectively gaining access to particularly suitable candidates for vaccine vehicles [30,31]. tumors, due to their small size and modifiability [16]. Nano- Two distinct approaches are accessible for vaccination. In the sized particles have similarly been used for vaccine construc- first approach, an antigen or epitope is engineered onto the tion against microbial agents [17]. Typically, nanoparticles are spore coat by fusing a spore coat with the antigen se- defined as particles between 0.1 and 100 nm [18]. Nanopar- quence [32–34]. In the second, the antigen is expressed con- ticles are formulated out of a variety of substances and fabri- stitutively in the vegetative cell by fusion of the antigen gene cated to carry an array of substances in a controlled and to the transcriptional and translational sequences of a suitable targeted mode [19]. Nanocarriers are prepared to take ad- bacillus gene [88–90]. Spores carrying this modified gene are vantage of fundamental cancer morphologies and modes of used for oral delivery and then germinate in the gastrointesti- development, such as rapid cellular proliferation, antigen nal system [91–93]. Consequently, the spore offers unique capa- expression, and leaky tumor vasculature [20]. Additionally, bilities for nanovaccine development that could ultimately lead nanocarriers are promising as the delivery systems for DNA to improved public health, both in developed and developing vaccines [21]. Nanoemulsions or nanosized aerosol vaccines countries. are also under development [22,23]. Although many reports have shown that safe, biocompatible materials could be en- 2.2. Virus-like particles gineered into nanoparticles that contain drugs or vaccines, researchers are trying to develop new materials for vectors Virus-Like Particles (VLPs) structurally mimic the viral cap- that interact specifically and predictably with cells. Nanosys- sid, and have been extensively and successfully employed as tems will be designed to evade normal barriers and stimulate nanovaccines [94]. The capability of VLPs to include nucleic antigen-presenting cells of the immune system [24–26]. Sci- acids and small biomolecules has also made them novel vehi- entists are also looking to these ‘‘smart’’ targetable nanoparti- cles for gene and vaccine deliveries [95]. The repetitive surface cles as magic bullets that could seek out and destroy individual of VLPs has regularly been used as a template for bionanofab- cancer cells while bypassing healthy ones [27]. Hence, various rication [96]. Recent progress has been shaped towards the de- types of nano-scaled material have been employed for the de- velopment of virus capsids, along with the precise spacing of sign and fabrication of nanovaccines (Table 1), based on nature surface peptide domains, which enables the modification of P. Gill / Scientia Iranica, Transactions F: Nanotechnology 20 (2013) 1003–1013 1005

Table 1: Characteristics of nanocarriers as nanovaccines.

Nanocarrier Source Size (nm) Immune response Ref. no.

Spore Bacterial Various Humoral-cellular [29–34] Proteosome Membrane protein-based 20–800 Humoral-cellular [35–41] Exosome Cellular 50–100 Cellular [42–44] Liposome Lipid-based Various Humoral-cellular [45–50] Virosome Liposome + viral env proteins Various Humoral-cellular [51–54] SuperFluid Biodegradable polymer 25–250 Humoral-cellular [55–59] Nanobead Inert nanomaterial 40 Humoral-cellular [60–63] VLP Viral Various Humoral [64–68] Phage Bacterial Various Humoral-cellular [69–73] the capsid shield through the fabrication of repetitive chemi- the phage nanovaccines might be made more efficient via cal adducts on a nanoscale platform [97]. Recently, capsid en- decorating their coats with phage fusion proteins displaying gineering has been improved for bacteriophage and virus immunogenic antigens for dendritic cells [71]. This could be capsids, with an extension to purified VLPs [98]. VLPs of vari- accomplished using a peptide that has an attaching epitope for ous have been confirmed as very immunogenic and are the CD40 receptor, which has numerous functions in the ac- already in use as nanovaccines in humans or [99]. For tivation of antigen-presenting cells [72]. This requirement for instance, the immunization of young healthy women with VLPs the peptide display by phage vaccines can be enhanced by ap- composed of the major structural protein, L1, of HPV16, induced plication of a developed dual expression system that permits high titer neutralizing antibodies, and protected them from HPV the generation of phages with mosaic heads that can accom- infection and associated cervical dysplasia [64]. The vaccination modate recalcitrant recombinant fusion proteins. This system mode decreases the incidence of cervical cancer; however, a employed bacteria carrying a that is deficient in a longer follow-up is essential to confirm this action. VLPs have major head protein, along with the carrying for- additionally been employed to deliver immunogenic epitopes eign head protein for both wild-type and recombinant of other pathogens. They have several advantages as immuno- phages [73]. gens in the form of short peptides [65]. The linking of VLPs to adjuvant molecules was also shown to 2.4. Exosomes improve the immunogenicity of the nanobioparticles [66]. For instance, the nontoxic subunit of cholera toxin (CTB) was chem- Exosomes are small (50–100 nm), spherical vesicles man- ically linked to VLPs via biotin-streptavidin [67]. There was a ufactured and released by most cells, like B cells [115], T higher level of Env-specific serum IgG1, secretory IgA antibod- cells [116], mast cells [117], epithelial cells [118] and Dendritic ies and cellular immune responses than in either the absence Cells (DCs) [119], to facilitate intercellular communication. of CTB or when CTB was just co-injected [68]. As there is in- These vesicles are of an endosomal origin, which are secreted creasing awareness of the VLP structure, it is expected that the in the extracellular milieu following the fusion of late endoso- development of VLPs as nanovaccines, as carriers for delivering mal multivesicular bodies with the plasma membrane [120]. small molecules, and as the basis for nanoarrays, will continue Exosomes have a defined protein composition, which con- with even greater potency [100–102]. fers specific biological activities contingent on the nature of the producing cell [121]. In the multivesicular bodies of anti- gen presenting cells, exogenous antigens are loaded onto MHC 2.3. Bacteriophages molecules and when the multivesicular bodies fuse with the cell membrane, the MHC molecules are incorporated into the cell While the study of the interaction of phage nanobioparticles membrane [122]. The internal vesicles are simultaneously re- and animals is still in its infancy, the capacity of a few of the leased as extracellular exosomes [123]. well-studied phage strains to deliver antigenic gene products Exosomes from antigen presenting cells bear not only MHC has been investigated [103–105]. Phage vaccines introduced class I and II, but also costimulatory molecules, such as CD54, in 1985 showed that it was possible to manufacture bacterio- CD80 and CD86, and they are enriched in tetraspanin proteins, phages that display foreign proteins fused to their normal coat like CD63 and CD81 [124,125]. Tetraspanin proteins have been proteins [106]. Following the description of phage display tech- proposed to have a modulatory role in several different cell pro- nology, its power was greatly improved using the affinity se- cesses, including adhesion, migration and proliferation [126]. lection to isolate phages that displayed specific peptides from Exosomes have been confirmed to be present in bronchoalve- random peptide collections [107–109]. Employment of this olar lavage [127], human plasma [128], malignant effusions technology for the development of vaccines and diagnostics has [129,130] and on the surface of follicular dendritic cells [131]; become an industry [110–112]. Additionally, it was also pos- however, their physiological roles in vivo are still unclear. They sible to use specific epitopes that have been chosen on the have been suggested to participate both in T cell stimula- basis of biological experiments [113]. For instance, a vaccine tion [42,43] and in tolerance induction [44]. Studies in mice using a phage display system provided complete immunolog- have demonstrated the potential of exosomes as immunother- ical protection to mice nasally challenged with live respira- apeutic agents in infections [132], transplantations [133], and tory syncytial virus [114]. A similar approach was applied to cancer [134–136]. construct a phage carrying a gene product protection against The mechanisms for how exosomes stimulate T cells are un- Yersinia pestisinfections [69]. The phage IgG2 responses were known. Some reports indicate that the exosomes from antigen indicated to be significantly higher following intramuscular presenting cells can stimulate T cell clones, on their own [122] phage vaccinations with the phage vaccine than with the plas- and more efficiently, if the exosomes come from mature den- mid DNA vaccine [70]. Moreover, it was demonstrated that dritic cells [42,136]. Other studies confirmed that exosomes 1006 P. Gill / Scientia Iranica, Transactions F: Nanotechnology 20 (2013) 1003–1013 require the presence of dendritic cells to efficiently stimulate Application of SuperFluids reduces the exposure of viral anti- T lymphocytes [43,137,138]. Some reports have employed T gens, such as HIV and influenza, to potentially denaturing or- cells [43,139], hybridomas [43,136] or clones [122,42,137,138] ganic solvents, such as methylenechloride and ethyl acetate, when looking at the capability of exosomes to stimulate T cells and improves the stability of protein antigens in the body at an in an antigen-specific manner. These studies demonstrate that ambient temperature for a long time. Therefore, the nanocar- + riers enhance the capability of nanoencapsulated vaccine anti- exosomes can directly stimulate viral-specific peripheral CD8 T cells [140]. gens to induce the production of protective and neutralizing Although exosomes express tumor antigens, leading to antibodies [55,56]. their proposed utility as nanovaccines, they also can suppress This controlled release nanovaccine delivery system has the T-cell-signaling molecules and induce apoptosis [141]. The first capability to deliver different types and combinations of HIV or phase I clinical trial using autologous exosomes has indicated influenza vaccine candidates (such as whole inactivated virus the feasibility of large-scale exosome production and the safety particles, DNA plasmids, and subunit protein antigens) [57–59]. of exosome administration [142]. Exosomes manufactured by The polymer nanoencapsulation technology will decrease cost dendritic cells are named ‘‘dexosomes’’ and contain essential by eliminating unnecessary processing steps, while improving components to activate both adaptive and innate immune re- the manufacturing environment [162]. The nanocarrier tech- sponses [143]. For instance, dexosome nanovaccines have been nology is portable, inexpensive, and amenable to large-scale developed that employ patient-specific dexosomes loaded with processing [163]. Several enabling nanotechnologies have been developed, tumor antigen-derived peptides to treat cancer [144]. The Ex- according to SuperFluids, for the enhanced delivery of nanovac- osome Display Technology manufactured by Anosys provides cines and nanodrugs, ranging from potent anticancer therapeu- the capability to manipulate exosome composition, and tailor tics to large recombinant proteins. One of these technologies exosomes, with new desirable characteristics, opening up op- is protein nanocarriers [164]. Protein nanocarriers can be gen- portunities in the field of recombinant vaccine and monoclonal erated without reduction in protein integrity and efficacy in a antibody preparation [145]. This is gained via generating gene solvent-free process, based on a SuperFluids rapid expansion coding for the chimeric proteins, attaching an exosome address- mechanism for the enhanced drug delivery of protein macro- ing sequence to antigens or biologically active proteins [146]. molecules. This technology can be applied to the pulmonary The resulting proteins are targeted to the exosomal compart- delivery of proteins and polypeptides. It also is used for oral ment and released in the extracellular milieu enclosed to ex- or depot delivery of vaccine antigens. Another form of these osomes [147]. Exosome research continues to reveal unique technologies is polymer nanospheres [165,166]. Biodegradable potential that broadens its field of application. polymeric nanospheres can be manufactured utilizing Super- Fluids technologies for the controlled delivery and release of 3. Man-made nanocarriers proteins through reverse engineering of the rapid expansion mechanism [167]. This technology can be employed for oral or 3.1. Proteosomes depot delivery of proteins and controlled-release adjuvants of vaccine antigens for infectious diseases [168]. The other gen- Proteosomes are vaccine delivery vehicles by virtue of their eration of these technologies is phospholipid nanosomes [169]. nanoparticulate nature, forming vesicles and vesicle clusters Phospholipid nanosomes or small uniform liposomes for the en- comparable to the size of small viruses [148,149]. Proteosome hanced delivery of hydrophilic and hydrophobic drugs can be has been used to describe preparations of the outer membrane produced without the use of toxic organic solvents, using a sim- proteins from microorganisms [150–154]. Proteosomes are de- ilar mechanism [170]. This technology can be employed for scribed as comparable in size to certain virus particles that intravenous and topical administration of small hydrophilic are hydrophobic and safe for human application [155]. Proteo- nanodrugs, proteins and genes [171]. somes are said to be useful in formulating vaccines with a va- riety of proteins and peptides [156,157]. Proteosome vaccine 3.3. Nanobeads vesicles and vesicle clusters may range in size from 20 to 800 nm, based on the type and amount of antigen encapsulated with Nanoparticle-based nanobeads serve as nanovaccine fab- the proteosomes [158]. The hydrophobic nature of the proteo- rication and more effective immunization [172]. Although a some porin proteins also contributes to vaccine delivery capa- number of adjuvants are currently confirmed for use in bilities by facilitating interaction of the vaccine nanoparticles , only alum has been widely employed in humans [173]. with, and uptake of, the nanovaccines by cells that initiate im- While it induces strong antibody responses, cell-mediated re- mune responses [159]. The fact that proteosomes are effective sponses are often low, and inflammatory reactions at the site nasal vaccines is considered to be particularly related to this en- of injection are usual [174]. Immunological characteristics of a novel nanobead adjuvant have been investigated in a large- hanced recognition and uptake afforded by the particulate and animal sheep model [175]. Contrarily to alum, the antigen co- hydrophobic nature of proteosomes [160]. This nanocarrier is valently bound to nanobeads induced substantial cell-mediated being applied to develop nanovaccines for influenza [35,36] al- responses, along with moderate humoral responses [60]. No ad- lergies [37], plague [38], Shigellosis [39], respiratory syncytial verse results were recognized at the site of immunization in virus [40], and AIDS [41]. the sheep [61]. Thus, nanobead adjuvants in veterinary species may be useful for the induction of immunity to viral pathogens, 3.2. SuperFluids where a cell-mediated response is favorable [62]. These find- ings also highlight the useful ability of nanobead vaccines for SuperFluids are being developed for nanoencapsulating po- intracellular pathogens in humans [63]. tent viral antigens in biodegradable polymer nanospheres for Nanobeads measure 40 nm [176]. Most adjuvants only stim- controlled release. These nanocarriers are supercritical, critical, ulate antibodies against a particular disease. Nanobead technol- or near-critical fluids, with or without polar cosolvents [161]. ogy gives the immune system a further boost, also producing T P. Gill / Scientia Iranica, Transactions F: Nanotechnology 20 (2013) 1003–1013 1007 cells which are required to eliminate viruses or cancer [177]. The action of influenza virosomes is likely to involve both The size of 40 nm is critical, as it is similar in size to many delivery of the enclosed antigen to the cytosol of antigen pre- viruses, where the nanobeads are taken up abundantly by the senting cells and the powerful helper activity of the viroso- immune system and tricked into manufacturing high levels of mal hemagglutinin. Although the immune responses elicited by many types of T-cell [178]. Covalently attached peptide to inert DNA-virosomes are moderate, they are promising and warrant carrier nano-beads can induce both cellular and humoral im- further research to ultimately develop effective DNA-based vi- munity in large outbreed animals, such as sheep [179]. This fact rosomal nanovaccines [199]. The presence of virus proteins not indicates that combining several peptides in the peptide-nano- only allows the liposome to target a particular cell, but also al- bead based vaccine approach can improve immunogenicity and lows it to fuse with the cell, ensuring direct delivery of the in- may be beneficial when dealing with a highly variable pathogen corporated material [200]. like foot-and-mouth disease virus [180]. The key feature of the immunopotentiating reconstituted in- fluenza virosomes is cytoplasmic delivery of the molecules en- 3.4. Virosomes capsulated in or associated with the nanoparticle, as opposed to plain liposomes being a prerequisite for efficient immune acti- Virosomes were developed from liposomes by combining vation [201]. Two human nanovaccines based on this technol- liposomes with fusogenic viral envelope proteins [181]. Unlike ogy (i.e., hepatitis A virus and influenza) have been successfully viruses, virosomes are not capable of replication but are pure transferred to the market. Several additional candidates are also fusion-active vesicles [182]. Due to the presence of specialized under development [202–204]. The excellent immunogenicity, viral proteins on the surface of virosomes, they can be applied as well as superior tolerability, of virosomal nanovaccines has to the active targeting and delivery of their content at the target been clinically approved. In the trivalent influenza nanovaccine, site [183]. Viruses have developed the ability to fuse with cells the virosome components (hemagglutinin and neuraminidase), during the course of evolution, allowing for release of their which are isolated from virus strains yearly recommended by contents directly into the cell [184]. This is because of the the World Health Organization, correspond to the nanovaccine presence of fusogenic proteins on the viral surface that facilitate protective antigens [202]. this fusion. If these fusogenic viral proteins are reconstituted Furthermore, virosomes have been shown to induce cyto- on the surface of a liposome, then, the liposome also acquires toxic T cell responses specific to encapsulated peptides. There- the ability to fuse with cells [185]. This is an extremely useful fore, they may also be employed to incorporate other unrelated tool in active transport, because it allows the direct release of antigens in the virosomal membrane; for example, a viroso- the liposomal contents into the cell. As there is no diffusion mal hepatitis A virus nanovaccine is currently on the market of bioactive material involved, it results in a more effective [203,204]. delivery. In another words, virosomes are reconstituted viral Further development of virosomal nanovaccines will include envelopes that retain the receptor binding and membrane the formulation of peptide epitopes (e.g. malaria, and hepatitis fusion activities of the virus from which they are derived [186]. C) [205,206] and approaches based on the use of recombinant Upon endocytosis, the low pH in the endosomes induces protein [52] or [53]. Because virosomal nanovac- fusion of the virosomal membrane with the endosomal cines can induce both cellular and humoral responses, these ef- membrane, causing the release of the contents of the virosome forts are not limited to prophylactic purposes, but aim also for into the cytoplasm of the cell [187]. The fusion process is therapeutic applications in the field of chronic infectious dis- mediated by hemagglutinin, the major envelope glycoprotein eases and cancer [54]. of the influenza virus [188]. Virosomes can be manufactured by detergent solubilization of the membrane of an enveloped 3.5. Liposomes virus, sedimentation of the viral nucleocapsid, and subsequent selective removal of the detergent from the supernatant, to Liposomes are vesicles of varying size consisting of a spher- produce reconstituted membrane vesicles consisting of viral ical lipid bilayer and an aqueous inner compartment that is envelope lipids and glycoproteins [189]. The most common produced in vitro [207]. First, liposomes were employed as a viruses used in the fabrication of virosomes are: Sendai, Semliki model to study the effect of narcotics on lipid bilayer mem- Forest, influenza, herpes simplex, and vesicular stomatitis branes [208]. Later, the liposomal structures were used as an [190–194]. The size and surface characteristics of virosomes immunological adjuvant [209]. Since these early experiments, can be measured through microscopic visualization. Almeida liposomes have become an established carrier and delivery ve- and colleagues published the first report on the generation hicle in the field of pharmaco- and immuno-therapy. of lipid vesicles containing viral spike proteins derived from Various aspects of immune responses mediated by lipo- the influenza virus [195]. They succeeded in producing somes in vivo show their promise in the development of membrane vesicles with spike proteins protruding from the optimized and subunit-defined nanovaccines [210]. The exten- vesicle surface using preformed liposomes and hemagglutinin sive literature demonstrates that liposomes are endocytosed and neuraminidase, purified from the influenza virus [196]. and they undergo processing through a very well character- Visualization of these vesicles by electron microscopy revealed ized endocytic pathway, delivering their contents, including that they very much resembled the native influenza virus. peptide antigens [211] to lysosomes [212]. Liposomes are a Consequently, they were called virosomes [197]. unique among delivery systems in that they are capable of + + Reconstituted virosomes or artificial viral envelopes appear modulating the generation of both CD4 [45] and CD8 [46] to be ideally suited as nanovaccine formulations for the deliv- mediated immune responses, and generation of Th1, Th2 or ery of protein antigens to the cytosol of antigen presenting cells, Th1/Th2 [47] phenotypes, and may possess the capacity to mod- and thus, for the introduction of antigenic peptides into the ulate a Th1/Th2 switch [48]. The induction of cell-mediated im- MHC class I presentation pathway [198]. Cytotoxic T-cell activ- mune responses clearly proposes the occurrence of intracellu- ity can be induced by immunization of mice with an antigenic lar delivery of antigen to antigen presenting cells via the classi- peptide or an entire protein encapsulated in virosomes [51]. cal MHC I and MHC II pathways. Additionally, liposomes might 1008 P. Gill / Scientia Iranica, Transactions F: Nanotechnology 20 (2013) 1003–1013 act as adjuvants through the nonclassical pathway mediated nano-sized were identical to the nanoparticles that by CD1 molecules [49]. Liposomes have also been reported to are obtained when CaCO3 precipitates [229]. Further investiga- promote the development of T-cell independent immune re- tions indicated that varying the levels of CO2 and NaHCO3 in the sponses [213], and are recognized for their potential to promote growth medium that was used to incubate the human serum long-term immunity through the development of T-cell mem- could change the appearance of the nanobacteria-like particles ory [214]. and, so, it could be concluded that the nanobacteria are CaCO3 The findings outlined above show the versatility and capa- precipitates [230]. In another study, the nanobacteria strain bility of liposomes for cell-mediated immunization. However, Nanobacterium sp. strain Seralab 901045 was analyzed [231]. the specific mechanisms by which they modulate antigen pre- This strain was submitted to a comprehensive battery of tests, sentation remains unclear [215], and their delivery mechanisms and the researchers concluded that there was no evidence have not been optimized for immunization purposes. It has to support the theory that nanobacteria are living organisms, been suggested that immunization by the use of liposomes with and instead proposed that they are self-propagating nanopar- entrapped DNA could circumvent the request for muscle in- ticles that comprise a fetuin-containing mineral–protein com- volvement and facilitate [216], instead, the uptake of DNA by plex [232]. antigen presenting cells infiltrating the site of injection or in On the other hand, nanobacteria are cytopathic in cells and the lymphatics, at the same time protecting DNA from extra- invade mammalian cells in a distinctive pathway. Nanobacte- cellular degradation [217]. Moreover, transfection of antigen ria trigger mammalian cells that are not commonly phagocytic presenting cells with the liposomal DNA and subsequent im- to engulf them, subsequently [233]. The nano-organisms are mune responses to the expressed antigen could be promoted one of the agents for cell vacuolization, poor thriving and un- by the judicious choice of vesicle surface charge, size, and lipid expected cell lysis, problems generally encountered in mam- composition or by the coentrapment of DNA with the plasmids malian cell cultures. Electron microscopy and FITC staining with expressing appropriate cytokines or immuno-stimulatory se- specific monoclonal antibodies revealed that nanobacteria were quences [218]. bound on the surface of the fibroblasts [234]. This means that Procedures have been now developed, by which DNA can be quantitatively entrapped into large [219] or nanobacteria were internalized, either by receptor-mediated small [220] neutral, anionic, or cationic liposomes that are ca- endocytosis or in a closely related manner. Fibroblasts indicated pable of transfecting cells in vitro with varying efficiency [221]. apoptotic abnormalities and death after internalization, if sub- Using liposomes, immunization of inbred or outbred mice by a jected to a high dose of nanobacteria per cell [233,234]. variety of routes, including the oral route [222], with cationic In summary, nanobacteria are non-detectable organisms liposomal DNA, led to much greater humoral and cell-mediated with present sterility testing procedures; however, they are de- immune responses, including cytotoxic T cell [50] immune re- tectable with advanced culture and immunoassays. They are sponses to the encoded antigen, than those gained with naked commonly present in serum and blood products and subse- DNA or DNA complexed to preformed similar liposomes. This quently in cell cultures and antigens (such as nanovaccines de- approach to genetic immunization mimics the way by which rived therefrom). Also, they may be present in antibody and immunity is acquired in viral infections, where both the viral gammaglobulin products. Nanobacteria are a potential risk be- DNA and the envelope proteins it encodes contribute to the im- cause of their cytotoxic properties and capability of infecting mune responses against the virus [223]. This technology has fetuses. Thus, their pathogenicity should be scrutinized [235]. been further advanced by the finding that coating liposomes containing the DNA and protein nanovaccines with mannose 5. Conclusions residues, via incorporation into the bilayers of a mannosylated lipid, further potentiates immune responses to the nanovaccine, presumably by the targeting of such liposomes to the mannose Nanobiotechnology in nanomedicine is in its infancy, hav- receptors on the surface of antigen presenting cells [224]. ing the potential to change medical research dramatically in the twenty-first century. Nanocarriers can be employed for analytical, nanoimaging [236], nanodiagnostic [237–239], nan- 4. Nanobacteria otherapeutic [240], and nanovaccination (Table 1)[241] goals. Objectives, such as targeting cancer [242], drug delivery [243], Nanobacteria are novel self-replicating- and mineralizing agents. The nanobioparticles have been purported to be living improving cell-material interaction [244], scaffolds for tissue organisms that are 80–500 nm in size [225]. Nanobacteria have engineering [245], gene delivery systems [246], and provid- been identified by National Aeronautics and Space Adminis- ing innovative opportunities in the fight against incurable tration (NASA) scientists as a potential culprit in kidney stone diseases [247], will improve the use of nanobiotechnologies. formation among astronauts. With the potential for future ex- Having more knowledge about nanobiotechnological tools and ploratory space missions to the Moon and , longer mis- techniques [248,249], there has been great progress on rec- sions, and exposure to the elements of outer space, health is a ognizing the function of biological nanostructures and their major concern for astronauts [226]. The concept that nanobac- interaction and integration with several non-living systems. teria are living organisms is still controversial, because research However, there are still open issues to be considered, mainly into their putative nucleic acid has not been completed yet. related to the biocompatibility of the nanomaterials which are However, researchers have provided additional clues to under- entered into the body [250]. Many novel nano-sized materials standing nanobacteria and its link to pathologic calcification- and nanocarriers are expected to be used, with an enormous related diseases [227]. positive impact on human health. In fact, the side-effects can Nanobacteria are, in fact, an abiotic phenomenon [228]. First be significantly reduced by delivering the vaccine agents us- reports stated that the investigators could obtain nanobacteria- ing nanocarriers. For example, the vision is to improve health like particles from healthy human serum by following a by enhancing the efficacy and safety of vaccination using previously published protocol, but they observed that these nanobiotechnological approaches [251]. P. Gill / Scientia Iranica, Transactions F: Nanotechnology 20 (2013) 1003–1013 1009

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Pooria Gill received his Ph.D. degree in Nanobiotechnology from Tarbiat [228] Hoover, R.B. ‘‘Mineralized remains of morphotypes of filamentous Modares University, Tehran, Iran. He is Assistant Professor of Nanobiotechnol- in carbonaceous meteorites’’, Proc. SPIE-Int. Soc. Opt. Eng., ogy and Nanomedicine, and Head of the Department of Medical Nanotechnol- 5906, pp. 1–17 Art. no. 59060J (2005). ogy in the Faculty of Advanced Medical Technologies at Golestan University of [229] Martel, J. and Young, J.D.-E. ‘‘Purported nanobacteria in human blood Medical Sciences, Gorgan, Iran. He is also researcher at the Institute for Ad- as nanoparticles’’, Proc. Natl. Acad. Sci. USA., 105(14), vanced Medical Technologies (IAMT), Tehran University of Medical Sciences pp. 5549–5554 (2008). (TUMS), Tehran, Iran.