Patent Application: US-201314388663-A

Abstract:
the present invention is based on the identification of the adjuvant properties of inorganic nanoparticles . as such , the invention provides an adjuvant comprising , consisting or consisting essentially of inorganic nanoparticle . the invention also provides compositions and vaccines comprising the same as well as methods which exploit the adjuvant inorganic nanoparticles .

Description:
the present invention will now be described in detail with reference to the following figures which show : fig1 : the shape and size of co 3 o 4 np ( a ) and i - alum ( b ). particles were dispersed in distilled water and measured by a transmission electron microscopy . fig2 : cytotoxicity of nps or i - alum to raw264 . 7 cells . particles were added ( concentrations given were in μg / ml ) to raw264 . 7 cells for 24 h with or without 1 % ova . levels of lactate dehydrogenase ( ldh ) were measured for cytotoxicity . note that i - alum showed around 6 times more cytotoxicity compared to co 3 o 4 np and ova increased the toxicity of particles . data are mean ± sem ( n = 4 per group ). the cytotoxicity data of particles treated with 1 % ova or without ova were compared with their respective controls . *** p & lt ; 0 . 001 . fig3 : ova - specific igg1 levels in serum 7 days after second subcutaneous sensitisation . other immunoglobulins ( igg2a , ige , iga , and igm ) showed no increases ( data not shown ). data are mean ± sem ( n = 5 per group ). fig4 : ova - specific immunoglobulin levels present in serum 7 days after intraperitoneal challenge with ovalbumin . immunoglobulins tested for were ( a ) igg1 , ( b ) igg2a , ( c ) ige , ( d ) iga , and ( e ) igm . data are mean ± sem ( representative data , n = 5 per group ). ( f ) proliferation of spleen cells 7 days after intraperitoneal challenge with ova . 3 h - thymidine incorporation of cells cultured with medium only or in the presence of 10 μm ova . fig5 : ova - specific igg1 levels in peritoneal lavage fluid 7 days after challenge with ovalbumin . other immunoglobulins ( igg2a , ige , iga , and igm ) showed no increases ( data not shown ). data are mean ± sem ( n = 5 per group ). fig6 : levels of cytokine in the peritoneal lavage fluid collected 7 days after challenge . ( a ) ifn - γ , ( b ) il - 1β . data are mean ± sem ( n = 5 per group ). * p & lt ; 0 . 05 compared to pbs treatment group . fig7 : differential cell counts of the peritoneal lavage fluid collected 7 days after challenge . data are mean ± sem ( n = 5 per group ). * p & lt ; 0 . 05 , ** p & lt ; 0 . 01 , and *** p & lt ; 0 . 001 compared to pbs treatment group . fig8 : the representative histology of injection site 7 days after challenge . a , pbs ; b , np - ova ; c , i - alum - ova ; d , np alone . note that np - ova and np alone group showed mild inflammation ( arrow ) along with the deposition of the nps ( black spots ) whilst i - alum - ova showed massive inflammation and necrosis ( arrow ). fig9 : graph showing the number of dendritic cells expressing certain markers . fig1 : graph showing the cytotoxic response to cobalt oxide nanoparticles fig1 : graph showing the cytotoxic response to imject alum fig1 : graph showing cytotoxicity of dc in response to cobalt oxide np ± 1 % ova . fig1 : graph showing cytotoxicity of dc in response to imject alum ± 1 % ova . fig1 : graph showing il - 1β generation by dc treated with cobalt oxide np ± 1 % ova . fig1 : graph showing il - 1β generation by dc treated with imject alum ± 1 % ova . as so very few adjuvants are capable of eliciting a th1 response it was considered that nps could be utilized to improve th1 immunity against a model antigen , ovalbumin ( ova ). we selected co 3 o 4 np as a candidate adjuvant because co 3 o 4 np produced a th1 response without the severe delayed - type hypersensitivity pathology seen with nionp [ 19 ]. the aim was to determine whether co 3 o 4 np could be suitable for use as an adjuvant by inducing a balanced th1 and th2 response to the model antigen ova given subcutaneously to female c57bl / 6 mice . to this end we compared anti - ova responses induced by co 3 o 4 np to those induced by a commercially available aluminium containing adjuvant . the adjuvant chosen was imject alum ( i - alum ), a metal particulate adjuvant widely used for murine experiments , which contains a 50 / 50 mixture of aluminium hydroxide and magnesium hydroxide and inactive stabilisers . in addition to ovalbumin - specific responses , toxicity was also assessed at the injection site and cytotoxicity for antigen presenting cells was evaluated using a mouse macrophage cell line . all chemicals and reagents were purchased from sigma - aldrich ( poole , uk ) unless otherwise stated . the size of co 3 o 4 np ( nanostructural and amorphous materials , tx , usa ) and imject alum ( thermo scientific , cramlington , uk ) was measured using a transmission electron microscopy ( jem - 1200ex ii , jeol , tokyo , japan ). the hydrodynamic sizes of particles were measured using a particle size analyser ( 90plus / bi - mas ; brookhaven instruments corporation , new york , usa ) in the presence of various concentrations of a dispersion medium . for in vivo study , hen egg ovalbumin ( grade v ) was used as a dispersion medium for co 3 o 4 np + ova ( np - ova ) and i - alum + ova ( i - alum - ova ) whilst heat - inactivated serum ( final concentration : 5 %) collected from healthy c57bl / 6 mice was used for np - alone . the levels of endotoxin in the particle suspensions were measured using a limulus amebocyte lysate assay ( cambrex , md ., usa ). the solubility of co 3 o 4 np and i - alum was tested in the acidic artificial lysosomal fluid ( ph 5 . 5 ) or basic artificial interstitial fluid ( ph 7 . 4 ) according the previously described method [ 19 ]. based on our previous study , we selected the dose of i - alum as 2 . 0 mg per mouse [ 17 ]. in contrast , the dose of co 3 o 4 np was selected as 25 μg per mouse based on the inflammogenicity in the lung ; instillation of 419 μg / rat of co 3 o 4 np showed acute neutrophilic inflammation and chronic lymphocytic / neutrophilic inflammation with alveolar lipoproteinosis [ 19 ]. mouse macrophage cell line , raw264 . 7 was cultured at 37 ° c . with 5 % co 2 in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) with l - glutamine , antibiotics ( penicillin and streptomycin ) and 10 % heat - inactivated foetal bovine serum ( fbs ). cells were detached using an accutase and seeded at 5 × 10 5 in 12 - well plates ( corning , n . y ., usa ) for experiments . the cytotoxic effects of nps and alum were measured using a lactate dehydrogenase ( ldh ) assay kit ( roche applied science , sussex , uk ) according to the manual instruction . as benchmark particles , tio 2 np ( 30 . 5 ± 1 . 8 nm ) ( nanostructural and amorphous materials , tx , usa ) were used . np or i - alum solutions with or without ova ( 1 %) were treated to raw264 . 7 cells for 24 h . each np was tested at concentrations of 10 , 30 , 100 and 500 μg / ml whilst i - alum was tested at concentrations of 80 , 240 , and 800 μg / ml . after incubation , cell - free culture supernatants were centrifuged at 17000 × g for 20 min to get rid of particles . the cytotoxicity was calculated as percentage compared to the positive control ( 0 . 1 % triton - x ). six - week old female c57bl / 6 mice were obtained from harlan laboratories ( hillcrest , uk ). all animals were maintained and handled under a specific license granted by the uk home office to one of the authors ( semh ) that ensures humane treatment and alleviation of suffering in all animal experiments . after 1 week to acclimatise , mice ( 5 mice per group ) were immunised twice at 2 weeks interval . each mouse was sensitised by subcutaneous injection at the base of tail with a 100 μl dose containing 25 μl pbs , 50 μl ( 100 μg ) endotoxin - free ovalbumin ( worthington biochemical corporation , nj , usa ), and 25 μl of adjuvant ( i - alum : 2 . 0 mg ; co 3 o 4 np : 25 μg ). pbs ( 100 μl ) and co 3 o 4 np ( 25 μg in pbs with 5 % mouse serum ) were used for negative controls . then , 50 μg of ova in 100 μl of pbs was injected into the peritoneum as a challenge . at 7 days after the second sensitisation , blood was collected from the tail vein . at 7 days after a challenge , the blood was collected by cardiac puncture and peritoneal lavage was performed three times using a 2 ml of 0 . 9 % saline . the first lavage fluid was kept for immunoglobulins and cytokines elisa , whilst cells from three lavages were pooled for counting cells . the site of injection was also fixed with 10 % neutral buffered formalin for histological analysis . blood samples collected 1 wk after immunisation and 1 wk after challenge were analysed for the presence of ova - specific immunoglobulins of iga , ige , igg1 , igg2c ( formerly known as igg2a b ) and igm subclasses . serum and peritoneal lavage samples were diluted as appropriate in pbs with 1 % bsa . biotin - conjugated secondary antibodies were diluted as follows : anti - iga — 1 : 1000 ; anti - igg1 — 1 : 8000 ; anti - igg2c — 1 : 1000 ; anti - igm — 1 : 1000 ; anti - ige — 1 : 250 . before measuring ige , serum was incubated for an hour with protein - g coupled beads to remove igg [ 17 ; 18 ]. to evaluate the cytokine profile , th1 type cytokine ( ifn - γ ), th2 type cytokines ( il - 10 and il - 13 ), and macrophage - derived pro - inflammatory cytokine ( il - 113 ) were tested in the peritoneal lavage . all kits were purchased from r & amp ; d systems ( abingdon , uk ) and cytokines were measured according to the manufacturer &# 39 ; s instruction . total cell number in lavage fluid was assessed by use of a cell nucleus counter ( chemometec , surrey , uk ). cells were cytospined onto glass slides ( thermo scientific , cramlington , uk ), fixed with methanol and stained with diff - quik ( raymond lamb , eastbourne , uk ). around 300 - 500 cells per slide were counted under a light microscope according their morphology . spleens were removed from mice and single cell suspensions made by passing through 40 μm filters . cells were layered onto lympholyte - m ™ ( fisher scientific , loughborough , uk ) density gradient separation medium and spun at 2000 × g for 15 minutes to remove dead cells , erythrocytes and granulocytes . the mononuclear cell layer at the interface was removed washed and resuspended in tissue culture medium ( rpmi 1640 supplemented with 10 % fcs , 100 u / ml penicillin , 100 μg / ml streptomycin , 2 mm l - glutamine and 50 μm 2 - me ; sigma , uk ). viable cells were counted and triplicate cultures containing 4 × 10 5 cells in 200 μl containing no or 10 μm endotoxin - free ovalbumin were plated in 96 - well plates . cultures were incubated for 48 hrs and 0 . 5 μci 3 - h - thymidine was added to each well overnight . cultures were harvested and incorporated thymidine counted in a betaplate scintillation counter ( wallac uk , milton keynes , uk ). supernatants from parallel non - labelled cultures were harvested at 72 hrs for cytokine analysis . paraffin embedded formalin fixed tissues from the injection site were cut into 3 μm sections and stained with haematoxylin and eosin . data were analysed with graphpad prism software ( version 5 , graphpad software inc ., la jolla , calif ., usa ). to compare treatment groups , one - way analysis of variance with post hoc tukey &# 39 ; s pairwise comparisons was applied . p & lt ; 0 . 05 was considered to be statistically significant . co 3 o 4 np showed spherical shapes and the primary size was 18 . 4 ± 5 . 0 nm . i - alum showed more heterogeneous shapes and the average size was 88 . 7 ± 32 . 4 nm for length and 28 . 9 ± 19 . 7 nm for width ( fig1 ). endotoxin levels of both particles were below the detection limit ( 0 . 1 eu / ml ). co 3 o 4 np showed large agglomerates without a dispersion medium but small agglomerates with ovalbumin as a dispersant ( table 1 ). i - alum showed small agglomerates even without dispersion medium . most i - alum was dissolved within 3 days in the acidic artificial lysosomal fluid ( ph 5 . 5 ) whilst minimally dissolved in the artificial interstitial fluid ( ph 7 . 4 ) ( data not shown ). co 3 o 4 np showed minimal dissolution in both conditions ( data not shown ). i - alum was by far the most toxic of the nps tested on mouse macrophages ( fig2 ). when particles were dispersed with ova , particles were more toxic . igg1 was the only immunoglobulin subclass measurable at this stage . fig3 shows that both i - alum - ova and np - ova induced igg1 production although i - alum - ova was more potent than np - ova . levels of ova - specific immunoglobulins induced by np - alone were similar to the vehicle control ( pbs ). immunoglobulin levels after challenge demonstrated a wider array of responses . both np - ova and i - alum - ova produced increases in the igg1 response although again i - alum - ova showed higher responses than np - ova ( fig4 a ). the igg1 levels in both np - ova and i - alum - ova groups showed a marked increase when compared to those of post - sensitisation . in addition , np - ova increased in igg2c levels ( fig4 b ). i - alum - ova induced higher ige , igm , and iga levels than np - ova ( fig4 c - 4e ). spleen mononuclear cells were challenged in vitro with ova 7 days after challenge . fig4 f shows that splenocytes from both i - alum and np - ova challenged mice proliferated . both np - ova and i - alum - ova showed increases in the igg1 levels ( fig5 ). i - alum - ova showed higher igg1 levels compared to np - ova . other immunoglobulins ( igg2c , ige , iga , and igm ) were detected only at very low concentrations similar with pbs group ( data not shown ). the peritoneal lavage fluid collected from the mice was also used to measure cytokine expression . levels of ifn - γ were significantly increased by the np - ova whilst other groups showed no significant responses ( fig6 a ). levels of il - 1β were significantly increased by i - alum - ova only ( fig6 b ). il - 10 and il - 13 showed no significant changes compared to controls ( data not shown ). number of total cells and lymphocytes was significantly increased by both np - ova and i - alum - ova whilst np - alone was comparable to vehicle control ( fig7 a and 7b ). number of total granulocytes was significantly increased only by i - alum - ova ( fig7 c ). histology samples taken from the injection site at 7 days after challenge provided sharply contrasting results between the two particles tested . the i - alum - ova treatment group showed marked inflammation and necrosis with no obvious particle deposition whilst the np - ova and np - alone group showed minimal inflammation with surrounding particle deposition ( fig8 ). determine the toxicity of cobalt oxide nanoparticles and i - alum to pbmc derived dendritic cells using the lactate dehydrogenase cytotoxicity assay determine the dose required to induce a good cytokine response of il - 1β , il - 10 and il - 12 without inducing toxicity in the cells treat bovine pbmc derived dendritic cells at the optimum concentrations of np / i - alum ± ag85 . ag85 is highly conserved and the protein is identical in the organism which causes human tuberculosis ( mtb ) and the related organism ( mb ) that causes tb in cattle . this will allow us to identify the profile of immunomodulatory cytokines produced by the cells by elisa and assess cell surface maturation markers and mhc class 11 expression by flow cytometric analysis successfully determined the cell surface marker profile for resting dcs . this was then compared to dcs treated with cobalt oxide nanoparticles and imject alum ± ag85 to address changes in activation status and mhc class 11 expression using the lactate dehydrogenase cytotoxity assay we determined the levels of toxicity against increasing doses of cobalt oxide nanoparticles and imject alum . the concentration of each will be 50 μg / ml looked at the profile of immunomodulatory cytokines produced by the cells by elisa . increased levels of il - 1β being produced in response to greater doses of imject alum . there is no difference in the levels of cellular toxicity in dendritic cells treated with cobalt oxide np &# 39 ; s and i - alum (± 1 % ova ) the levels of il - 1β in the supernatants from dendritic cells treated with cobalt oxide np &# 39 ; s and i - alum are comparable for both the cobalt oxide and imject alum treated dcs the addition of 1 % ova leads to increased levels of il - 1 beta being produced with imject alum + 1 % ova showing higher levels compared to cobalt oxide np + 1 % ova . the safety of adjuvants to be used on human is a key issue in their development . recent studies have shown that the licensed adjuvant activates human monocytes and macrophages [ 20 ], suggesting that what would be deemed as toxicity is potentially a part of the mechanism by which alum adjuvant works . in this study , we hypothesised that co 3 o 4 np may produce a balanced th1 and th2 response with less toxicity than alum , suggesting that they may be a safer option . co 3 o 4 np showed relatively homogenous spherical shape of particles . interestingly , i - alum was more heterogeneous and less than 100 nm in both dimensions . the heterogeneous shape might be due to the composition of i - alum comprising a mixture of aluminium hydroxide and magnesium hydroxide . therefore , both co 3 o 4 np and i - alum were within the definition of nanoparticles and classified as metal oxide nps [ 21 ; 22 ]. as previous studies have shown that size matters in adjuvanticity , optimal dispersion of nps might be beneficial [ 12 ]. ova acted as an excellent dispersant for both particles . co 3 o 4 np showed small agglomerates when dispersed with ova which forms a protein corona providing repulsive force between particles [ 23 ]. in contrast to co 3 o 4 np , i - alum was relatively well - dispersed in saline which might be because of the stabilisers added during manufacturing . adjuvants are believed to work by activating antigen presenting cells but there is little information on their toxicity for such cells . using a murine macrophage cell line , the ldh cytotoxicity study showed that i - alum by itself was approximately 6 times more toxic than an equivalent amount of co 3 o 4 np . dispersal in ova showed increased toxicity of both co 3 o 4 np and alum which might be due to the cytotoxicity of ova itself or to facilitating recognition and phagocytosis of particles by forming a protein corona . the post - sensitisation data showed that both i - alum - ova and np - ova increased serum ova - specific igg1 levels ( which is marker for th2 response [ 3 ]) whilst other immunoglobulins were not increased . however , the potential for igg1 production by i - alum - ova was about three times higher than that of np - ova . the post - challenge data showed that a t cell response was generated , confirmed by proliferation of spleen cells cultured with ova in vitro . further boosted immunoglobulin responses were also seen in mice immunized with either adjuvant . the presence of antigen specific class - switched immunoglobulins ( igg and ige ) in serum indicate that both th1 and th2 ova - specific cd4 + t lymphocytes have been activated by ova after sensitisation with the antigen using co 3 o 4 np as adjuvant . in serum i - alum - ova produced mainly th2 type ( igg1 ) and allergic antibody responses ( ige ) whilst np - ova produced a more balanced th1 ( igg2c ) and th2 ( igg1 ) type response with much less ige . this suggests that co 3 o 4 np as an adjuvant maybe more likely to provide robust ‘ all - round ’ immunity and less likely to induce unwanted allergic / side effects . this was further supported by the post - challenge peritoneal lavage fluid analysis . th2 dependent igg1 levels were significantly increased by np - ova and i - alum - ova with the same pattern as seen in serum . peritoneal lavage ifn - γ a th1 type cytokine [ 24 ], was increased only in the np - ova immunised group . the differential cell counts of the peritoneal lavage fluid showed that both np - ova and alum - ova induced lymphocytic inflammation which is important for adjuvanticity [ 5 ] but np - ova induced far less granulocytic inflammation . this granulocytic inflammation by i - alum - ova was consistent with the increased il - 1β in the peritoneal lavage which is one of the macrophage derived pro - inflammatory cytokines . the pattern of response induced by i - alum - ova is consistent with that seen in other studies [ 25 ; 26 ]. at 7 days after challenge at the doses used , i - alum induced prolonged massive inflammation and necrosis in the subcutaneous and muscular layer of the injected skin whilst co 3 o 4 np showed mild inflammation . the greater inflammation induced by i - alum compared to co 3 o 4 np was consistent with increased peritoneal il - 1β secretion and number of granulocytes recruited to the peritoneal cavity by i - alum indicating inflammatory responses at both sensitisation and challenge sites . in addition the i - alum induced more allergic anti - ova ige serum antibodies . this provides a more rounded result with co 3 o 4 np than a previous report where tio 2 np showed a higher ige response than al ( oh ) 3 , though in that case al ( oh ) 3 produced the higher granulocyte recruitment concurring with our data [ 27 ]. in addition , co 3 o 4 np showed a more balanced th1 and th2 response even with 80 times less mass administered than i - alum . the absence of cell death and inflammation at the injection site of np - ova group indicates that it will cause less local pain . in addition from a veterinary point of view in food animals , less cell death and inflammation at the injection site means less and hide meat spoilage and may have improved economic benefits . within 3 days of incubation , most of the i - alum was dissolved in the acidic ( ph 5 . 5 ) solution which is the same ph of the lysosomal fluid of alveolar macrophages [ 28 ] whilst there was minimal dissolution in the basic ( ph 7 . 4 ) solution . however , co 3 o 4 np showed minimal dissolution in both conditions . compositional ions released in the acidic lysosomal condition would be likely to play a role in cell death and inflammation for high - solubility nps such as cuonp [ 29 ] and znonp [ 30 ]. therefore , al 3 + dissolved in the lysosomes of phagocytic cells might contribute to cell death and further inflammation by trojan - horse type mechanism [ 31 ]. co 3 o 4 np had not changed in solubility and this was consistent with persistence at the injection site . this suggests that the co 3 o 4 np is very insoluble and stable . there is a concern regarding the solubility of co 3 o 4 np , as cobalt ions are being released very slowly into cells and their long - term effect is unknown [ 19 ]. cobalt poisoning is not common but it can occur . there are reports of hip replacements ( which contain cobalt and chromium ) that corrode over time and release metal ions into the body causing severe illness [ 32 ]. this seems an unlikely side - effect here for co 3 o 4 np due to their small dose and volume of injection and insoluble nature . in our previous study , deposition of co 3 o 4 np inside of lung causes delayed - type hypersensitivity and pulmonary alveolar lipoproteinosis [ 19 ]. however considering the lung has much more active immune response than the skin and muscle , the subcutaneous side effects might be more limited and indeed only mild inflammation was produced in the injection site at 5 weeks after two injections . however if multiple repeated vaccinations were to be administered , build - up of co 3 o 4 np and release of cobalt ions may cause health problems . accumulation of the co 3 o 4 np within the subcutaneous and muscular layers indicates that co 3 o 4 np may provide a continuous source of desorbed antigen which is one of the modes of actions proposed for alum adjuvant [ 33 ]. the comparison between i - alum and co 3 o 4 np is suitable for measuring a response in mice . imject alum is not licensed for use in human and has a different physicochemistry to clinically approved products [ 33 ]. to be able to fully evaluate co 3 o 4 np in long - term study as a prospective human adjuvant further studies are required but we believe that co 3 o 4 np shows great promise in inducing both th1 and th2 mediated responses . in conclusion , co 3 o 4 np stimulated less allergic antibody production and in vivo inflammation ( at both sensitisation and challenge sites ) than a standard dose of the alum - containing adjuvant imject alum . together with the evidence that they also produced lower in vitro toxicity whilst stimulating both th1 and th2 in vivo antibody responses , this indicates that co 3 o 4 np would be suitable for use as a vaccine adjuvant . 1 . gupta r k , siber g r . adjuvants for human vaccines — current status , problems and future prospects . vaccine . 13 , 1263 - 1276 ( 1995 ). 2 . schijns v e , brewer j m . new views on immunopotentiators in modern vaccines . expert . rev . vaccines . 7 , 877 - 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