Nanocarriers, nanovaccines, and nanobacteria as nanobiotechnological concerns in modern vaccines

==Abstract==

This review briefly describes the concerns of nanobiotechnology in the design and development of novel vaccines using the most known nanocarriers, including nature-made nanocarriers (such as bacterial spores, virus-like particles, exosomes, and bacteriophages), man-made nanocarriers (such as Proteosomes, liposomes, virosomes, SuperFluids, and nanobeads), and their applications in therapeutic and protective immunization, as well as their advantages and disadvantages. Here, we focus on the development of nano-based vaccines as “nanovaccines” for inducing immune systems, and the foreseeable promises and problems when compared with existing vaccines. Also, we review a potential nano-hazard for vaccines, so-called nanobacterial contamination.

==Keywords==

Nanocarrier ; Nanovaccine ; Bacterial spore ; Proteosome ; Exosome ; Liposome ; Virosome ; SuperFluid ; Nanobead ; Virus-like particle (VLP) ; Bacteriophage ; Nanobacteria

==1. Introduction==

Vaccination is the most effective strategy for reducing high morbidity and mortality rates, as well as diminishing the enormous social and economic impact associated with disorders. Vaccines are utilized to stimulate the immune system to initiate an immune response against specific targets, such as cancer cells and microbial agents  [[#br000005|[1]]]  and [[#br000010|[2]]] . Various types of vaccine may be used to treat cancers, such as whole tumor cell vaccines, dendritic cell vaccines, idiotype vaccines, antigen or adjuvant vaccines, viral vectors, and DNA vaccines  [[#br000015|[3]]] , [[#br000020|[4]]] , [[#br000025|[5]]] , [[#br000030|[6]]] , [[#br000035|[7]]]  and [[#br000040|[8]]] . Although the immunologic effect is not always sufficient to reverse the progression of cancer, vaccines are generally well tolerated and provide useful anticancer effects in some situations  [[#br000045|[9]]] . Antigen cancer vaccines are typically composed of a cancer associated antigen, not normally present on healthy cells, along with other components (i.e. adjuvants) utilized to stimulate the immune responses against the antigens, resulting in the destruction of malignant cells, without harming normal cells, and preventing recurrence  [[#br000050|[10]]] .      

On the other hand, infection can protect against subsequent disease by induction of both humoral and cellular immunity, but, inert protein-based vaccines are not as effective  [[#br000055|[11]]] . CD<sub>8</sub>  T cells play a vital role in protective immunity against many intracellular pathogens and cancer, but are notoriously difficult to activate by vaccination and immunotherapy  [[#br000060|[12]]] . Vaccines that require a specific immunization sequence with repeated doses (such as prime-boost) are very efficient, but complicated to administer, where logistic considerations of vaccine distribution are critical  [[#br000065|[13]]] .      

As a novel approach, simple inert nanocarriers able to generate strong protection after a single dose could be useful for a variety of applications  [[#br000070|[14]]] . These nanomaterials could be classified as nature-made and man-made nanocarriers/nanoparticles. In fact, nanobiotechnology is particularly used in developing a new generation of more effective vaccines (i.e., nanovaccines) capable of overcoming the many biological, biophysical, and biomedical barriers that the body builds against standard intervention  [[#br000075|[15]]] . Nanoparticles show much promise in cancer therapy, by selectively gaining access to tumors, due to their small size and modifiability  [[#br000080|[16]]] . Nano-sized particles have similarly been used for vaccine construction against microbial agents  [[#br000085|[17]]] . Typically, nanoparticles are defined as particles between 0.1 and 100 nm  [[#br000090|[18]]] . Nanoparticles are formulated out of a variety of substances and fabricated to carry an array of substances in a controlled and targeted mode  [[#br000095|[19]]] . Nanocarriers are prepared to take advantage of fundamental cancer morphologies and modes of development, such as rapid cellular proliferation, antigen expression, and leaky tumor vasculature  [[#br000100|[20]]] . Additionally, nanocarriers are promising as the delivery systems for DNA vaccines  [[#br000105|[21]]] . Nanoemulsions or nanosized aerosol vaccines are also under development  [[#br000110|[22]]]  and [[#br000115|[23]]] . Although many reports have shown that safe, biocompatible materials could be engineered into nanoparticles that contain drugs or vaccines, researchers are trying to develop new materials for vectors that interact specifically and predictably with cells. Nanosystems will be designed to evade normal barriers and stimulate antigen-presenting cells of the immune system  [[#br000120|[24]]] , [[#br000125|[25]]]  and [[#br000130|[26]]] . Scientists are also looking to these “smart” targetable nanoparticles as magic bullets that could seek out and destroy individual cancer cells while bypassing healthy ones  [[#br000135|[27]]] . Hence, various types of nano-scaled material have been employed for the design and fabrication of nanovaccines ([[#t000005|Table 1]] ), based on nature or man-made nanocarriers. Bacterial spores, Proteosomes, exosomes, liposomes, virosomes, SuperFluids, nanoparticle-based nanobeads, virus-like particles and bacteriophages are some instances of nano-sized vehicles that are reviewed here as potential nanocarriers for vaccine delivery to immune systems  [[#br000140|[28]]] . Nanobacteria are another important subject for review here, because of their role in the contamination of vaccines and some complicated disorders  [[#br000370|[74]]] . Because nanobacteria have been found to be a contaminant in previously assumed-to-be-sterile medical products, we also aim to discuss it as a nano-hazard in this review  [[#br000375|[75]]] .

<span id='t000005'></span>

{| class="wikitable" style="min-width: 60%;margin-left: auto; margin-right: auto;"
|+

Table 1.

Characteristics of nanocarriers as nanovaccines.

|-

! Nanocarrier
! Source
! Size (nm)
! Immune response
! Ref. no.
|-

| '''Spore'''
| Bacterial
| Various
| Humoral-cellular
| [[#br000145|[29]]] , [[#br000150|[30]]] , [[#br000155|[31]]] , [[#br000160|[32]]] , [[#br000165|[33]]]  and [[#br000170|[34]]]
|-

| '''Proteosome'''
| Membrane protein-based
| 20–800
| Humoral-cellular
| [[#br000175|[35]]] , [[#br000180|[36]]] , [[#br000185|[37]]] , [[#br000190|[38]]] , [[#br000195|[39]]] , [[#br000200|[40]]]  and [[#br000205|[41]]]
|-

| '''Exosome'''
| Cellular
| 50–100
| Cellular
| [[#br000210|[42]]] , [[#br000215|[43]]]  and [[#br000220|[44]]]
|-

| '''Liposome'''
| Lipid-based
| Various
| Humoral-cellular
| [[#br000225|[45]]] , [[#br000230|[46]]] , [[#br000235|[47]]] , [[#br000240|[48]]] , [[#br000245|[49]]]  and [[#br000250|[50]]]
|-

| '''Virosome'''
| Liposome + viral env proteins
| Various
| Humoral-cellular
| [[#br000255|[51]]] , [[#br000260|[52]]] , [[#br000265|[53]]]  and [[#br000270|[54]]]
|-

| '''SuperFluid'''
| Biodegradable polymer
| 25–250
| Humoral-cellular
| [[#br000275|[55]]] , [[#br000280|[56]]] , [[#br000285|[57]]] , [[#br000290|[58]]]  and [[#br000295|[59]]]
|-

| '''Nanobead'''
| Inert nanomaterial
| 40
| Humoral-cellular
| [[#br000300|[60]]] , [[#br000305|[61]]] , [[#br000310|[62]]]  and [[#br000315|[63]]]
|-

| '''VLP'''
| Viral
| Various
| Humoral
| [[#br000320|[64]]] , [[#br000325|[65]]] , [[#br000330|[66]]] , [[#br000335|[67]]]  and [[#br000340|[68]]]
|-

| '''Phage'''
| Bacterial
| Various
| Humoral-cellular
| [[#br000345|[69]]] , [[#br000350|[70]]] , [[#br000355|[71]]] , [[#br000360|[72]]]  and [[#br000365|[73]]]
|}

==2. Nature-made nanocarriers==

===2.1. Bacterial spores===

Through evolution, nature has produced highly complex nanoarchitectures using macromolecules, especially polysaccharides, nucleic acids, and proteins  [[#br000380|[76]]] . Understanding the principle of how these macromolecules interact to fabricate nano-scaled structures, it should be possible to exploit this science in the design and development of novel artificial structures and tools  [[#br000385|[77]]] . The advantage of this “learning from nature” approach is that it well defines the bionanofabrication processes that already exist in bacteria  [[#br000390|[78]]] . Microorganisms have novel and interesting structures, such as bacterial spore coats  [[#br000395|[79]]] , which have protective characteristics  [[#br000400|[80]]] . Bacterial spores are dormant life forms with formidable resistance properties. These nanostructures attribute multiple layers of protein, which encase the spores in a protective and flexible shell [[#br000405|[81]]] , [[#br000410|[82]]] , [[#br000415|[83]]] , [[#br000420|[84]]]  and [[#br000425|[85]]] . The shield has features that are pertinent for merging with nanobiotechnological fields, such as self-assembling protomers, and a capacity for engineering and delivering foreign molecules  [[#br000430|[86]]] . In fact, the spore coat could be employed not only as a delivery vehicle for various molecules, but also as a source of novel self assembling biomolecules  [[#br000435|[87]]] . The coat can act as a device for heterologous antigen presentation and prophylactic immunization  [[#br000145|[29]]] .      

There are a number of attributes that make bacillus spores particularly suitable candidates for vaccine vehicles  [[#br000150|[30]]]  and [[#br000155|[31]]] . Two distinct approaches are accessible for vaccination. In the first approach, an antigen or epitope is engineered onto the spore coat by fusing a spore coat gene with the antigen sequence  [[#br000160|[32]]] , [[#br000165|[33]]]  and [[#br000170|[34]]] . In the second, the antigen is expressed constitutively in the vegetative cell by fusion of the antigen gene to the transcriptional and translational sequences of a suitable bacillus gene  [[#br000440|[88]]] , [[#br000445|[89]]]  and [[#br000450|[90]]] . Spores carrying this modified gene are used for oral delivery and then germinate in the gastrointestinal system  [[#br000455|[91]]] , [[#br000460|[92]]]  and [[#br000465|[93]]] . Consequently, the spore offers unique capabilities for nanovaccine development that could ultimately lead to improved public health, both in developed and developing countries.      

===2.2. Virus-like particles===

Virus-Like Particles (VLPs) structurally mimic the viral capsid, and have been extensively and successfully employed as nanovaccines  [[#br000470|[94]]] . The capability of VLPs to include nucleic acids and small biomolecules has also made them novel vehicles for gene and vaccine deliveries  [[#br000475|[95]]] . The repetitive surface of VLPs has regularly been used as a template for bionanofabrication  [[#br000480|[96]]] . Recent progress has been shaped towards the development of virus capsids, along with the precise spacing of surface peptide domains, which enables the modification of the capsid shield through the fabrication of repetitive chemical adducts on a nanoscale platform  [[#br000485|[97]]] . Recently, capsid engineering has been improved for bacteriophage and plant virus capsids, with an extension to purified VLPs  [[#br000490|[98]]] . VLPs of various viruses have been confirmed as very immunogenic and are already in use as nanovaccines in humans or animals  [[#br000495|[99]]] . For instance, the immunization of young healthy women with VLPs composed of the major structural protein, L<sub>1</sub> , of HPV <sub>16</sub> , induced high titer neutralizing antibodies, and protected them from HPV infection and associated cervical dysplasia  [[#br000320|[64]]] . The vaccination mode decreases the incidence of cervical cancer; however, a longer follow-up is essential to confirm this action. VLPs have additionally been employed to deliver immunogenic epitopes of other pathogens. They have several advantages as immunogens in the form of short peptides  [[#br000325|[65]]] .      

The linking of VLPs to adjuvant molecules was also shown to improve the immunogenicity of the nanobioparticles  [[#br000330|[66]]] . For instance, the nontoxic subunit of cholera toxin (CTB) was chemically linked to VLPs via biotin-streptavidin  [[#br000335|[67]]] . There was a higher level of Env-specific serum IgG<sub>1</sub> , secretory IgA antibodies and cellular immune responses than in either the absence of CTB or when CTB was just co-injected  [[#br000340|[68]]] . As there is increasing awareness of the VLP structure, it is expected that the development of VLPs as nanovaccines, as carriers for delivering small molecules, and as the basis for nanoarrays, will continue with even greater potency  [[#br000500|[100]]] , [[#br000505|[101]]]  and [[#br000510|[102]]] .      

===2.3. Bacteriophages===

While the study of the interaction of phage nanobioparticles and animals is still in its infancy, the capacity of a few of the well-studied phage strains to deliver antigenic gene products has been investigated  [[#br000515|[103]]] , [[#br000520|[104]]]  and [[#br000525|[105]]] . Phage vaccines introduced in 1985 showed that it was possible to manufacture bacteriophages that display foreign proteins fused to their normal coat proteins  [[#br000530|[106]]] . Following the description of phage display technology, its power was greatly improved using the affinity selection to isolate phages that displayed specific peptides from random peptide collections  [[#br000535|[107]]] , [[#br000540|[108]]]  and [[#br000545|[109]]] . Employment of this technology for the development of vaccines and diagnostics has become an industry  [[#br000550|[110]]] , [[#br000555|[111]]]  and [[#br000560|[112]]] . Additionally, it was also possible to use specific epitopes that have been chosen on the basis of biological experiments  [[#br000565|[113]]] . For instance, a vaccine using a phage display system provided complete immunological protection to mice nasally challenged with live respiratory syncytial virus  [[#br000570|[114]]] . A similar approach was applied to construct a phage carrying a gene product protection against ''Yersinia pestis'' infections   [[#br000345|[69]]] . The phage IgG<sub>2</sub>  responses were indicated to be significantly higher following intramuscular phage vaccinations with the phage vaccine than with the plasmid DNA vaccine  [[#br000350|[70]]] . Moreover, it was demonstrated that the phage nanovaccines might be made more efficient via decorating their coats with phage fusion proteins displaying immunogenic antigens for dendritic cells  [[#br000355|[71]]] . This could be accomplished using a peptide that has an attaching epitope for the CD<sub>40</sub>  receptor, which has numerous functions in the activation of antigen-presenting cells  [[#br000360|[72]]] . This requirement for the peptide display by phage vaccines can be enhanced by application of a developed dual expression system that permits the generation of phages with mosaic heads that can accommodate recalcitrant recombinant fusion proteins. This system employed bacteria carrying a prophage that is deficient in a major head protein, along with the plasmids carrying foreign head protein genes for both wild-type and recombinant phages  [[#br000365|[73]]] .      

===2.4. Exosomes===

Exosomes are small (50–100 nm), spherical vesicles manufactured and released by most cells, like B cells  [[#br000575|[115]]] , T cells  [[#br000580|[116]]] , mast cells  [[#br000585|[117]]] , epithelial cells  [[#br000590|[118]]]  and Dendritic Cells (DCs)  [[#br000595|[119]]] , to facilitate intercellular communication. These vesicles are of an endosomal origin, which are secreted in the extracellular milieu following the fusion of late endosomal multivesicular bodies with the plasma membrane  [[#br000600|[120]]] . Exosomes have a defined protein composition, which confers specific biological activities contingent on the nature of the producing cell  [[#br000605|[121]]] . In the multivesicular bodies of antigen presenting cells, exogenous antigens are loaded onto MHC molecules and when the multivesicular bodies fuse with the cell membrane, the MHC molecules are incorporated into the cell membrane  [[#br000610|[122]]] . The internal vesicles are simultaneously released as extracellular exosomes  [[#br000615|[123]]] .      

Exosomes from antigen presenting cells bear not only MHC class I and II, but also costimulatory molecules, such as CD<sub>54</sub> ,CD<sub>80</sub>  and CD<sub>86</sub> , and they are enriched in tetraspanin proteins, like CD<sub>63</sub>  and CD<sub>81</sub>    [[#br000620|[124]]]  and [[#br000625|[125]]] . Tetraspanin proteins have been proposed to have a modulatory role in several different cell processes, including adhesion, migration and proliferation  [[#br000630|[126]]] . Exosomes have been confirmed to be present in bronchoalveolar lavage  [[#br000635|[127]]] , human plasma  [[#br000640|[128]]] , malignant effusions [[#br000645|[129]]]  and [[#br000650|[130]]]  and on the surface of follicular dendritic cells  [[#br000655|[131]]] ; however, their physiological roles in vivo are still unclear. They have been suggested to participate both in T cell stimulation  [[#br000210|[42]]]  and [[#br000215|[43]]]  and in tolerance induction  [[#br000220|[44]]] . Studies in mice have demonstrated the potential of exosomes as immunotherapeutic agents in infections  [[#br000660|[132]]] , transplantations  [[#br000665|[133]]] , and cancer  [[#br000670|[134]]] , [[#br000675|[135]]]  and [[#br000680|[136]]] .      

The mechanisms for how exosomes stimulate T cells are unknown. Some reports indicate that the exosomes from antigen presenting cells can stimulate T cell clones, on their own  [[#br000610|[122]]]  and more efficiently, if the exosomes come from mature dendritic cells  [[#br000210|[42]]]  and [[#br000680|[136]]] . Other studies confirmed that exosomes require the presence of dendritic cells to efficiently stimulate T lymphocytes  [[#br000215|[43]]] , [[#br000685|[137]]]  and [[#br000690|[138]]] . Some reports have employed T cells  [[#br000215|[43]]]  and [[#br000695|[139]]] , hybridomas  [[#br000215|[43]]]  and [[#br000680|[136]]]  or clones  [[#br000610|[122]]] , [[#br000210|[42]]] , [[#br000685|[137]]]  and [[#br000690|[138]]]  when looking at the capability of exosomes to stimulate T cells in an antigen-specific manner. These studies demonstrate that exosomes can directly stimulate viral-specific peripheral CD<sub>8</sub><sup>+</sup>  T cells  [[#br000700|[140]]] .      

Although exosomes express tumor antigens, leading to their proposed utility as nanovaccines, they also can suppress T-cell-signaling molecules and induce apoptosis  [[#br000705|[141]]] . The first phase I clinical trial using autologous exosomes has indicated the feasibility of large-scale exosome production and the safety of exosome administration  [[#br000710|[142]]] . Exosomes manufactured by dendritic cells are named “dexosomes” and contain essential components to activate both adaptive and innate immune responses  [[#br000715|[143]]] . For instance, dexosome nanovaccines have been developed that employ patient-specific dexosomes loaded with tumor antigen-derived peptides to treat cancer  [[#br000720|[144]]] . The Exosome Display Technology manufactured by Anosys provides the capability to manipulate exosome composition, and tailor exosomes, with new desirable characteristics, opening up opportunities in the field of recombinant vaccine and monoclonal antibody preparation  [[#br000725|[145]]] . This is gained via generating gene coding for the chimeric proteins, attaching an exosome addressing sequence to antigens or biologically active proteins  [[#br000730|[146]]] . The resulting proteins are targeted to the exosomal compartment and released in the extracellular milieu enclosed to exosomes  [[#br000735|[147]]] . Exosome research continues to reveal unique potential that broadens its field of application.      

==3. Man-made nanocarriers==

===3.1. Proteosomes===

Proteosomes are vaccine delivery vehicles by virtue of their nanoparticulate nature, forming vesicles and vesicle clusters comparable to the size of small viruses  [[#br000740|[148]]]  and [[#br000745|[149]]] . Proteosome has been used to describe preparations of the outer membrane proteins from microorganisms  [[#br000750|[150]]] , [[#br000755|[151]]] , [[#br000760|[152]]] , [[#br000765|[153]]]  and [[#br000770|[154]]] . Proteosomes are described as comparable in size to certain virus particles that are hydrophobic and safe for human application  [[#br000775|[155]]] . Proteosomes are said to be useful in formulating vaccines with a variety of proteins and peptides  [[#br000780|[156]]]  and [[#br000785|[157]]] . Proteosome vaccine vesicles and vesicle clusters may range in size from 20 to 800 nm, based on the type and amount of antigen encapsulated with the proteosomes  [[#br000790|[158]]] . The hydrophobic nature of the proteosome porin proteins also contributes to vaccine delivery capabilities by facilitating interaction of the vaccine nanoparticles with, and uptake of, the nanovaccines by cells that initiate immune responses  [[#br000795|[159]]] . The fact that proteosomes are effective nasal vaccines is considered to be particularly related to this enhanced recognition and uptake afforded by the particulate and hydrophobic nature of proteosomes  [[#br000800|[160]]] . This nanocarrier is being applied to develop nanovaccines for influenza  [[#br000175|[35]]]  and [[#br000180|[36]]]  allergies  [[#br000185|[37]]] , plague  [[#br000190|[38]]] , Shigellosis  [[#br000195|[39]]] , respiratory syncytial virus  [[#br000200|[40]]] , and AIDS  [[#br000205|[41]]] .      

===3.2. SuperFluids===

SuperFluids are being developed for nanoencapsulating potent viral antigens in biodegradable polymer nanospheres for controlled release. These nanocarriers are supercritical, critical, or near-critical fluids, with or without polar cosolvents  [[#br000805|[161]]] . Application of SuperFluids reduces the exposure of viral antigens, such as HIV and influenza, to potentially denaturing organic solvents, such as methylenechloride and ethyl acetate, and improves the stability of protein antigens in the body at an ambient temperature for a long time. Therefore, the nanocarriers enhance the capability of nanoencapsulated vaccine antigens to induce the production of protective and neutralizing antibodies  [[#br000275|[55]]]  and [[#br000280|[56]]] .      

This controlled release nanovaccine delivery system has the capability to deliver different types and combinations of HIV or influenza vaccine candidates (such as whole inactivated virus particles, DNA plasmids, and subunit protein antigens)  [[#br000285|[57]]] , [[#br000290|[58]]]  and [[#br000295|[59]]] . The polymer nanoencapsulation technology will decrease cost by eliminating unnecessary processing steps, while improving the manufacturing environment  [[#br000810|[162]]] . The nanocarrier technology is portable, inexpensive, and amenable to large-scale processing  [[#br000815|[163]]] .      

Several enabling nanotechnologies have been developed, according to SuperFluids, for the enhanced delivery of nanovaccines and nanodrugs, ranging from potent anticancer therapeutics to large recombinant proteins. One of these technologies is protein nanocarriers  [[#br000820|[164]]] . Protein nanocarriers can be generated without reduction in protein integrity and efficacy in a solvent-free process, based on a SuperFluids rapid expansion mechanism for the enhanced drug delivery of protein macromolecules. This technology can be applied to the pulmonary delivery of proteins and polypeptides. It also is used for oral or depot delivery of vaccine antigens. Another form of these technologies is polymer nanospheres  [[#br000825|[165]]]  and [[#br000830|[166]]] . Biodegradable polymeric nanospheres can be manufactured utilizing SuperFluids technologies for the controlled delivery and release of proteins through reverse engineering of the rapid expansion mechanism  [[#br000835|[167]]] . This technology can be employed for oral or depot delivery of proteins and controlled-release adjuvants of vaccine antigens for infectious diseases  [[#br000840|[168]]] . The other generation of these technologies is phospholipid nanosomes  [[#br000845|[169]]] . Phospholipid nanosomes or small uniform liposomes for the enhanced delivery of hydrophilic and hydrophobic drugs can be produced without the use of toxic organic solvents, using a similar mechanism  [[#br000850|[170]]] . This technology can be employed for intravenous and topical administration of small hydrophilic nanodrugs, proteins and genes  [[#br000855|[171]]] .      

===3.3. Nanobeads===

Nanoparticle-based nanobeads serve as nanovaccine fabrication and more effective immunization  [[#br000860|[172]]] . Although a number of adjuvants are currently confirmed for use in animal species, only alum has been widely employed in humans  [[#br000865|[173]]] . While it induces strong antibody responses, cell-mediated responses are often low, and inflammatory reactions at the site of injection are usual  [[#br000870|[174]]] . Immunological characteristics of a novel nanobead adjuvant have been investigated in a large-animal sheep model  [[#br000875|[175]]] . Contrarily to alum, the antigen covalently bound to nanobeads induced substantial cell-mediated responses, along with moderate humoral responses  [[#br000300|[60]]] . No adverse results were recognized at the site of immunization in the sheep  [[#br000305|[61]]] . Thus, nanobead adjuvants in veterinary species may be useful for the induction of immunity to viral pathogens, where a cell-mediated response is favorable  [[#br000310|[62]]] . These findings also highlight the useful ability of nanobead vaccines for intracellular pathogens in humans  [[#br000315|[63]]] .      

Nanobeads measure 40 nm  [[#br000880|[176]]] . Most adjuvants only stimulate antibodies against a particular disease. Nanobead technology gives the immune system a further boost, also producing T cells which are required to eliminate viruses or cancer  [[#br000885|[177]]] . The size of 40 nm is critical, as it is similar in size to many viruses, where the nanobeads are taken up abundantly by the immune system and tricked into manufacturing high levels of many types of T-cell  [[#br000890|[178]]] . Covalently attached peptide to inert carrier nano-beads can induce both cellular and humoral immunity in large outbreed animals, such as sheep  [[#br000895|[179]]] . This fact indicates that combining several peptides in the peptide-nano-bead based vaccine approach can improve immunogenicity and may be beneficial when dealing with a highly variable pathogen like foot-and-mouth disease virus  [[#br000900|[180]]] .      

===3.4. Virosomes===

Virosomes were developed from liposomes by combining liposomes with fusogenic viral envelope proteins  [[#br000905|[181]]] . Unlike viruses, virosomes are not capable of replication but are pure fusion-active vesicles  [[#br000910|[182]]] . Due to the presence of specialized viral proteins on the surface of virosomes, they can be applied to the active targeting and delivery of their content at the target site  [[#br000915|[183]]] . Viruses have developed the ability to fuse with cells during the course of evolution, allowing for release of their contents directly into the cell  [[#br000920|[184]]] . This is because of the presence of fusogenic proteins on the viral surface that facilitate this fusion. If these fusogenic viral proteins are reconstituted on the surface of a liposome, then, the liposome also acquires the ability to fuse with cells  [[#br000925|[185]]] . This is an extremely useful tool in active transport, because it allows the direct release of the liposomal contents into the cell. As there is no diffusion of bioactive material involved, it results in a more effective delivery. In another words, virosomes are reconstituted viral envelopes that retain the receptor binding and membrane fusion activities of the virus from which they are derived  [[#br000930|[186]]] .      

Upon endocytosis, the low pH in the endosomes induces fusion of the virosomal membrane with the endosomal membrane, causing the release of the contents of the virosome into the cytoplasm of the cell  [[#br000935|[187]]] . The fusion process is mediated by hemagglutinin, the major envelope glycoprotein of the influenza virus  [[#br000940|[188]]] . Virosomes can be manufactured by detergent solubilization of the membrane of an enveloped virus, sedimentation of the viral nucleocapsid, and subsequent selective removal of the detergent from the supernatant, to produce reconstituted membrane vesicles consisting of viral envelope lipids and glycoproteins  [[#br000945|[189]]] . The most common viruses used in the fabrication of virosomes are: Sendai, Semliki Forest, influenza, herpes simplex, and vesicular stomatitis [[#br000950|[190]]] , [[#br000955|[191]]] , [[#br000960|[192]]] , [[#br000965|[193]]]  and [[#br000970|[194]]] . The size and surface characteristics of virosomes can be measured through microscopic visualization. Almeida and colleagues published the first report on the generation of lipid vesicles containing viral spike proteins derived from the influenza virus  [[#br000975|[195]]] . They succeeded in producing membrane vesicles with spike proteins protruding from the vesicle surface using preformed liposomes and hemagglutinin and neuraminidase, purified from the influenza virus  [[#br000980|[196]]] . Visualization of these vesicles by electron microscopy revealed that they very much resembled the native influenza virus. Consequently, they were called virosomes  [[#br000985|[197]]] .      

Reconstituted virosomes or artificial viral envelopes appear to be ideally suited as nanovaccine formulations for the delivery of protein antigens to the cytosol of antigen presenting cells, and thus, for the introduction of antigenic peptides into the MHC class I presentation pathway  [[#br000990|[198]]] . Cytotoxic T-cell activity can be induced by immunization of mice with an antigenic peptide or an entire protein encapsulated in virosomes  [[#br000255|[51]]] . The action of influenza virosomes is likely to involve both delivery of the enclosed antigen to the cytosol of antigen presenting cells and the powerful helper activity of the virosomal hemagglutinin. Although the immune responses elicited by DNA-virosomes are moderate, they are promising and warrant further research to ultimately develop effective DNA-based virosomal nanovaccines  [[#br000995|[199]]] . The presence of virus proteins not only allows the liposome to target a particular cell, but also allows it to fuse with the cell, ensuring direct delivery of the incorporated material  [[#br001000|[200]]] .      

The key feature of the immunopotentiating reconstituted influenza virosomes is cytoplasmic delivery of the molecules encapsulated in or associated with the nanoparticle, as opposed to plain liposomes being a prerequisite for efficient immune activation  [[#br001005|[201]]] . Two human nanovaccines based on this technology (i.e., hepatitis A virus and influenza) have been successfully transferred to the market. Several additional candidates are also under development  [[#br001010|[202]]] , [[#br001015|[203]]]  and [[#br001020|[204]]] . The excellent immunogenicity, as well as superior tolerability, of virosomal nanovaccines has been clinically approved. In the trivalent influenza nanovaccine, the virosome components (hemagglutinin and neuraminidase), which are isolated from virus strains yearly recommended by the World Health Organization, correspond to the nanovaccine protective antigens  [[#br001010|[202]]] .      

Furthermore, virosomes have been shown to induce cytotoxic T cell responses specific to encapsulated peptides. Therefore, they may also be employed to incorporate other unrelated antigens in the virosomal membrane; for example, a virosomal hepatitis A virus nanovaccine is currently on the market [[#br001015|[203]]]  and [[#br001020|[204]]] .      

Further development of virosomal nanovaccines will include the formulation of peptide epitopes (e.g. malaria, and hepatitis C)  [[#br001025|[205]]]  and [[#br001030|[206]]]  and approaches based on the use of recombinant protein  [[#br000260|[52]]]  or nucleic acid  [[#br000265|[53]]] . Because virosomal nanovaccines can induce both cellular and humoral responses, these efforts are not limited to prophylactic purposes, but aim also for therapeutic applications in the field of chronic infectious diseases and cancer  [[#br000270|[54]]] .      

===3.5. Liposomes===

Liposomes are vesicles of varying size consisting of a spherical lipid bilayer and an aqueous inner compartment that is produced ''in vitro''     [[#br001035|[207]]] . First, liposomes were employed as a model to study the effect of narcotics on lipid bilayer membranes  [[#br001040|[208]]] . Later, the liposomal structures were used as an immunological adjuvant  [[#br001045|[209]]] . Since these early experiments, liposomes have become an established carrier and delivery vehicle in the field of pharmaco- and immuno-therapy.      

Various aspects of immune responses mediated by liposomes ''in vivo''  show their promise in the development of optimized and subunit-defined nanovaccines   [[#br001050|[210]]] . The extensive literature demonstrates that liposomes are endocytosed and they undergo processing through a very well characterized endocytic pathway, delivering their contents, including peptide antigens  [[#br001055|[211]]]  to lysosomes  [[#br001060|[212]]] . Liposomes are a unique among delivery systems in that they are capable of modulating the generation of both CD<sub>4</sub><sup>+</sup>    [[#br000225|[45]]]  and CD<sub>8</sub><sup>+</sup>    [[#br000230|[46]]]  mediated immune responses, and generation of Th<sub>1</sub> ,Th<sub>2</sub>  or Th<sub>1</sub> / Th<sub>2</sub>    [[#br000235|[47]]]  phenotypes, and may possess the capacity to modulate a Th<sub>1</sub> / Th<sub>2</sub>  switch  [[#br000240|[48]]] . The induction of cell-mediated immune responses clearly proposes the occurrence of intracellular delivery of antigen to antigen presenting cells via the classical MHC I and MHC II pathways. Additionally, liposomes might act as adjuvants through the nonclassical pathway mediated by CD<sub>1</sub>  molecules  [[#br000245|[49]]] . Liposomes have also been reported to promote the development of T-cell independent immune responses  [[#br001065|[213]]] , and are recognized for their potential to promote long-term immunity through the development of T-cell memory  [[#br001070|[214]]] .      

The findings outlined above show the versatility and capability of liposomes for cell-mediated immunization. However, the specific mechanisms by which they modulate antigen presentation remains unclear  [[#br001075|[215]]] , and their delivery mechanisms have not been optimized for immunization purposes. It has been suggested that immunization by the use of liposomes with entrapped DNA could circumvent the request for muscle involvement and facilitate  [[#br001080|[216]]] , instead, the uptake of DNA by antigen presenting cells infiltrating the site of injection or in the lymphatics, at the same time protecting DNA from extracellular degradation  [[#br001085|[217]]] . Moreover, transfection of antigen presenting cells with the liposomal DNA and subsequent immune responses to the expressed antigen could be promoted by the judicious choice of vesicle surface charge, size, and lipid composition or by the coentrapment of DNA with the plasmids expressing appropriate cytokines or immuno-stimulatory sequences  [[#br001090|[218]]] .      

Procedures have been now developed, by which plasmid DNA can be quantitatively entrapped into large  [[#br001095|[219]]]  or small  [[#br001100|[220]]]  neutral, anionic, or cationic liposomes that are capable of transfecting cells ''in vitro''  with varying efficiency   [[#br001105|[221]]] . Using liposomes, immunization of inbred or outbred mice by a variety of routes, including the oral route  [[#br001110|[222]]] , with cationic liposomal DNA, led to much greater humoral and cell-mediated immune responses, including cytotoxic T cell  [[#br000250|[50]]]  immune responses to the encoded antigen, than those gained with naked DNA or DNA complexed to preformed similar liposomes. This approach to genetic immunization mimics the way by which immunity is acquired in viral infections, where both the viral DNA and the envelope proteins it encodes contribute to the immune responses against the virus  [[#br001115|[223]]] . This technology has been further advanced by the finding that coating liposomes containing the DNA and protein nanovaccines with mannose 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 receptors on the surface of antigen presenting cells  [[#br001120|[224]]] .      

==4. Nanobacteria==

Nanobacteria are novel self-replicating- and mineralizing agents. The nanobioparticles have been purported to be living organisms that are 80–500 nm in size  [[#br001125|[225]]] . Nanobacteria have been identified by National Aeronautics and Space Administration (NASA) scientists as a potential culprit in kidney stone formation among astronauts. With the potential for future exploratory space missions to the Moon and Mars, longer missions, and exposure to the elements of outer space, health is a major concern for astronauts  [[#br001130|[226]]] . The concept that nanobacteria are living organisms is still controversial, because research into their putative nucleic acid has not been completed yet. However, researchers have provided additional clues to understanding nanobacteria and its link to pathologic calcification-related diseases  [[#br001135|[227]]] .      

Nanobacteria are, in fact, an abiotic phenomenon  [[#br001140|[228]]] . First reports stated that the investigators could obtain nanobacteria-like particles from healthy human serum by following a previously published protocol, but they observed that these nano-sized organisms were identical to the nanoparticles that are obtained when CaCO<sub>3</sub>  precipitates  [[#br001145|[229]]] . Further investigations indicated that varying the levels of CO<sub>2</sub>  and NaHCO<sub>3</sub>  in the growth medium that was used to incubate the human serum could change the appearance of the nanobacteria-like particles and, so, it could be concluded that the nanobacteria are CaCO<sub>3</sub>  precipitates  [[#br001150|[230]]] . In another study, the nanobacteria strain ''Nanobacterium sp. strain Seralab 901045''  was analyzed   [[#br001155|[231]]] . This strain was submitted to a comprehensive battery of tests, and the researchers concluded that there was no evidence to support the theory that nanobacteria are living organisms, and instead proposed that they are self-propagating nanoparticles that comprise a fetuin-containing mineral–protein complex  [[#br001160|[232]]] .      

On the other hand, nanobacteria are cytopathic in cells and invade mammalian cells in a distinctive pathway. Nanobacteria trigger mammalian cells that are not commonly phagocytic to engulf them, subsequently  [[#br001165|[233]]] . The nano-organisms are one of the agents for cell vacuolization, poor thriving and unexpected cell lysis, problems generally encountered in mammalian cell cultures. Electron microscopy and FITC staining with specific monoclonal antibodies revealed that nanobacteria were bound on the surface of the fibroblasts  [[#br001170|[234]]] . This means that nanobacteria were internalized, either by receptor-mediated endocytosis or in a closely related manner. Fibroblasts indicated apoptotic abnormalities and death after internalization, if subjected to a high dose of nanobacteria per cell  [[#br001165|[233]]]  and [[#br001170|[234]]] .      

In summary, nanobacteria are non-detectable organisms with present sterility testing procedures; however, they are detectable with advanced culture and immunoassays. They are commonly present in serum and blood products and subsequently in cell cultures and antigens (such as nanovaccines derived therefrom). Also, they may be present in antibody and gammaglobulin products. Nanobacteria are a potential risk because of their cytotoxic properties and capability of infecting fetuses. Thus, their pathogenicity should be scrutinized  [[#br001175|[235]]] .      

==5. Conclusions==

Nanobiotechnology in nanomedicine is in its infancy, having the potential to change medical research dramatically in the twenty-first century. Nanocarriers can be employed for analytical, nanoimaging  [[#br001180|[236]]] , nanodiagnostic  [[#br001185|[237]]] , [[#br001190|[238]]]  and [[#br001195|[239]]] , nanotherapeutic  [[#br001200|[240]]] , and nanovaccination ([[#t000005|Table 1]] )  [[#br001205|[241]]]  goals. Objectives, such as targeting cancer  [[#br001210|[242]]] , drug delivery  [[#br001215|[243]]] , improving cell-material interaction  [[#br001220|[244]]] , scaffolds for tissue engineering  [[#br001225|[245]]] , gene delivery systems  [[#br001230|[246]]] , and providing innovative opportunities in the fight against incurable diseases  [[#br001235|[247]]] , will improve the use of nanobiotechnologies. Having more knowledge about nanobiotechnological tools and techniques  [[#br001240|[248]]]  and [[#br001245|[249]]] , there has been great progress on recognizing the function of biological nanostructures and their interaction and integration with several non-living systems. However, there are still open issues to be considered, mainly related to the biocompatibility of the nanomaterials which are entered into the body  [[#br001250|[250]]] . Many novel nano-sized materials and nanocarriers are expected to be used, with an enormous positive impact on human health. In fact, the side-effects can be significantly reduced by delivering the vaccine agents using nanocarriers. For example, the vision is to improve health by enhancing the efficacy and safety of vaccination using nanobiotechnological approaches  [[#br001255|[251]]] .      

On the other hand, the safety of nanovaccines is equally important. This concept has been found to be useful in employing nanovaccines. However, with nanovaccines, the particles are cleared slowly over a prolonged time period and this may induce toxicity  [[#br001255|[251]]] ; thus, nanobacterial contamination would be possible. In recent years, some scientists have evaluated different nanomaterials with different assays to show their heterogeneous responses and their activities in life systems  [[#br001260|[252]]] . They also predicted the nanotoxicity of nanomaterials and their safety in human studies. Their efforts will help to speed up nanotoxicological tracking of nanomaterials by applying high-throughput assays and evaluation acceleration using nanomaterials for clinical trials, to manage disorders in the near future  [[#br001255|[251]]]  and [[#br001265|[253]]] .      

In conclusion, “Nanobiotechnology” will play a key role in the medicine of tomorrow (i.e., Nanomedicine) providing revolutionary opportunities for therapeutic and protective ways to improve health and enhance human physical abilities, and enable precise and effective modern nanovaccines tailored to the patient  [[#br001270|[254]]] .

==Acknowledgments==

The present study was partly supported by Golestan University of Medical Sciences (GOUMS)  and the Iranian Nanotechnology Initiative Council (INIC) .

==References==

<ol style='list-style-type: none;margin-left: 0px;'><li><span id='br000005'></span>
[[#br000005|[1]]] J.F.T. Griffin; A strategic approach to vaccine development: animal models, monitoring vaccine efficacy, formulation and delivery; Adv. Drug Deliv. Rev., 54 (6) (2002), pp. 851–861</li>
<li><span id='br000010'></span>
[[#br000010|[2]]] R. Medzhitov, C. Janeway; Innate immunity: the virtues of a nonclonal system of recognition; Cell, 91 (3) (1997), pp. 295–298</li>
<li><span id='br000015'></span>
[[#br000015|[3]]] M. Shi, S. Hao, L. Su, X. Zhang, J. Yuan, X. Guo, C. Zheng, J. Xiang; Vaccine of engineered tumor cells secreting stromal cell-derived factor-1 induces T-cell dependent antitumor responses; Cancer Biother. Radio., 20 (4) (2005), pp. 401–409</li>
<li><span id='br000020'></span>
[[#br000020|[4]]] S. Prasad, V. Cody, J.K. Saucier-Sawyer, W.M. Saltzman, C.T. Sasaki, R.L. Edelson, M.A. Birchall, D.J. Hanlon; Polymer nanoparticles containing tumor lysates as antigen delivery vehicles for dendritic cell-based antitumor immunotherapy; Nanomed. Nanotech. Biol. Med., 7 (1) (2011), pp. 1–10</li>
<li><span id='br000025'></span>
[[#br000025|[5]]] H. Mahaseth, J.D. Brody, R. Sinha, P.J. Shenoy, C.R. Flowers; Idiotype vaccine strategies for treatment of follicular lymphoma; Future Oncol., 7 (1) (2011), pp. 111–122</li>
<li><span id='br000030'></span>
[[#br000030|[6]]] A. Bolhassani, S. Safaiyan, S. Rafati; Improvement of different vaccine delivery systems for cancer therapy; Mol. Cancer, 10 (2011) Art. no. 3</li>
<li><span id='br000035'></span>
[[#br000035|[7]]] C.A. Klebanoff, N. Acquavella, Z. Yu, N.P. Restifo; Therapeutic cancer vaccines: are we there yet?; Immunol. Rev., 239 (1) (2011), pp. 27–44</li>
<li><span id='br000040'></span>
[[#br000040|[8]]] M.A. Liu; DNA vaccines: an historical perspective and view to the future; Immunol. Rev., 239 (1) (2011), pp. 62–84</li>
<li><span id='br000045'></span>
[[#br000045|[9]]] N.P. Praetorius, T.K. Mandal; Engineered nanoparticles in cancer therapy; Recent Pat. Drug Deliv. Formul., 1 (1) (2007), pp. 37–51</li>
<li><span id='br000050'></span>
[[#br000050|[10]]] T. Fifis, P. Mottram, V. Bogdanoska, J. Hanley, M. Plebanski; Short peptide sequences containing MHC class I and/or class II epitopes linked to nano-beads induce strong immunity and inhibition of growth of antigen-specific tumour challenge in mice; Vaccine, 23 (2) (2004), pp. 258–266</li>
<li><span id='br000055'></span>
[[#br000055|[11]]] T. Fifis, A. Gamvrellis, B. Crimeen-Irwin, G.A. Pietersz, J. Li, P.L. Mottram, I.F.C. McKenzie, M. Plebanski; Size-dependent immunogenicity: Therapeutic and protective properties of nano-vaccines against tumors; J. Immunol., 173 (5) (2004), pp. 3148–3154</li>
<li><span id='br000060'></span>
[[#br000060|[12]]] M.E. Truckenmiller, C.C. Norbury; Viral vectors for inducing CD8+ T cell responses; Expert Opin. Biol. Ther., 4 (6) (2004), pp. 861–868</li>
<li><span id='br000065'></span>
[[#br000065|[13]]] T. Mashal, K. Nakamura, M. Kizuki, K. Seino, T. Takano; Impact of conflict on infant immunisation coverage in Afghanistan: a countrywide study 2000–2003; Int. J. Health Geogr. Categories, 6 (2007) Art. no. 23</li>
<li><span id='br000070'></span>
[[#br000070|[14]]] V.J. Mohanraj, T.J. Barnes, C.A. Prestidge; Silica nanoparticle coated liposomes: A new type of hybrid nanocapsule for proteins; Int. J. Pharm., 392 (1–2) (2010), pp. 285–293</li>
<li><span id='br000075'></span>
[[#br000075|[15]]] M.R. Mozafari, A. Pardakhty, S. Azarmi, J.A. Jazayeri, A. Nokhodchi, A. Omri; Role of nanocarrier systems in cancer nanotherapy; J. Liposome Res., 19 (4) (2009), pp. 310–321</li>
<li><span id='br000080'></span>
[[#br000080|[16]]] L.C. Kennedy, L.R. Bickford, N.A. Lewinski, A.J. Coughlin, Y. Hu, E.S. Day, J.L. West, R.A. Drezek; A new era for cancer treatment: gold-nanoparticle-mediated thermal therapies; Small, 7 (2) (2011), pp. 169–183</li>
<li><span id='br000085'></span>
[[#br000085|[17]]] C.I. Champion, V.A. Kickhoefer, G. Liu, R.J. Moniz, A.S. Freed, L.L. Bergmann, D. Vaccari, S. Raval-Fernandes, A.M. Chan, L.H. Rome, K.A. Kelly; A vault nanoparticle vaccine induces protective mucosal immunity; PLoS ONE, 4 (4) (2009) Art. no. e5409</li>
<li><span id='br000090'></span>
[[#br000090|[18]]] M. Ozkan; Quantum dots and other nanoparticles: what can they offer to drug discovery?; Drug Discov. Today, 9 (24) (2004), pp. 1065–1071</li>
<li><span id='br000095'></span>
[[#br000095|[19]]] J.E. Smith, K.E. Sapsford, W. Tan, F.S. Ligler; Optimization of antibody-conjugated magnetic nanoparticles for target preconcentration and immunoassays; Anal. Biochem., 410 (1) (2011), pp. 124–132</li>
<li><span id='br000100'></span>
[[#br000100|[20]]] P.K. Jain, I.H. ElSayed, M.A. El-Sayed; Au nanoparticles target cancer; Nano Today, 2 (1) (2007), pp. 18–29</li>
<li><span id='br000105'></span>
[[#br000105|[21]]] J.I. Rodriguez-Devora, S. Ambure, Z.-D. Shi, Y. Yuan, W. Sun, T. Xu; Physically facilitating drug-delivery systems; Ther. Deliv., 3 (1) (2012), pp. 125–139</li>
<li><span id='br000110'></span>
[[#br000110|[22]]] M.-H. Huang, S.-C. Lin, C.-H. Hsiao, H.-J. Chao, H.-R. Yang, C.-C. Liao, P.-W. Chuang, H.-P. Wu, C.-Y. Huang, C.-H. Leng, S.-J. Liu, H.-W. Chen, A.-H. Chou, A.Y.-C. Hu, P. Chong; Emulsified nanoparticles containing inactivated influenza virus and CpG oligodeoxynucleotides critically influences the host immune responses in mice; PLoS ONE, 5 (8) (2010) Art. no. e12279</li>
<li><span id='br000115'></span>
[[#br000115|[23]]] B.S. Zolnik, Á González-Fernández, N. Sadrieh, M.A. Dobrovolskaia; Minireview: nanoparticles and the immune system; Endocrinology, 151 (2) (2010), pp. 458–465</li>
<li><span id='br000120'></span>
[[#br000120|[24]]] C.I. Champion, V.A. Kickhoefer, G. Liu, R.J. Moniz, A.S. Freed, L.L. Bergmann, D. Vaccari, S. Raval-Fernandes, A.M. Chan, L.H. Rome, K.A. Kelly; A vault nanoparticle vaccine induces protective mucosal immunity; PLoS ONE, 4 (4) (2009) Art. no. e5409</li>
<li><span id='br000125'></span>
[[#br000125|[25]]] R.Y. Sweeney, E.Y. Park, B.L. Iverson, G. Georgiou; Assembly of multimeric phage nanostructures through leucine zipper interactions; Biotechnol. Bioeng., 95 (3) (2006), pp. 539–545</li>
<li><span id='br000130'></span>
[[#br000130|[26]]] R. Klippstein, D. Pozo; Nanotechnology-based manipulation of dendritic cells for enhanced immunotherapy strategies; Nanomed. Nanotech. Biol. Med., 6 (4) (2010), pp. 523–529</li>
<li><span id='br000135'></span>
[[#br000135|[27]]] E.H. Chowdhury; pH-sensitive nano-crystals of carbonate apatite for smart and cell-specific transgene delivery; Expet. Opin. Drug Deliv., 4 (3) (2007), pp. 193–196</li>
<li><span id='br000140'></span>
[[#br000140|[28]]] K.K. Jain; The Handbook of Nanomedicine; Humana Press (2008)</li>
<li><span id='br000145'></span>
[[#br000145|[29]]] L.H. Duc, H.A. Hong, S.M. Cutting; Germination of the spore in the gastrointestinal tract provides a novel route for heterologous antigen delivery; Vaccine, 21 (27–30) (2003), pp. 4215–4224</li>
<li><span id='br000150'></span>
[[#br000150|[30]]] E. Ricca, S.M. Cutting; Emerging applications of bacterial spores in nanobiotechnology; J. Nanobiotechnol., 1 (2003) Art. no. 6</li>
<li><span id='br000155'></span>
[[#br000155|[31]]] L.H. Duc, H.A. Hong, N.Q. Uyen, S.M. Cutting; Immunogenicity and intracellular fate of ''B. subtilis'' spores                                        ; Vaccine, 22 (15-16) (2004), pp. 1873–1885</li>
<li><span id='br000160'></span>
[[#br000160|[32]]] R. Isticato, G. Cangiano, H.T. Tran, A. Ciabattini, D. Medaglini, M.R. Oggioni, M. De Felice, G. Pozzi, E. Ricca; Surface display of recombinant proteins on ''Bacillus subtilis'' spores                                        ; J. Bacteriol., 183 (21) (2001), pp. 6294–6301</li>
<li><span id='br000165'></span>
[[#br000165|[33]]] L. Zheng, W.P. Donovan, P.C. Fitz-James, R. Losick; Gene encoding a morphogenic protein required in the assembly of the outer coat of the ''Bacillus subtilis'' endospore                                        ; Genes Dev., 2 (21) (1988), pp. 1047–1054</li>
<li><span id='br000170'></span>
[[#br000170|[34]]] M. Sacco, E. Ricca, R. Losick, S. Cutting; An additional GerEcontrolled gene encoding an abundant spore coat protein from ''Bacillus subtilis''; J. Bacteriol., 177 (2) (1995), pp. 372–377</li>
<li><span id='br000175'></span>
[[#br000175|[35]]] R. Levi, E. Aboud-Pirak, C. Leclerc, G.H. Lowell, R. Arnon; Intranasal immunization of mice against influenza with synthetic peptides anchored to proteosomes; Vaccine, 13 (14) (1995), pp. 1353–1359</li>
<li><span id='br000180'></span>
[[#br000180|[36]]] R. Dalseg, E. Wedege, J. Holst, I.L. Haugen, E.A. Høiby, B. Haneberg; Outer membrane vesicles from group B meningococci are strongly immunogenic when given intranasally to mice; Vaccine, 17 (19) (1999), pp. 2336–2345</li>
<li><span id='br000185'></span>
[[#br000185|[37]]] Burt, D.S. and White, G.L. “Proteosome vaccines against toxins, allergy and cancer”, EP Patent 1419784 (2004).</li>
<li><span id='br000190'></span>
[[#br000190|[38]]] X.-F. Wu, Y.-G. Xie, Y. Yuan, D. Wang, S.-K. Yu, X.-L. Wang; Proteosome adjuvant and its application in anti-plague immunity induced by recombinant F1-V protein; Chin. J. Microbiol. Immunol., 27 (3) (2007), pp. 247–250</li>
<li><span id='br000195'></span>
[[#br000195|[39]]] N. Orr, R. Arnon, G. Rubin, D. Cohen, H. Bercovier, G.H. Lowell; Enhancement of anti-Shigella lipopolysaccharide (LPS) response by addition of the cholera toxin B subunit to oral and intranasal proteosome-Shigella flexneri 2a LPS vaccines; Infect. Immun., 62 (11) (1994), pp. 5198–5200</li>
<li><span id='br000200'></span>
[[#br000200|[40]]] S.L. Cyr, T. Jones, I. Stoica-Popescu, A. Brewer, S. Chabot, M. Lussier, D. Burt, B.J. Ward; Intranasal proteosome-based respiratory syncytial virus (RSV) vaccines protect BALB/c mice against challenge without eosinophilia or enhanced pathology; Vaccine, 25 (29) (2007), pp. 5378–5389</li>
<li><span id='br000205'></span>
[[#br000205|[41]]] P. Kalantari, O.F. Harandi, P.A. Hankey, A.J Henderson; HIV-1 Tat mediates degradation of RON receptor tyrosine kinase, a regulator of inflammation; J. Immunol., 181 (2) (2008), pp. 1548–1555</li>
<li><span id='br000210'></span>
[[#br000210|[42]]] I. Hwang, X. Shen, J. Sprent; Direct stimulation of naive T cells by membrane vesicles from antigen-presenting cells: distinct roles for CD54 and B7 molecules; Proc. Natl. Acad. Sci. USA., 100 (11) (2003), pp. 6670–6675</li>
<li><span id='br000215'></span>
[[#br000215|[43]]] H. Vincent-Schneider, P. Stumptner-Cuvelette, D. Lankar, S. Pain, G. Raposo, P. Benaroch, C. Bonnerot; Exosomes bearing HLA-DR1 molecules need dendritic cells to efficiently stimulate specific T cells; Int. Immunol., 14 (7) (2002), pp. 713–722</li>
<li><span id='br000220'></span>
[[#br000220|[44]]] M. Karlsson, S. Lundin, U. Dahlgren, H. Kahu, I. Pettersson, E. Telemo; Tolerosomes are produced by intestinal epithelial cells; Eur. J. Immunol., 31 (10) (2001), pp. 2892–2900</li>
<li><span id='br000225'></span>
[[#br000225|[45]]] M.L. Saxton, D.L. Longo, H.E. Wetzel, H. Tribble, W.G. Alvord, L.W. Kwak, A.S. Leonard, C.D. Ullmann, B.D. Curti, A.C. Ochoa; Adoptive transfer of anti-CD3-activated CD4+ T cells plus cyclophosphamide and liposome-encapsulated interleukin-2 cure murine MC-38 and 3LL tumors and establish tumor-specific immunity; Blood, 89 (7) (1997), pp. 2529–2536</li>
<li><span id='br000230'></span>
[[#br000230|[46]]] A. Takagi, M. Matsui, S. Ohno, H. Duan, O. Moriya, N. Kobayashi, H. Oda, M. Mori, A. Kobayashi, M. Taneichi, T. Uchida, T. Akatsuka; Highly efficient antiviral CD8+ T-cell induction by peptides coupled to the surfaces of liposomes; Clin. Vaccine Immunol., 16 (10) (2009), pp. 1383–1392</li>
<li><span id='br000235'></span>
[[#br000235|[47]]] L.A. Myc, A. Gamian, A. Myc; Cancer vaccines. any future?; Arch. Immunol. Ther. Exp. (Warsz), 59 (4) (2011), pp. 249–259</li>
<li><span id='br000240'></span>
[[#br000240|[48]]] M. Henriksen-Lacey, K.S. Korsholm, P. Andersen, Y. Perrie, D. Christensen; Liposomal vaccine delivery systems; Exp. Opin. Drug Deliv., 8 (4) (2011), pp. 505–519</li>
<li><span id='br000245'></span>
[[#br000245|[49]]] J. Inoue, R. Ideue, D. Takahashi, M. Kubota, Y. Kumazawa; Liposomal glycosphingolipids activate natural killer T cell-mediated immune responses through the endosomal pathway; J. Controlled Release, 133 (1) (2009), pp. 18–23</li>
<li><span id='br000250'></span>
[[#br000250|[50]]] R.A. Schwendener, B. Ludewig, A. Cerny, O. Engler; Liposome-based vaccines; Methods Mol. Biol., 605 (2010), pp. 163–175</li>
<li><span id='br000255'></span>
[[#br000255|[51]]] L. Bungener, J. Idema, W. Ter Veer, A. Huckriede, T. Daemen, J. Wilschut; Virosomes in vaccine development: induction of cytotoxic T lymphocyte activity with virosome-encapsulated protein antigens; J. Lipos. Res., 12 (1–2) (2002), pp. 155–163</li>
<li><span id='br000260'></span>
[[#br000260|[52]]] D. Felnerova, J.-F. Viret, R. Glück, C. Moser; Liposomes and virosomes as delivery systems for antigens, nucleic acids and drugs; Curr. Opin. Biotechnol., 15 (6) (2004), pp. 518–529</li>
<li><span id='br000265'></span>
[[#br000265|[53]]] M.E. Christopher, J.P. Wong; Recent developments in delivery of nucleic acid-based antiviral agents; Curr. Pharmaceut. Design., 12 (16) (2006), pp. 1995–2006</li>
<li><span id='br000270'></span>
[[#br000270|[54]]] Y. Kaneda; Virosome: a novel vector to enable multi-modal strategies for cancer therapy; Adv. Drug Deliv. Rev., 64 (8) (2012), pp. 730–738</li>
<li><span id='br000275'></span>
[[#br000275|[55]]] U. Gupta, N.K. Jain; Non-polymeric nano-carriers in HIV/AIDS drug delivery and targeting; Adv. Drug Deliv. Rev., 62 (4–5) (2010), pp. 478–490</li>
<li><span id='br000280'></span>
[[#br000280|[56]]] Castor, T.P. “Polymer nanospheres for improved drug delivery of protein therapeutics and vaccine antigens”, ''2007 NSTI Nanotechnol. Conf. Trade Show - NSTI Nanotech 2007, Technical Proceedings'' , 2, pp. 362–365 (2007).                                    </li>
<li><span id='br000285'></span>
[[#br000285|[57]]] E.M. Martin Del Valle, M.A. Galan; Supercritical fluid technique for particle engineering: Drug delivery applications; Rev. Chem. Eng., 21 (1) (2005), pp. 33–69</li>
<li><span id='br000290'></span>
[[#br000290|[58]]] Castor, T.P. and Ilynskii, P.O. “Inactivated vaccines for aids and other infectious diseases”, US Patent 7033813 (2006).</li>
<li><span id='br000295'></span>
[[#br000295|[59]]] Castor, T.P. and Ilynskii, P.O. “Inactivated vaccines for aids and other infectious diseases”, US Patent 2003108918 (2003).</li>
<li><span id='br000300'></span>
[[#br000300|[60]]] M. Kalkanidis, G.A. Pietersz, S.D. Xiang, P.L. Mottram, B. Crimeen-Irwin, K. Ardipradja, M. Plebanski; Methods for nano-particle based vaccine formulation and evaluation of their immunogenicity; Methods, 40 (1) (2006), pp. 20–29</li>
<li><span id='br000305'></span>
[[#br000305|[61]]] J.-P.Y. Scheerlinck, D.L.V. Greenwood; Virus-sized vaccine delivery systems; Drug Discov. Today, 13 (19–20) (2008), pp. 882–887</li>
<li><span id='br000310'></span>
[[#br000310|[62]]] J.-P.Y. Scheerlinck, D.L.V. Greenwood; Particulate delivery systems for animal vaccines; Methods, 40 (1) (2006), pp. 118–124</li>
<li><span id='br000315'></span>
[[#br000315|[63]]] J.-P.Y. Scheerlinck, S. Gekas, H.-H. Yen, S. Edwards, M. Pearse, A. Coulter, P. Sutton; Local immune responses following nasal delivery of an adjuvanted influenza vaccine; Vaccine, 24 (18) (2006), pp. 3929–3936</li>
<li><span id='br000320'></span>
[[#br000320|[64]]] L.A. Koutsky, K.A. Ault, C.M. Wheeler, D.R. Brown, E. Barr, F.B. Alvarez, L.M. Chiacchierini, K.U. Jansen; A controlled trial of a human papillomavirus type 16 vaccine; N. Engl. J. Med., 347 (21) (2002), pp. 1645–1651</li>
<li><span id='br000325'></span>
[[#br000325|[65]]] A.C. Tissot, R. Renhofa, N. Schmitz, I. Cielens, E. Meijerink, V. Ose, G.T. Jennings, P. Saudan, P. Pumpens, M.F. Bachmann; Versatile virus-like particle carrier for epitope based vaccines; PloS ONE, 5 (3) (2010), p. e9809</li>
<li><span id='br000330'></span>
[[#br000330|[66]]] F.M. Buonaguro, M.L. Tornesello, L. Buonaguro; New adjuvants in evolving vaccine strategies; Expert Opin. Biol. Ther., 11 (7) (2011), pp. 827–832</li>
<li><span id='br000335'></span>
[[#br000335|[67]]] S.-M. Kang, Q. Yao, L. Guo, R.W. Compans; Mucosal immunization with virus-like particles of simian immunodeficiency virus conjugated with cholera toxin subunit B; J. Virol., 77 (18) (2003), pp. 9823–9830</li>
<li><span id='br000340'></span>
[[#br000340|[68]]] S.-M. Kang, R.W. Compans; Enhancement of mucosal immunization with virus-like particles of simian immunodeficiency virus; J. Virol., 77 (6) (2003), pp. 3615–3623</li>
<li><span id='br000345'></span>
[[#br000345|[69]]] A.S. Chernyavskaya, I.V. Morozova, S.A. Lebedeva, M.I. Zarenkov; Construction of variants of vaccinal strain Yersinia pestis EV76 (RIEG line) differing in antibiotic resistance spectra with stage-by-stage transduction of R-transposons; Antibiot. Khimioter., 50 (7) (2005), pp. 13–17</li>
<li><span id='br000350'></span>
[[#br000350|[70]]] J.B. March, J.R. Clark, C.D. Jepson; Genetic immunisation against hepatitis B using whole bacteriophage <math display="inline">\lambda </math>  particles                                        ; Vaccine, 22 (13–14) (2004), pp. 1666–1671</li>
<li><span id='br000355'></span>
[[#br000355|[71]]] J.R. Clark, J.B. March; Bacteriophages and biotechnology: vaccines, gene therapy and antibacterials; Trends Biotechnol., 24 (5) (2006), pp. 212–218</li>
<li><span id='br000360'></span>
[[#br000360|[72]]] J.L. Richards, J.R. Abend, M.L. Miller, S. Chakraborty-Sett, S. Dewhurst, L.E. Whetter; A peptide containing a novel FPGN CD40-binding sequence enhances adenoviral infection of murine and human dendritic cells; Eur. J. Biochem., 270 (10) (2003), pp. 2287–2294</li>
<li><span id='br000365'></span>
[[#br000365|[73]]] C.N. Zanghi, H.A. Lankes, B. Bradel-Tretheway, J. Wegman, S. Dewhurst; A simple method for displaying recalcitrant proteins on the surface of bacteriophage lambda; Nucleic Acid Res., 33 (18) (2005), pp. 1–7</li>
<li><span id='br000370'></span>
[[#br000370|[74]]] A.P. Sommer, M. Milankovits, A.R. Mester; Nanobacteria, HIV and magic bullets — update of perspectives; Chemotherapy, 52 (2) (2006), pp. 95–97</li>
<li><span id='br000375'></span>
[[#br000375|[75]]] Åkerman, K.K., Kuikka, J.T., Çiftçioglu, N., Parkkinen, J., Bergström, K.A., Kuronen, I. and Kajander, E.O. “Radiolabeling and in vivo distribution of nanobacteria in rabbit”, ''Proc. SPIE-Int. Soc. Opt. Eng.'' , 3111, pp. 436–442 (1997).                                    </li>
<li><span id='br000380'></span>
[[#br000380|[76]]] C.M. Niemeyer, C.A. Mirkin; Nanobiotechnology: Concepts, Applications and Perspectives; WILEY-VCH Verlag GmbH & Co, KGaA, Weinheim (2004)</li>
<li><span id='br000385'></span>
[[#br000385|[77]]] C.A. Mirkin, C.M. Niemeyer; Nanobiotechnology II: More Concepts and Applications; WILEY-VCH Verlag GmbH & Co, KGaA, Weinheim (2007)</li>
<li><span id='br000390'></span>
[[#br000390|[78]]] D.S. Goodsell; Bionanotechnology: Lessons from Nanture; Wiley-Liss, Inc., Hoboken, New Jersey (2004)</li>
<li><span id='br000395'></span>
[[#br000395|[79]]] R.J. Cano, M. Borucki; Revival and identification of bacterial spores in 25- to 40-million-year-old Dominican amber; Science, 268 (5213) (1995), pp. 1060–1064</li>
<li><span id='br000400'></span>
[[#br000400|[80]]] M.R. Oggion, A. Ciabattini, A.M. Cuppone, G. Pozzi; Bacillus spores for vaccine delivery; Vaccine, 21 (Suppl. 2) (2003), pp. S96–S101</li>
<li><span id='br000405'></span>
[[#br000405|[81]]] W.J. Nicholson, N. Munakata, G. Horneck, H.J. Melosh, P. Setlow; Resistance of ''Bacillus'' endospores to extreme terrestrial and extraterrestrial environments                                        ; Microbiol. Mol. Biol. Rev., 64 (3) (2000), pp. 548–572</li>
<li><span id='br000410'></span>
[[#br000410|[82]]] J. Errington; ''Bacillus subtilis'' sporulation: regulation of gene expression and control of morphogenesis                                        ; Microbiol. Rev., 57 (1) (1993), pp. 1–33</li>
<li><span id='br000415'></span>
[[#br000415|[83]]] P.J. Piggot, J.G. Coote; Genetic aspects of bacterial endospore formation; Bacteriological Rev., 40 (4) (1976), pp. 908–962</li>
<li><span id='br000420'></span>
[[#br000420|[84]]] A.I. Aronson, P. Fitz-James; Structure and morphogenesis of the bacterial spore coat; Bacteriological Rev., 40 (2) (1976), pp. 360–402</li>
<li><span id='br000425'></span>
[[#br000425|[85]]] A. Driks; ''Bacillus subtilis'' spore coat                                        ; Microbiol. Mol. Biol. Rev., 63 (1) (1999), pp. 1–20</li>
<li><span id='br000430'></span>
[[#br000430|[86]]] A.O. Henriques, C.P. Moran; Structure and assembly of the bacterial endospore coat; Methods, 20 (1) (2000), pp. 95–110</li>
<li><span id='br000435'></span>
[[#br000435|[87]]] L.H. Duc, H.A. Hong, N. Fairweather, E. Ricca, S.M. Cutting; Bacterial spores as vaccine vehicles; Infect. Immun., 71 (5) (2003), pp. 2810–2818</li>
<li><span id='br000440'></span>
[[#br000440|[88]]] S.M. Cutting, P.B. Vander-Horn; Genetic analysis; C.R. Harwood, S.M. Cutting (Eds.), Molecular Biological Methods for Bacillus, John Wiley & Sons Ltd., Chichester, England (1990), pp. 27–74</li>
<li><span id='br000445'></span>
[[#br000445|[89]]] S.J. Challacombe; Salivary antibodies and systemic tolerance in mice after oral immunisation with bacterial antigens; Ann. New York Acad. Sci., 409 (1983), pp. 177–192</li>
<li><span id='br000450'></span>
[[#br000450|[90]]] A.S. De Boer, B. Diderichsen; On the safety of ''Bacillus subtilis'' and ''B. amyloliquefacien'' s: a review                                        ; Appl. Microbiol. Biotechnol., 36 (1) (1991), pp. 1–4</li>
<li><span id='br000455'></span>
[[#br000455|[91]]] G. Casula, S.M. Cutting; ''Bacillus'' probiotics: spore germination in the gastrointestinal tract                                        ; App. Envron. Microbiol., 68 (5) (2002), pp. 2344–2352</li>
<li><span id='br000460'></span>
[[#br000460|[92]]] R. Fuller; Probiotics in man and animals; J. Appl. Bacteriol., 66 (5) (1989), pp. 365–378R. Fuller Probiotics in human medicine; Gut, 32 (4) (1991), pp. 439–442</li>
<li><span id='br000465'></span>
[[#br000465|[93]]] T.T. Hoa, L.H. Duc, R. Isticato, L. Baccigalupi, E. Ricca, P.H. Van, S.M. Cutting; Fate and dissemination of ''Bacillus subtilis'' spores in a murine model                                        ; App. Envron. Microbiol., 67 (9) (2001), pp. 3819–3823</li>
<li><span id='br000470'></span>
[[#br000470|[94]]] E.M. Plummer, M. Manchester; Viral nanoparticles and virus-like particles: platforms for contemporary vaccine design; Wiley Interdiscip Rev Nanomed Nanobiotechnol., 3 (2) (2011), pp. 174–196</li>
<li><span id='br000475'></span>
[[#br000475|[95]]] R.L. Garcea, L. Gissmann; Virus-like particles as vaccines and vessels for the delivery of small molecules; Curr. Opin. Biotechnol., 15 (6) (2004), pp. 513–517</li>
<li><span id='br000480'></span>
[[#br000480|[96]]] C. Mao, D.J. Solis, B.D. Reiss, S.T. Kottmann, R.Y. Sweeney, A. Hayhurst, G. Georgiou, B. Iverson, A.M. Belcher; Virus-based toolkit for the directed synthesis of magnetic and semiconducting nanowires; Science, 303 (5655) (2004), pp. 213–217</li>
<li><span id='br000485'></span>
[[#br000485|[97]]] Q. Wang, T. Lin, L. Tang, J.E. Johnson, M.G. Finn; Icosahedral virus particles as addressable nanoscale building blocks; Angew. Chem. Int. Ed., 41 (3) (2002), pp. 459–462</li>
<li><span id='br000490'></span>
[[#br000490|[98]]] M. Herbst-Kralovetz, H.S. Mason, Q. Chen; Norwalk virus-like particles as vaccines; Expert. Rev. Vaccines, 9 (3) (2010), pp. 299–307</li>
<li><span id='br000495'></span>
[[#br000495|[99]]] R. Noad, P. Roy; Virus-like particles as immunogens; Trends Microbiol., 11 (9) (2003), pp. 438–444</li>
<li><span id='br000500'></span>
[[#br000500|[100]]] J.K. Pokorski, N.F. Steinmetz; The art of engineering viral nanoparticles; Mol. Pharmaceut., 8 (1) (2011), pp. 29–43</li>
<li><span id='br000505'></span>
[[#br000505|[101]]] M.G. Mateu; Virus engineering: Functionalization and stabilization; Protein Eng. Des. Sel., 24 (1–2) (2011), pp. 53–63</li>
<li><span id='br000510'></span>
[[#br000510|[102]]] A. Roldão, M.C.M. Mellado, L.R. Castilho, M.J.T. Carrondo, P.M. Alves; Virus-like particles in vaccine development; Expert Rev. Vaccines, 9 (10) (2010), pp. 1149–1176</li>
<li><span id='br000515'></span>
[[#br000515|[103]]] J. Gao, Y. Wang, Z. Liu, Z. Wang; Phage display and its application in vaccine design; Ann. Microbiol., 60 (1) (2010), pp. 13–19</li>
<li><span id='br000520'></span>
[[#br000520|[104]]] A. Ghaemi, H. Soleimanjahi, P. Gill, Z.M. Hassan, F. Roohvand; Recombinant lambda-phage nanobioparticles for tumor therapy in mice models; Genet. Vaccines Ther., 8 (3) (2010), pp. 1–7</li>
<li><span id='br000525'></span>
[[#br000525|[105]]] A. Ghaemi, H. Soleimanjahi, P. Gill, Z.M. Hassan, S. Razeghi, M. Fazeli, S-M.H. Razavinikoo; Protection of mice by a <math display="inline">\lambda </math> -based therapeutic vaccine against cancer associated with human papillomavirus type 16                                        ; Intervirol, 54 (3) (2011), pp. 105–112</li>
<li><span id='br000530'></span>
[[#br000530|[106]]] G.P. Smith; Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface; Science, 228 (4705) (1985), pp. 1315–1317</li>
<li><span id='br000535'></span>
[[#br000535|[107]]] J.K. Scott, G.P. Smith; Searching for peptide ligands with an epitope library; Science, 249 (4967) (1990), pp. 386–390</li>
<li><span id='br000540'></span>
[[#br000540|[108]]] J.J. Devlin, L.C. Panganiban, P.E. Devlin; Random peptide libraries: a source of specific protein binding molecules; Science, 249 (4967) (1990), pp. 404–406</li>
<li><span id='br000545'></span>
[[#br000545|[109]]] S.E. Cwirla, E.A. Peters, R.W. Barrett, W.J. Dower; Peptides on phage: a vast library of peptides for identifying ligands; Proc. Natl. Acad. Sci. USA., 87 (16) (1990), pp. 6378–6382</li>
<li><span id='br000550'></span>
[[#br000550|[110]]] M.B. Irving, O. Pan, J.K. Scott; Random-peptide libraries and antigen-fragment libraries for epitope mapping and the development of vaccines and diagnostics; Curr. Opin. Chem. Biol., 5 (3) (2001), pp. 314–324</li>
<li><span id='br000555'></span>
[[#br000555|[111]]] T.J. Curiel, C. Morris, M. Brumlik, S.J. Landry, K. Finstad, A. Nelson, V. Joshi, C. Hawkins, X. Alarez, A. Lackner, M. Mohamadzadeh; Peptides identified through phage display direct immunogenic antigen to dendritic cells; J. Immunol., 172 (12) (2004), pp. 7425–7431</li>
<li><span id='br000560'></span>
[[#br000560|[112]]] L.-F. Wang, M. Yu; Epitope identification and discovery using phage display libraries: applications in vaccine development and diagnostics; Curr. Drug Targets, 5 (1) (2004), pp. 1–15</li>
<li><span id='br000565'></span>
[[#br000565|[113]]] B. Shields, J. Mills, R. Ghildyal, P. Gooley, J. Meanger; Multiple heparin binding domains of respiratory syncytial virus G mediate binding to mammalian cells; Arch. Virol., 148 (10) (2003), pp. 1987–2003</li>
<li><span id='br000570'></span>
[[#br000570|[114]]] N. Bastien, M. Trudel, C. Simard; Protective immune responses induced by the immunization of mice with a recombinant bacteriophage displaying an epitope of the human respiratory syncytial virus; Virology, 234 (1) (1997), pp. 118–122</li>
<li><span id='br000575'></span>
[[#br000575|[115]]] G. Raposo, H.W. Nijman, W. Stoorvogel, R. Liejendekker, C.V. Harding, C.J. Melief, H.J. Geuze; B lymphocytes secrete antigen-presenting vesicles; J. Exp. Med., 183 (3) (1996), pp. 1161–1172</li>
<li><span id='br000580'></span>
[[#br000580|[116]]] N. Blanchard, D. Lankar, F. Faure, A. Regnault, C. Dumont, G. Raposo, C. Hivroz; TCR activation of human Tcells induces the production of exosomes bearing the TCR/CD3/zeta complex; J. Immunol., 168 (7) (2002), pp. 3235–3241</li>
<li><span id='br000585'></span>
[[#br000585|[117]]] G. Raposo, D. Tenza, S. Mecheri, R. Peronet, C. Bonnerot, C. Desaymard; Accumulation of major histocompatibility complex class II molecules in mast cell secretory granules and their release upon degranulation; Mol. Biol. Cell, 8 (12) (1997), pp. 2631–2645</li>
<li><span id='br000590'></span>
[[#br000590|[118]]] G. van Niel, G. Raposo, C. Candalh, M. Boussac, R. Hershberg, N. Cerf- Bensussan, M. Heyman; Intestinal epithelial cells secrete exosome-like vesicles; Gastroenterology, 121 (2) (2001), pp. 337–349</li>
<li><span id='br000595'></span>
[[#br000595|[119]]] L. Zitvogel, A. Regnault, A. Lozier, J. Wolfers, C. Flament, D. Tenza, P. Ricciardi-Castagnoli, G. Raposo, S. Amigorena; Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes; Nat. Med., 4 (5) (1998), pp. 594–600</li>
<li><span id='br000600'></span>
[[#br000600|[120]]] B. Février, G. Raposo; Exosomes: endosomal-derived vesicles shipping extracellular messages; Curr. Opin. Cell Biol., 16 (4) (2004), pp. 415–421</li>
<li><span id='br000605'></span>
[[#br000605|[121]]] S.I. Buschow, J.M.P. Liefhebber, R. Wubbolts, W. Stoorvogel; Exosomes contain ubiquitinated proteins; Blood Cells Mol. Dis., 35 (3) (2005), pp. 398–403</li>
<li><span id='br000610'></span>
[[#br000610|[122]]] M. Zhu, Y. Li, J. Shi, W. Feng, G. Nie, Y. Zhao; Exosomes as extrapulmonary signaling conveyors for nanoparticle-induced systemic immune activation; Small, 8 (3) (2012), pp. 404–412</li>
<li><span id='br000615'></span>
[[#br000615|[123]]] B.T. Pan, K. Ten, C. Wu, M. Adam, R.M. Johnstone; Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes; J. Cell Biol., 101 (3) (1985), pp. 942–948</li>
<li><span id='br000620'></span>
[[#br000620|[124]]] A. Clayton, J. Court, H. Navabi, M. Adams, M.D. Mason, J.A. Hobot, G.R. Newman, B. Jasani; Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry; J. Immunol. Methods, 247 (1–2) (2001), pp. 163–174</li>
<li><span id='br000625'></span>
[[#br000625|[125]]] J.M. Escola, M.J. Kleijmeer, W. Stoorvogel, J.M. Griffith, O. Yoshie, H.J. Geuze; Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B lymphocytes; J. Biol. Chem., 273 (32) (1998), pp. 20121–20127</li>
<li><span id='br000630'></span>
[[#br000630|[126]]] M.D. Wright, G.W. Moseley, A.B. van Spriel; Tetraspanin microdomains in immune cell signalling and malignant disease; Tissue Antigens, 64 (5) (2004), pp. 533–542</li>
<li><span id='br000635'></span>
[[#br000635|[127]]] C. Admyre, J. Grunewald, J. Thyberg, S. Gripenback, G. Tornling, A. Eklund, A. Scheynius, S. Gabrielsson; Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present in human BAL fluid; Eur. Respir. J., 22 (4) (2003), pp. 578–583</li>
<li><span id='br000640'></span>
[[#br000640|[128]]] M.P. Caby, D. Lankar, C. Vincendeau-Scherrer, G. Raposo, C. Bonnerot; Exosomal-like vesicles are present in human blood plasma; Int. Immunol., 17 (7) (2005), pp. 879–887</li>
<li><span id='br000645'></span>
[[#br000645|[129]]] F. Andre, N.E.C. Schartz, M. Movassagh, C. Flament, P. Pautier, P. Morice, C. Pomel, C. Lhomme, B. Escudier, T. Le Chevalier, T. Tursz, S. Amigorena, G. Raposo, E. Angevin, L. Zitvogel; Malignant effusions and immunogenic tumour-derived exosomes; Lancet, 360 (9329) (2002), pp. 295–305</li>
<li><span id='br000650'></span>
[[#br000650|[130]]] M.P. Bard, J.P. Hegmans, A. Hemmes, T.M. Luider, R. Willemsen, L.A. Severijnen, J.P. Van Meerbeeck, S.A. Burgers, H.C. Hoogsteden, B.N. Lambrecht; Proteomic analysis of exosomes isolated from human malignant pleural effusions; Am. J. Respir. Cell Mol. Biol., 31 (1) (2004), pp. 114–121</li>
<li><span id='br000655'></span>
[[#br000655|[131]]] K. Denzer, M. van Eijk, M.J. Kleijmeer, E. Jakobson, C. de Groot, H.J. Geuze; Follicular dendritic cells carry MHC class II-expressing microvesicles at their surface; J. Immunol., 165 (3) (2000), pp. 1259–1265</li>
<li><span id='br000660'></span>
[[#br000660|[132]]] F. Aline, D. Bout, S. Amigorena, P. Roingeard, I. Dimier-Poisson; Toxoplasma gondii antigen-pulsed-dendritic cell-derived exosomes induce a protective immune response against T. gondii infection; Infect. Immun., 72 (7) (2004), pp. 4127–4137</li>
<li><span id='br000665'></span>
[[#br000665|[133]]] H. Peche, M. Heslan, C. Usal, S. Amigorena, M.C. Cuturi; Presentation of donor major histocompatibility complex antigens by bone marrow dendritic cell-derived exosomes modulates allograft rejection; Transplantation, 76 (10) (2003), pp. 1503–1510</li>
<li><span id='br000670'></span>
[[#br000670|[134]]] B. Escudier, T. Dorval, N. Chaput, F. André, M.-P. Caby, S. Novault, C. Flament, C. Leboulaire, C. Borg, S. Amigorena, C. Boccaccio, C. Bonnerot, O. Dhellin, M. Movassagh, S. Piperno, C. Robert, V. Serra, N. Valente, J.-B. Le Pecq, A. Spatz, O. Lantz, T. Tursz, E. Angevin, L. Zitvogel; Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of the first phase I clinical trial; J. Transl. Med., 3 (10) (2005), p. 13</li>
<li><span id='br000675'></span>
[[#br000675|[135]]] M.A. Morse, J. Garst, T. Osada, S. Khan, A. Hobeika, T.M. Clay, N. Valente, R. Shreeniwas, M.A. Sutton, A. Delcayre, D.-H. Hsu, J.-B. Le Pecq, H.K. Lyerly; A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer; J. Transl. Med., 3 (9) (2005), p. 8</li>
<li><span id='br000680'></span>
[[#br000680|[136]]] S. Utsugi-Kobukai, H. Fujimaki, C. Hotta, M. Nakazawa, M. Minami; MHC class I-mediated exogenous antigen presentation by exosomes secreted from immature and mature bone marrow derived dendritic cells; Immunol. Lett., 89 (2–3) (2003), pp. 125–131</li>
<li><span id='br000685'></span>
[[#br000685|[137]]] C. Thery, L. Duban, E. Segura, P. Veron, O. Lantz, S. Amigorena; Indirect activation of naive CD4+ T cells by dendritic cell-derived exosomes; Nat. Immunol., 3 (12) (2002), pp. 1156–1162</li>
<li><span id='br000690'></span>
[[#br000690|[138]]] F. André, N. Chaput, N.E.C. Schartz, C. Flament, N. Aubert, J. Bernard, F. Lemonnier, G. Raposo, B. Escudier, D.-H. Hsu, T. Tursz, S. Amigorena, E. Angevin, L. Zitvogel; Exosomes as potent cell-free peptide-based vaccine. I. dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells; J. Immunol., 172 (4) (2004), pp. 2126–2136</li>
<li><span id='br000695'></span>
[[#br000695|[139]]] D.H. Hsu, P. Paz, G. Villaflor, A. Rivas, A. Mehta-Damani, E. Angevin, L. Zitvogel, J.B. Le Pecq; Exosomes as a tumor vaccine: enhancing potency through direct loading of antigenic peptides; J. Immunother., 26 (5) (2003), pp. 440–450</li>
<li><span id='br000700'></span>
[[#br000700|[140]]] C. Admyre, S.M. Johansson, S. Paulie, S. Gabrielsson; Direct exosome stimulation of peripheral human T cells detected by ELISPOT; Eur. J. Immunol., 36 (7) (2006), pp. 1772–1781</li>
<li><span id='br000705'></span>
[[#br000705|[141]]] D.D. Taylor, C. Gerçel-Taylor; Tumour-derived exosomes and their role in cancer-associated T-cell signalling defects; Br. J. Cancer, 92 (2) (2005), pp. 305–311</li>
<li><span id='br000710'></span>
[[#br000710|[142]]] A. Tan, H. de la Peña, A.M. Seifalian; The application of exosomes as a nanoscale cancer vaccine; Int. J. Nanomedicine, 5 (1) (2010), pp. 889–900</li>
<li><span id='br000715'></span>
[[#br000715|[143]]] A. Delcayre, H. Shu, J.-B. Le Pecq; Dendritic cell-derived exosomes in cancer immunotherapy: exploiting nature’s antigen delivery pathway; Expert Rev. Anticancer Ther., 5 (3) (2005), pp. 537–547</li>
<li><span id='br000720'></span>
[[#br000720|[144]]] J.-B. Le Pecq; Dexosomes as a therapeutic cancer vaccine: from bench to bedside; Blood Cell Mol. Dis., 35 (2) (2005), pp. 129–135</li>
<li><span id='br000725'></span>
[[#br000725|[145]]] X. Chen, C.-H. Chang, D.M. Goldenberg; Novel strategies for improved cancer vaccines; Expert Rev. Vaccines, 8 (5) (2009), pp. 567–576</li>
<li><span id='br000730'></span>
[[#br000730|[146]]] A. De Gassart, B. Trentin, M. Martin, A. Hocquellet, P. Bette-Bobillo, R. Mamoun, M. Vidal; Exosomal sorting of the cytoplasmic domain of bovine leukemia virus TM Env protein; Cell Biol. Int., 33 (1) (2009), pp. 36–48</li>
<li><span id='br000735'></span>
[[#br000735|[147]]] L. Blanc, M. Vidal; Reticulocyte membrane remodeling: contribution of the exosome pathway; Curr. Opin. Hematol., 17 (3) (2010), pp. 177–183</li>
<li><span id='br000740'></span>
[[#br000740|[148]]] A. Singh, R. Misra, C. Mohanty, S.K. Sahoo; Applications of nanotechnology in vaccine delivery; Int. J. Green Nanotechnol. Biomedicine, 2 (1) (2010), pp. B25–B45</li>
<li><span id='br000745'></span>
[[#br000745|[149]]] J.M. Langley, S.A. Halperin, S. McNeil, B. Smith, T. Jones, D. Burt, C.P. Mallett, G.H. Lowell, L. Fries; Safety and immunogenicity of a Proteosome™-trivalent inactivated influenza vaccine, given nasally to healthy adults; Vaccine, 24 (10) (2006), pp. 1601–1608</li>
<li><span id='br000750'></span>
[[#br000750|[150]]] G.H. Lowell, L.F. Smith, R.C. Seid, W.D. Zollinger; Peptides bound to proteosomes via hydrophobic feet become highly immunogenic without adjuvants; J. Exp. Med., 167 (2) (1988), pp. 658–663</li>
<li><span id='br000755'></span>
[[#br000755|[151]]] G.H. Lowell, W.R. Ballou, L.F. Smith, R.A. Wirtz, W.D. Zollinger, W.T. Hockmeyer; Proteosome-lipopeptide vaccines: enhancement of immunogenicity for malaria CS peptide; Science, 240 (4853) (1988), pp. 800–802</li>
<li><span id='br000760'></span>
[[#br000760|[152]]] M.-N. Kweon; Shigellosis: the current status of vaccine development; Curr. Opin. Infect. Dis., 21 (3) (2008), pp. 313–318</li>
<li><span id='br000765'></span>
[[#br000765|[153]]] Lowell, G.H. “Immuno-potentiating systems for preparation of immunogenic materials”, US Patent 5726292 (1998).</li>
<li><span id='br000770'></span>
[[#br000770|[154]]] Zollinger, W. and Boslego, J. “Process for the preparation of detoxified polysaccharide-outer membrane protein complexes, and their use as antibacterial vaccines”, US Patent 4707543 (1987).</li>
<li><span id='br000775'></span>
[[#br000775|[155]]] S. Sharma, T.K.S. Mukkur, H.A.E. Benson, Y. Chen; Pharmaceutical aspects of intranasal delivery of vaccines using particulate systems; J. Pharm. Sci., 98 (3) (2009), pp. 812–843</li>
<li><span id='br000780'></span>
[[#br000780|[156]]] J. Weber; Peptide vaccines for cancer; Cancer Invest., 20 (2) (2002), pp. 208–221</li>
<li><span id='br000785'></span>
[[#br000785|[157]]] G.B. Lesinski, M.A.J. Westerink; Novel vaccine strategies to T-independent antigens; J. Microbiol. Meth., 47 (2) (2001), pp. 135–149</li>
<li><span id='br000790'></span>
[[#br000790|[158]]] S. Chabot, A. Brewer, G. Lowell, M. Plante, S. Cyr, D.S. Burt, B.J. Ward; A novel intranasal Protollin™-based measles vaccine induces mucosal and systemic neutralizing antibody responses and cell-mediated immunity in mice; Vaccine, 23 (11) (2005), pp. 1374–1383</li>
<li><span id='br000795'></span>
[[#br000795|[159]]] L.M. Wetzler, M.S. Blake, K. Barry, E.C. Gotschlich; Gonococcal porin vaccine evaluation: comparison of Por proteosomes, liposomes, and blebs isolated from rmp deletion mutants; J. Infect. Dis., 166 (3) (1992), pp. 551–555</li>
<li><span id='br000800'></span>
[[#br000800|[160]]] J. Treanor, C. Nolan, D. O’Brien, D. Burt, G. Lowell, J. Linden, L. Fries; Intranasal administration of a proteosome-influenza vaccine is well-tolerated and induces serum and nasal secretion influenza antibodies in healthy human subjects; Vaccine, 24 (3) (2006), pp. 254–262</li>
<li><span id='br000805'></span>
[[#br000805|[161]]] T.P. Castor; Phospholipid nanosomes; Curr. Drug Deliv., 2 (4) (2005), pp. 329–340</li>
<li><span id='br000810'></span>
[[#br000810|[162]]] Castor, T.P. “Polymer microspheres/nanospheres and encapsulating therapeutic proteins therein”, US Patent 2010233308 (2010).</li>
<li><span id='br000815'></span>
[[#br000815|[163]]] Castor, T.P. “Polymer microspheres/nanospheres and encapsulating therapeutic proteins therein”, US Patent 2006033224 (2006).</li>
<li><span id='br000820'></span>
[[#br000820|[164]]] “Protein Nanoparticles”, ''Technical Proceedings of the 2005 NSTI Nanotechnol. Conf. Trade Show, Volume 1, Chapter 3: Drug delivery'' , Nanotech 2005, pp. 172–175 (2005).                                    </li>
<li><span id='br000825'></span>
[[#br000825|[165]]] P. Debbage; Targeted drugs and nanomedicine: present and future; Curr. Pharm. Des., 15 (2) (2009), pp. 153–172</li>
<li><span id='br000830'></span>
[[#br000830|[166]]] A.F. Soares, R. Carvalho, A. de, F. Veiga; Oral administration of peptides and proteins: nanoparticles and cyclodextrins as biocompatible delivery systems; Nanomedicine, 2 (2) (2007), pp. 183–202</li>
<li><span id='br000835'></span>
[[#br000835|[167]]] Castor, T.P. “Polymer microspheres/nanospheres and encapsulating therapeutic proteins therein”, US Patent 2010074961 (2010).</li>
<li><span id='br000840'></span>
[[#br000840|[168]]] A.C. Lima, P. Sher, J.F. Mano; Production methodologies of polymeric and hydrogel particles for drug delivery applications; Expert Opin. Drug Deliv., 9 (2) (2012), pp. 231–248</li>
<li><span id='br000845'></span>
[[#br000845|[169]]] F. Alexis, E.M. Pridgen, R. Langer, O.C. Farokhzad; Nanoparticle technologies for cancer therapy; M.R. Mozafari (Ed.), Handbook of Experimental Pharmacology, Springer (2006), pp. 55–86</li>
<li><span id='br000850'></span>
[[#br000850|[170]]] T. Yasuji, H. Takeuchi, Y. Kawashim; Particle design of poorly water-soluble drug substances using supercritical fluid technologies; Adv. Drug Deliv. Rev., 60 (3) (2008), pp. 388–398</li>
<li><span id='br000855'></span>
[[#br000855|[171]]] Á. Martín, S. Varona, A. Navarrete, M.J. Cocero; Encapsulation and co-precipitation processes with supercritical fluids: applications with essential oils; The Open Chem. Eng. J., 4 (1) (2010), pp. 31–41</li>
<li><span id='br000860'></span>
[[#br000860|[172]]] M. Skwarczynski, I. Toth; Peptide-based subunit nanovaccines; Curr. Drug Deliv., 8 (3) (2011), pp. 282–289</li>
<li><span id='br000865'></span>
[[#br000865|[173]]] M.L. Mbow, E. De Gregorio, J.B. Ulmer; Alum’s adjuvant action: grease is the word; Nat. Med., 17 (4) (2011), pp. 415–416</li>
<li><span id='br000870'></span>
[[#br000870|[174]]] V.E.J.C. Schijns, E.C. Lavelle; Trends in vaccine adjuvants; Expert Rev. Vaccines, 10 (4) (2011), pp. 539–550</li>
<li><span id='br000875'></span>
[[#br000875|[175]]] J.-P.Y. Scheerlinck, S. Gloster, A. Gamvrellis, P.L. Mottram, M. Plebanski; Systemic immune responses in sheep, induced by a novel nano-bead adjuvant; Vaccine, 24 (8) (2006), pp. 1124–1131</li>
<li><span id='br000880'></span>
[[#br000880|[176]]] J.-U.A.H. Junghanns, R.H. Müller; Nanocrystal technology, drug delivery and clinical applications; Int. J. Nanomedicine, 3 (3) (2008), pp. 295–309</li>
<li><span id='br000885'></span>
[[#br000885|[177]]] A.S. Cavallaro, D. Mahony, M. Commins, T.J. Mahony, N. Mitter; Endotoxin-free purification for the isolation of Bovine Viral Diarrhoea Virus E2 protein from insoluble inclusion body aggregates; Microb. Cell Fact., 10 (2011) Art. no. 57</li>
<li><span id='br000890'></span>
[[#br000890|[178]]] S.D. Xiang, A. Scholzen, G. Minigo, C. David, V. Apostolopoulos, P.L. Mottram, M. Plebanski; Pathogen recognition and development of particulate vaccines: does size matter?; Methods, 40 (1) (2006), pp. 1–9</li>
<li><span id='br000895'></span>
[[#br000895|[179]]] D.L.V. Greenwood, K. Dynon, M. Kalkanidis, S. Xiang, M. Plebanski, J.-P.Y. Scheerlinck; Vaccination against foot-and-mouth disease virus using peptides conjugated to nano-beads; Vaccine, 26 (22) (2008), pp. 2706–2713</li>
<li><span id='br000900'></span>
[[#br000900|[180]]] T. Riedel, A. Ghasparian, K. Moehle, P. Rusert, A. Trkola, J.A. Robinson; Synthetic virus-like particles and conformationally constrained peptidomimetics in vaccine design; Chem. Bio. Chem., 12 (18) (2011), pp. 2829–2836</li>
<li><span id='br000905'></span>
[[#br000905|[181]]] R. Sharma, M. Yasir; Virosomes: a novel carrier for drug delivery; Int. J. Pharm. Tech. Res., 2 (4) (2010), pp. 2327–2339</li>
<li><span id='br000910'></span>
[[#br000910|[182]]] C. Ludwig, R. Wagner; Virus-like particles-universal molecular toolboxes; Curr. Opin. Biotechnol., 18 (6) (2007), pp. 537–545</li>
<li><span id='br000915'></span>
[[#br000915|[183]]] F. Canal, J. Sanchis, M.J. Vicent; Polymer-drug conjugates as nano-sized medicines; Curr. Opin. Biotechnol., 22 (6) (2011), pp. 894–900</li>
<li><span id='br000920'></span>
[[#br000920|[184]]] L. Wessels, K. Weninger; Physical aspects of viral membrane fusion; The Sci. World J., 9 (2009), pp. 764–780</li>
<li><span id='br000925'></span>
[[#br000925|[185]]] M. Owais, C.M. Gupta; Liposome-mediated cytosolic delivery of macromolecules and its possible use in vaccine development; Eur. J. Biochem., 267 (13) (2000), pp. 3946–3956</li>
<li><span id='br000930'></span>
[[#br000930|[186]]] L. Bungener, A. Huckriede, A. De Mare, J. De Vries-Idema, J. Wilschut, T. Daemen; Virosome-mediated delivery of protein antigens in vivo: efficient induction of class I MHC-restricted cytotoxic T lymphocyte activity; Vaccine, 23 (10) (2005), pp. 1232–1241</li>
<li><span id='br000935'></span>
[[#br000935|[187]]] K. Sasaki, K. Kogure, S. Chaki, Y. Nakamura, R. Moriguchi, H. Hamada, R. Danev, K. Nagayama, S. Futaki, H. Harashima; An artificial virus-like nano carrier system: Enhanced endosomal escape of nanoparticles via synergistic action of pH-sensitive fusogenic peptide derivatives; Anal. Bioanal. Chem., 391 (8) (2008), pp. 2717–2727</li>
<li><span id='br000940'></span>
[[#br000940|[188]]] J. Wilschut; Influenza vaccines: the virosome concept; Immunol. Lett., 122 (2) (2009), pp. 108–111</li>
<li><span id='br000945'></span>
[[#br000945|[189]]] R. Bron, A. Ortiz, J. Dijkstra, T. Stegmann, J. Wilschut; Preparation, properties, and applications of reconstituted influenza virus envelopes (virosomes); Methods Enzymol., 220 (1993), pp. 313–331</li>
<li><span id='br000950'></span>
[[#br000950|[190]]] E. Ponimaskin, K.K.H. Bareesel, K. Markgraf, R. Reszka, K. Lehmann, H.R. Gelderblom, M. Gawaz, M.F.G. Schmidt; Sendai virosomes revisited: reconstitution with exogenous lipids leads to potent vehicles for gene transfer; Virology, 269 (2) (2000), pp. 391–403</li>
<li><span id='br000955'></span>
[[#br000955|[191]]] M. Kumar, M.Q. Hassan, S.K. Tyagi, D.P. Sarkar; A 45,000-M(r) glycoprotein in the Sendai virus envelope triggers virus- cell fusion; J. Virol., 71 (9) (1997), pp. 6398–6406</li>
<li><span id='br000960'></span>
[[#br000960|[192]]] K. Ramani, Q. Hassan, B. Venkaiah, S.E. Hasnain, D.P. Sarkar; Site-specific gene delivery in vivo through engineered Sendai viral envelopes; Proc. Natl. Acad. Sci. USA., 95 (20) (1998), pp. 11886–11890</li>
<li><span id='br000965'></span>
[[#br000965|[193]]] E.G. Ponimaskin, M.F.G. Schmidt; Fusogenic viral envelopes as potent vehicles for gene transfer; Curr. Genomics, 2 (3) (2001), pp. 261–267</li>
<li><span id='br000970'></span>
[[#br000970|[194]]] T. Daemen, A. De Mare, L. Bungener, J. De Jonge, A. Huckriede, J. Wilschut; Virosomes for antigen and DNA delivery; Adv. Drug Deliv. Rev., 57 (3) (2005), pp. 451–463</li>
<li><span id='br000975'></span>
[[#br000975|[195]]] J.D. Almeida, C.M. Brand, D.C. Edwards, T.D. Heath; Formation of virosomes from influenza subunits and liposomes; Lancet, 2 (7941) (1975), pp. 899–901</li>
<li><span id='br000980'></span>
[[#br000980|[196]]] J.D. Almeida, L. Hoyle; The reaggregation of influenza virus sub-units produced by detergent treatment of virus particles; Micron, 3 (3) (1971), pp. 306–326</li>
<li><span id='br000985'></span>
[[#br000985|[197]]] J.D. Almeida, C.M. Brand; A morphological study of the internal component of influenza virus; J. Gen. Virol., 27 (3) (1975), pp. 313–318</li>
<li><span id='br000990'></span>
[[#br000990|[198]]] R. Glück, K.G. Burri, I. Metcalfe; Adjuvant and antigen delivery properties of virosomes; Curr. Drug Deliv., 2 (4) (2005), pp. 395–400</li>
<li><span id='br000995'></span>
[[#br000995|[199]]] M.G. Cusi, C. Terrosi, G.G. Savellini, G.D. Di Genova, R. Zurbriggen, P. Correale; Efficient delivery of DNA to dendritic cells mediated by influenza virosomes; Vaccine, 22 (5–6) (2004), pp. 735–739</li>
<li><span id='br001000'></span>
[[#br001000|[200]]] D.D. Lasic; Kinetic and thermodynamic effects in the formation of amphiphilic colloidal particles; J. Lipos. Res., 3 (2) (1993), pp. 257–273</li>
<li><span id='br001005'></span>
[[#br001005|[201]]] Y.-S. Zheng, H.-L. Liu, P. Zhao; Application of immunopotentiating reconstituted influenza virosomes in vaccine development; Chin. J. Biol., 22 (8) (2009), pp. 838–840</li>
<li><span id='br001010'></span>
[[#br001010|[202]]] P. Marchisio, S. Esposito, S. Bianchini, E. Dusi, M. Fusi, E. Nazzari, R. Picchi, C. Galeone, N. Principi; Efficacy of injectable trivalent virosomal-adjuvanted inactivated influenza vaccine in preventing acute otitis media in children with recurrent complicated or noncomplicated acute otitis media; Pediatr. Infect. Dis. J., 28 (10) (2009), pp. 855–859</li>
<li><span id='br001015'></span>
[[#br001015|[203]]] A. Arkema, A. Huckriede, P. Schoen, J. Wilschut, T. Daemen; Induction of cytotoxic T lymphocyte activity by fusion-active peptide- containing virosomes; Vaccine, 18 (14) (2000), pp. 1327–1333</li>
<li><span id='br001020'></span>
[[#br001020|[204]]] P.A. Bovier; Epaxal<sup>®</sup> : a virosomal vaccine to prevent hepatitis A infection                                        ; Expert Rev. Vaccines, 7 (8) (2008), pp. 1141–1150</li>
<li><span id='br001025'></span>
[[#br001025|[205]]] C.A. Daubenberger, G. Pluschke, R. Zurbriggen, N. Westerfeld; Development of influenza virosome-based synthetic malaria vaccines; Expert Opin. Drug Discovery, 3 (4) (2008), pp. 415–423</li>
<li><span id='br001030'></span>
[[#br001030|[206]]] N. Subramanian, P. Mani, S. Roy, S.V. Gnanasundram, D.P. Sarkar, S. Das; Targeted delivery of hepatitis C virus-specific short hairpin RNA in mouse liver using Sendai virosomes; J. Gen. Virol., 90 (8) (2009), pp. 1812–1819</li>
<li><span id='br001035'></span>
[[#br001035|[207]]] T.M. Taylor, P.M. Davidson, B.D. Bruce, J. Weiss; Liposomal nanocapsules in food science and agriculture; Crit. Rev. Food Sci. Nutr., 45 (7–8) (2005), pp. 587–605</li>
<li><span id='br001040'></span>
[[#br001040|[208]]] A.D. Bangham, M.M. Standish, N. Miller; Cation permeability of phospholipid model membranes: Effect of narcotics; Nature, 208 (5017) (1965), pp. 1295–1297</li>
<li><span id='br001045'></span>
[[#br001045|[209]]] A.C. Allison, G. Gregoriadis; Liposomes as immunological adjuvants; Nature, 252 (5480) (1974), p. 252</li>
<li><span id='br001050'></span>
[[#br001050|[210]]] M. Foldvari; Biphasic vesicles: a novel topical drug delivery system; J. Biomed. Nanotechnol., 6 (5) (2010), pp. 543–557</li>
<li><span id='br001055'></span>
[[#br001055|[211]]] S. Muro, M. Koval, V. Muzykantov; Endothelial endocytic pathways: Gates for vascular drug delivery; Curr. Vasc. Pharmacol., 2 (3) (2004), pp. 281–299</li>
<li><span id='br001060'></span>
[[#br001060|[212]]] S.L. Hart; Lipid carriers for gene therapy; Curr. Drug Deliv., 2 (4) (2005), pp. 423–428</li>
<li><span id='br001065'></span>
[[#br001065|[213]]] H. Koide, T. Asai, K. Hatanaka, S. Akai, T. Ishii, E. Kenjo, T. Ishida, H. Kiwada, H. Tsukada, N. Oku; T cell-independent B cell response is responsible for ABC phenomenon induced by repeated injection of PEGylated liposomes; Int. J. Pharm., 392 (1–2) (2010), pp. 218–223</li>
<li><span id='br001070'></span>
[[#br001070|[214]]] E. Shahum, H.-M. The’rien; Effect of liposomal antigens on the priming and activation of the immune system by dendritic cells; Int. Immunopharmacol., 2 (4) (2002), pp. 591–601</li>
<li><span id='br001075'></span>
[[#br001075|[215]]] P. Nordly, H.B. Madsen, H.M. Nielsen, C. Foged; Status and future prospects of lipid-based particulate delivery systems as vaccine adjuvants and their combination with immunostimulators; Exp. Opin. Drug Deliv., 6 (7) (2009), pp. 657–672</li>
<li><span id='br001080'></span>
[[#br001080|[216]]] G. Gregoriadis, R. Saffie, J.B. De Souza; Liposome-mediated DNA vaccination; FEBS Lett., 402 (2–3) (1997), pp. 107–110</li>
<li><span id='br001085'></span>
[[#br001085|[217]]] S.W. Dow; Liposome-nucleic acid immunotherapeutics; Exp. Opin. Drug Deliv., 5 (1) (2008), pp. 11–24</li>
<li><span id='br001090'></span>
[[#br001090|[218]]] M. Gürsel, S. Tunca, M. Özkan, G. Özcengiz, G. Alaeddinoglu; Immunoadjuvant action of plasmid DNA in liposomes; Vaccine, 17 (11–12) (1999), pp. 1376–1383</li>
<li><span id='br001095'></span>
[[#br001095|[219]]] M.G. Carstens, M.G.M. Camps, M. Henriksen-Lacey, K. Franken, T.H.M. Ottenhoff, Y. Perrie, J.A. Bouwstra, F. Ossendorp, W. Jiskoot; Effect of vesicle size on tissue localization and immunogenicity of liposomal DNA vaccines; Vaccine, 29 (29–30) (2011), pp. 4761–4770</li>
<li><span id='br001100'></span>
[[#br001100|[220]]] L. Zhu, R.I. Mahato; Lipid and polymeric carrier-mediated nucleic acid delivery; Exp. Opin. Drug Deliv., 7 (10) (2010), pp. 1209–1226</li>
<li><span id='br001105'></span>
[[#br001105|[221]]] Y.S. Tarahovsky; Cell transfection by DNA-lipid complexes — lipoplexes; Biochemistry (Moscow), 74 (12) (2009), pp. 1293–1304</li>
<li><span id='br001110'></span>
[[#br001110|[222]]] D.P. Vangasseri, Z. Cui, W. Chen, D.A. Hokey, L.D. Falo Jr., L. Huang; Immunostimulation of dendritic cells by cationic liposomes; Mol. Membr. Biol., 23 (5) (2006), pp. 385–395</li>
<li><span id='br001115'></span>
[[#br001115|[223]]] Y. Perrie, S. McNeil, A. Vangala; Liposome-mediated DNA immunisation via the subcutaneous route; J. Drug Target., 11 (8–10) (2003), pp. 555–563</li>
<li><span id='br001120'></span>
[[#br001120|[224]]] Y. Hattori, S. Kawakami, S. Suzuki, F. Yamashita, M. Hashida; Enhancement of immune responses by DNA vaccination through targeted gene delivery using mannosylated cationic liposome formulations following intravenous administration in mice; Biochem. Biophys. Res. Commun., 317 (4) (2004), pp. 992–999</li>
<li><span id='br001125'></span>
[[#br001125|[225]]] A.B. Simonetti, G.E. Englert, K. Campos, M. Mergener, C. De David, A.P. De Oliveira, P.M. Roehe; Nanobacteria-like particles: A threat to cell cultures; Braz. J. Microbiol., 38 (1) (2007), pp. 153–158</li>
<li><span id='br001130'></span>
[[#br001130|[226]]] Z. Zhou, L. Hong, X. Shen, X. Rao, X. Jin, G. Lu, L. Li, E. Xiong, W. Li, J. Zhang, Z. Chen, J. Pan, B. Song; Detection of nanobacteria infection in type III prostatitis; Urology, 71 (6) (2008), pp. 1091–1095</li>
<li><span id='br001135'></span>
[[#br001135|[227]]] B.C. Jeong, B.S. Kim, H.H. Kim; Association between nanobacteria and urinary calcium stone disease; Korean J. Urol., 48 (5) (2007), pp. 512–516</li>
<li><span id='br001140'></span>
[[#br001140|[228]]] R.B. Hoover; Mineralized remains of morphotypes of filamentous cyanobacteria in carbonaceous meteorites; Proc. SPIE-Int. Soc. Opt. Eng., 5906 (2005), pp. 1–17 Art. no. 59060J</li>
<li><span id='br001145'></span>
[[#br001145|[229]]] J. Martel, J.D.-E. Young; Purported nanobacteria in human blood as calcium carbonate nanoparticles; Proc. Natl. Acad. Sci. USA., 105 (14) (2008), pp. 5549–5554</li>
<li><span id='br001150'></span>
[[#br001150|[230]]] G. Pasquinelli, F. Papadopulos, M. Nigro; Nanobacteria and psammoma bodies: Ultrastructural observations in a case of pathological placental calcification; Ultrastruct. Pathol., 34 (6) (2010), pp. 344–350</li>
<li><span id='br001155'></span>
[[#br001155|[231]]] D. Raoult, M. Drancourt, S. Azza, C. Nappez, R. Guieu, J.-M. Rolain, P. Fourquet, B. Campagna, B. La Scola, J.-L. Mege, P. Mansuelle, E. Lechevalier, Y. Berland, J.-P. Gorvel, P. Renesto; Nanobacteria are mineralo fetuin complexes; PLoS Pathog., 4 (2) (2008), p. e41</li>
<li><span id='br001160'></span>
[[#br001160|[232]]] J.D. Young, J. Martel, D. Young, A. Young, C.-M. Hung, L. Young, Y.-J. Chao, J. Young, C.-Y. Wu; Characterization of granulations of calcium and apatite in serum as pleomorphic mineralo-protein complexes and as precursors of putative nanobacteria; PLoS ONE, 4 (5) (2009), p. e5421</li>
<li><span id='br001165'></span>
[[#br001165|[233]]] E.O. Kajander, R.J. Harvima, L. Kauppinen, K.K. Akerman, H. Martikainen, R.L. Pajula, S.O. Karenlampi; Effect of selenomethionine on cell growth and on S-adenosynmethionine metabolism in cultured malignant cells; Biochem. J., 267 (3) (1990), pp. 767–774</li>
<li><span id='br001170'></span>
[[#br001170|[234]]] N. Ciftcioglu, O. Kajander; Interaction of nanobacteria with cultured mammalian cells; Pathophysiology, 4 (1998), pp. 259–270</li>
<li><span id='br001175'></span>
[[#br001175|[235]]] J. Hodgson; To treat or not to treat: That is the question for serum; Nat. Biotechnol., 13 (4) (1995), pp. 333–343</li>
<li><span id='br001180'></span>
[[#br001180|[236]]] J. Liu, A.L. Levine, J.S. Mattoon, M. Yamaguchi, R.J. Lee, X. Pan, T.J. Rosol; Nanoparticles as image enhancing agents for ultrasonography; Phys. Med. Biol., 51 (9) (2006), pp. 2179–2189</li>
<li><span id='br001185'></span>
[[#br001185|[237]]] P. Gill, A-H. Alvandi, H. Abdul-Tehrani, M. Sadeghizadeh; Colorimetric detection of helicobacter pylori DNA using isothermal helicase-dependent amplification and gold nanoparticle probes; Diag. Microbiol. Infect. Dis., 62 (2) (2008), pp. 119–124</li>
<li><span id='br001190'></span>
[[#br001190|[238]]] P. Gill, M. Ghalami, A. Ghaemi, N. Mosavari, H. Abdul-Tehrani, M. Sadeghizadeh; Nanodiagnostic method for colorimetric detection of mycobacterium tuberculosis 16S rRNA; Nanobiotechnol., 4 (1–4) (2008), pp. 28–35</li>
<li><span id='br001195'></span>
[[#br001195|[239]]] P. Gill, A. Ghaemi; Nucleic acid isothermal amplification technologies — a review; Nucleosids Nucleotides Nucleic Acids (NNNA), 27 (3) (2008), pp. 224–243</li>
<li><span id='br001200'></span>
[[#br001200|[240]]] I.H. El-Sayed, X. Huang, M. El-Sayed; Selective laser photo-thermal therapy of epithelial carcinoma using anti-EGFR antibody conjugated gold nanoparticles; Cancer Lett., 239 (1) (2006), pp. 129–135</li>
<li><span id='br001205'></span>
[[#br001205|[241]]] Z. Cui, R.J. Mumper; Microparticles and nanoparticles as delivery systems for DNA vaccines; Crit. Rev. Ther. Drug Carrier Syst., 20 (2–3) (2003), pp. 103–137</li>
<li><span id='br001210'></span>
[[#br001210|[242]]] E. Wagner; Programmed drug delivery: nanosystems for tumor targeting; Expert Opin. Biol. Ther., 7 (5) (2007), pp. 587–593</li>
<li><span id='br001215'></span>
[[#br001215|[243]]] H. Devalapally, A. Chakilam, M.M. Amiji; Role of nanotechnology in pharmaceutical product development; J. Pharm. Sci., 96 (10) (2007), pp. 2547–2565</li>
<li><span id='br001220'></span>
[[#br001220|[244]]] S.K. Sahoo, S. Parveen, J.J. Panda; The present and future of nanotechnology in human health care; Nanomedicine, 3 (1) (2007), pp. 20–31</li>
<li><span id='br001225'></span>
[[#br001225|[245]]] K.M. Woo, V.J. Chen, P.X. Ma; Nano-fibrous scaffolding architecture selectively enhances protein adsorption contributing to cell attachment; J. Biomed. Mater. Res., 67 (2) (2003), pp. 531–537</li>
<li><span id='br001230'></span>
[[#br001230|[246]]] M.D. Bhavsar, M.M. Amiji; Polymeric nano- and microparticle technologies for oral gene delivery; Expert Opin. Drug Deliv., 4 (3) (2007), pp. 197–213</li>
<li><span id='br001235'></span>
[[#br001235|[247]]] J.R. Korzenic, D.K. Podolsky; Evolving knowledge and therapy of inflammatory bowel disease; Nat. Rev. Drug Discov., 5 (3) (2006), pp. 197–209</li>
<li><span id='br001240'></span>
[[#br001240|[248]]] B. Ranjbar, P. Gill; Circular dichroism techniques: biomolecular and nanostructural analyses-a review; Chem. Biol. Drug Des., 74 (2) (2009), pp. 101–120</li>
<li><span id='br001245'></span>
[[#br001245|[249]]] P. Gill, T-T. Moghadam, B. Ranjbar; Differential scanning calorimetry techniques: applications in biology and nanoscience; J. Biomol. Tech., 21 (4) (2010), pp. 167–193</li>
<li><span id='br001250'></span>
[[#br001250|[250]]] G. Kersten, H. Hirshberg; Antigen delivery systems; Expert Rev. Vaccines, 3 (4) (2004), pp. 453–462</li>
<li><span id='br001255'></span>
[[#br001255|[251]]] T.D. Nandedkar; Nanovaccines: recent developments in vaccination; J. Biosci., 34 (6) (2009), pp. 995–1003</li>
<li><span id='br001260'></span>
[[#br001260|[252]]] S.Y. Shaw, E.C. Westly, M.J. Pittet, A. Subramanian, S.L. Schreiber, R. Weissleder; Perturbational profi ling of nanomaterial biologic activity; Proc. Natl. Acad. Sci. USA, 105 (2008), pp. 7387–7392</li>
<li><span id='br001265'></span>
[[#br001265|[253]]] A. Nel, T. Xia, L. Madler, N. Li; Toxic potential at the nanolevel; Science, 311 (2006), pp. 622–627</li>
<li><span id='br001270'></span>
[[#br001270|[254]]] K.K. Jain; Nanomedicine: application of nanobiotechnology in medical practice; Med. Princ. Pract., 17 (2) (2008), pp. 89–101</li>
</ol>