,Joo Yeon Prk,Yun Shon, Gyong Shim,＊,＊

aCollege of Pharmacy and Research Institute of Pharmaceutical Sciences,Seoul National University,Seoul 151-742, Republic of Korea

bDepartment of Molecular Medicine and Biopharmaceutical Sciences,Graduate School of Convergence Science, Seoul National University,Daehak-dong,Gwanank-gu,Seoul,Republic of Korea

Review

Nanotechnology and vaccine development

Mi-Gyeong Kima,Joo Yeon Parka,Yuna Shona,Gunwoo Kimb, Gayong Shima,＊,Yu-Kyoung Oha,＊

aCollege of Pharmacy and Research Institute of Pharmaceutical Sciences,Seoul National University,Seoul 151-742, Republic of Korea

bDepartment of Molecular Medicine and Biopharmaceutical Sciences,Graduate School of Convergence Science, Seoul National University,Daehak-dong,Gwanank-gu,Seoul,Republic of Korea

A R T I C L E I N F O

Article history:

Received 28 March 2014

Received in revised form

7 June 2014

Accepted 9 June 2014

Available online 18 June 2014

Nanotechnology

Vaccine delivery

Liposomes

Emulsions

Polymeric nanocarriers

Carbon nanomaterials

Despite the progress of conventional vaccines,improvements are clearly required due to concerns about the weak immunogenicity of these vaccines,intrinsic instability in vivo, toxicity,and the need for multiple administrations.To overcome such problems,nanotechnologyplatformshaverecentlybeen incorporatedintovaccinedevelopment. Nanocarrier-based delivery systems offer an opportunity to enhance the humoral and cellular immune responses.This advantage is attributable to the nanoscale particle size, which facilitates uptake by phagocytic cells,the gut-associated lymphoid tissue,and the mucosa-associated lymphoid tissue,leading to eff i cient antigen recognition and presentation.Modifying the surfaces of nanocarriers with a variety of targeting moieties permits the delivery of antigens to specif i c cell surface receptors,thereby stimulating specif i c and selective immune responses.In this review,we introduce recent advances in nanocarrierbased vaccine delivery systems,with a focus on the types of carriers,including liposomes, emulsions,polymer-based particles,and carbon-based nanomaterials.We describe the remaining challenges and possible breakthroughs,including the development of needlefree nanotechnologies and a fundamental understanding of the in vivo behavior and stability of the nanocarriers in nanotechnology-based delivery systems.

? 2014 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/ licenses/by-nc-nd/3.0/).

1. Introduction

The seasonal outbreaks of pantropic infection diseases have elevated the development of effective vaccines to the status of a global healthcare concern.Vaccines have been developed using killed organisms[1],live attenuated organisms[2],or inactivated toxins[3].Recently,subunit vaccines[4],and DNA vaccines that encode antigenic pathogenic proteins[5]have been examined as new vaccine modalities.Although subunit vaccines and DNA vaccines have the advantages of a high safety prof i le over traditional vaccine,these vaccines suffer from a relatively lower immunogenicity.The immunogenicity may potentially be improved by modulating the vaccine formulation using nanotechnology.

The nanotechnologies developed for use in the f i eld of vaccines encompass nanocarriers having a variety of compositions,sizes,and surface properties[6].Numerous vaccine nanocarriers have been designed and investigated for their utility in the delivery of antigens and adjuvants to immune cells in an effort to promote a protective immune response. Unfortunately,although antigens may be taken up by the immune cells,insuff i cient adjuvant activity may result in limited immunogenicity.In some approaches,nanocarriers have been designed to co-deliver both an antigen and an adjuvant[7].Nanocarriers can facilitate the targeting and/or sustained releaseofantigensoradjuvantstoantigenpresenting cells[8,9].

Working mechanisms of nanotechnology-based vaccine formulations support the utility of nanocarriers in the vaccine f i elds.Particles smaller than 10 μm are readily taken up by phagocytic cells,such as macrophages and dendritic cells (DC).This property has been used to improve the cellular uptake of antigens,thereby increasing the eff i ciency of antigen recognition and presentation[10].Solid nanocarriers can protect protein-based antigen vaccines from degradation and facilitate entry into the gut-associated lymphoid tissue and mucosa-associated lymphoid tissues,rendering them appropriate for vaccine delivery via oral or mucosal routes[11]. Surface-modif i ed nanocarriers may assist the targeted delivery of antigens.Immune cells express a variety of surface receptors,including the mannose receptor,scavenger receptor,and toll-like receptors(TLR)[12].Nanocarriers coated with immune cell-targeting molecules,such as carbohydrates[13], antibodies[14],and peptides[15],may target these overexpressed receptors to improve the eff i ciency of antigen and adjuvant delivery toward the promotion of specif i c and selective immune responses in prophylactic vaccines.

This review provides an overview of recent advances in nanocarrier vaccine systems,including liposomes,emulsions, polymer-basednanodeliverysystems,andcarbon-based nanodelivery systems(Fig.1).The current status of in vivo applications of nanocarriers is summarized in Table 1.

2. Nanodelivery systems for vaccines

2.1. Liposomes

Since the f i rst report that liposomes can act as immunological adjuvants[16],liposome formulations(Fig.1A)have been extensively studied for use in vaccine delivery systems.As of this publication,at least 8 liposomal vaccines are in clinical trials or have been approved for human use[17].The physicochemical properties of liposomes,including their size,lipid composition,and structure,may be modulated according to the properties of the vaccine antigen to maximize immunogenicity.Liposomes are composed of biocompatible phospholipid bilayers and are capable of loading and delivering both hydrophilic and hydrophobic molecules.These properties enable the co-delivery of antigen and other molecules,such as adjuvants.The surfaces of liposomes may be easily modif i ed using the appropriate functionally active lipid as a component of the lipid bilayer.Surface-modif i ed liposomes have been designed to target immune cells, co-deliver immunostimulatory agents,and enhance both the humoral and cell-mediated immune responses simultaneously to improve the eff i cacy of liposomal vaccines.

Fig.1-Structure of nanocarriers for vaccine antigen delivery.(A)Liposomes,(B)emulsions,(C)polymeric nanoparticles,and(D)graphene oxide nanosheets.

The physicochemicalproperties of liposomes,such as their size and f l uidity,are important for the induction of an immune response.The vesicle sizes of liposomes composed of cationic dimethyldioctadecylammonium(DDA)can affect the cell-mediated immune response,but not the humoral immune response[18].Liposomes larger than 2 μm in diameter were found to effectively promote interleukin-10 production, whereas liposomes 500 nm in diameter promoted a higher level of interferon-γ production in splenocytes.

Small unilamellar vesicles composed of cationic DDA liposomes were found to produce higher CD8 T cell responses compared to the larger multilamellar vesicles[19].A recent study reported that rigid DDA lipid-based liposomes produced a Th1-directed immune response against antigens that was 100 times greater than the response produced by f l uidic dimethyldioleoylammonium(DODA)-based liposomes[20].

The liposomal delivery of protein antigens via surface adsorption methods may be optimized by tuning the surface antigen and lipid ratio.The protein surface antigen-to-lipid ratio can affect the aggregation behavior of liposomes and can impact general vaccine stability during storage[21].Surface modif i cations to antigen-carrying liposomes gearing polyethylene glycol(PEG)groups can reduce liposome aggregation.However,the retention of the liposomes at the injection sites was simultaneously reduced,thereby altering the Th1/Th2 immune response compared to the response produced by unmodif i ed liposomes[22].

The liposomal co-delivery of antigens and an immunostimulatory molecule can enhance the generation of a protective immuneresponse.Theentrapmentoftrehalose6,6-dibehenate (TDB),an immunostimulatory molecule,within a liposome did notaffectthephysicochemicalpropertiesofneutraldistearoylsn-glycero-3-phosphocholine(DSPC)or cationic DDA-based liposomes,and signif i cantly increased the production of IFN-γ after immunization[23].Monophosphoryl lipid A(MPL),a poorly soluble TLR 4 agonist,was added to the bilayers ofcationic liposomes composed of DDA and TBD to increase liposome membrane packing and reduce the surface charges. The MPL/DDA/TDB liposomal formulation carried ovalbumin (OVA)as a model antigen.In mice,the presence of MPL in liposomes did not affect the humoral immune response,but signif i cantly enhanced the antigen-specif i c CD8+T cell immune response[24].Some studies reported the use of an adjuvantformulation comprising liposomescontaining an immunostimulatory molecule.The co-administration of a trivalent inf l uenza vaccine and cationic liposomes containing TDB was found to augment both the humoral and the cellular immune responses[25].

Table 1-In vivo applications of nanocarriers for delivery of vaccines.

Interbilayer-crosslinked multilamellar vesicles were recently designed and tested as nanocarriers for protein antigens[26].The vesicles were formed by crosslinking the head groups of lipid bilayers within anionic mutilamellar vesicles composedof1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC), 1,2-di-(9Z-octadecenoyl)-snglycero-3-phospho-(1'-rac-glycerol) (DOPG)and maleimide-headgroup lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide(MPB).Interbilayer crosslinking via divalent magnesium cation-basedfusion permittedthestable entrapment of protein antigens within the core and lipophilic immunostimulatorymolecules withinthe lipid membranes.OVA was usedas amodelantigen todemonstratethattheinterbilayercrosslinked vesicles provided the sustained release of antigens and enhanced the antigen-specif i c CD8+T cell immune response in comparison with the uncrosslinked liposomes.

Nickel-chelating liposomes were examined for their potential use in His-tag-mediated antigen vaccine loading[27]. Nickel-chelating liposomes 100 nm in size were prepared using egg phosphatidylcholine(EPC)and a nickel-chelating lipid,1,2-dioleoyl-sn-glycero-3-[N(5-amino-1-carboxypentyl) iminodiacetic acid]succinyl nickel salt(DOGS-NTA-Ni),in a molar ratio of 95:5.The His-tagged heat shock protein from Candida albicans was loaded onto the surfaces of the nickelchelating liposomes via the formation of metallochelating bonds by incubating the antigens with the liposomes.

Nickel-chelating liposomes have also been prepared by incorporating nitrilotriacetic acid lipid derivatives into the liposomes to promote bonding to polyhistidinylated OVA antigens[28].The liposomes were composed of DOGS-NTANi, dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol(DMPG),cholesterol,and MPL.Although these metallochelating liposomes offer meaningful progress toward recombinant protein antigen loading without covalently modifying the antigens,the toxicity of the nickel-chelating lipids presents certain safety concerns for clinical applications.

Liposomes-in-oil adjuvant formulations were found to prolong the immune response after vaccination[29].Diphtheria toxoids were co-encapsulated with poly(I:C)in a liposome composed of lecithin and cholesterol(9:1 M ratio).The subcutaneous injection of the liposomes suspended in oil (Marcol 52:Montanide 888,9:1)generated a persistent immune response by reducing antigen transport to the draining lymph nodes.

Cationic liposomes have been examined for their ability to enhance the adjuvanticity of nucleic acid-based TLR agonists. Liposomes composed of the cationic lipid octadecenolyoxy [ethyl-2-heptadecenyl-3 hydroxyethyl]imidazolinium chloride and cholesterol were complexed to the TLR agonist CpG-based plasmid DNA[30].The intramuscular administration of the combination of lipoplexes and Fluzone,an inf l uenza vaccine,wasshowntoprovideanenhancedantibody response and cell immunity in mice[30]and in elderly rhesus macaques[31].

2.2. Emulsions

Emulsions have been long studied as adjuvant formulations and more recently studied as vaccine delivery systems. Emulsions are dispersions of two or more immiscible liquids composed of oil,emulsif i ers,and excipients(Fig.1B).Two broad classes of emulsions may be formed:water-in-oil emulsions and oil-in-water emulsions.The latter emulsion type is typically used in adjuvant formulations.

ThemostfamousvaccineadjuvantemulsionisMF59.MF59 consists of squalene oil,Span 85,and Tween 80 in a citrate buffer.The MF59-adjuvanted seasonal f l u vaccine(Fluad?) was approved in Europe in 1997[32].MF59 can effectively increase the immune response in infants and young children [33].A recent study investigated the role of each component of MF59 toward the adjuvant properties[34].Span 85 was found to activate the muscle transcriptome.However,Span 85 alone did not provide an eff i cient immune response comparable to that of MF59.

MF59was studied for its ability to augment the inductionof antibodies against meningococcal protein antigens[35]and the Neisseria meningitidis B vaccine[36].The delivery of recombinant meningococcal B protein antigens in conjunction with the TLR4 agonist,E6020,withinMF59was shown to cause a strong antigen-specif i c CD4 T-cell response after three staged intramuscular injections into CD-1 mice[35].

AF03,anothersqualene-basedemulsion,hasbeenusedasa vaccine adjuvant.AF03,which consists of squalene,sorbitan oleate,andcetheareth-12,ispresentinthemarketedinf l uenza vaccine,Humenza?.Recently,thephysicochemicalproperties of AF03 were characterized to evaluate the long-term stability of the formulation.The surfactants in AF03 were analyzed usingmassspectroscopy and high-performanceliquid chromatography-mass spectroscopy as quality control tests in emulsion manufacturing processes[37,38].

In addition to the squalene-based MF59 and AF03 emulsions,adjuvantnanoemulsion formulationshavebeen developedwithoutsqualene.Forexample,anaqueous dispersion of W805EC,composed of cetyl pyridinium chloride, Tween 80,ethanol,and soybean oil,in phosphate-buffered saline or 0.9%NaCl was found to promote an immune response to the model antigen OVA via intranasal administration[39].The nasal adjuvanticity of the W805EC nanoemulsion was attributed to dendritic cell engulfment of antigen-loaded epithelial cells[40].

Glucopyranosyl lipid A(GLA),a TLR4 agonist,has been tested for its adjuvanticity in the context of oil-in-water emulsion vaccine antigen formulations.For example,GMZ2, a fusion protein component in an anti-falciparum vaccine, was combined with GLA in an oil-in-water emulsion vaccine formulation[41].The immunostimulatory properties of GLA were then compared with the properties of several other immunostimulatory agents formulated with GMZ2.GLA was found to display the highest antigen-specif i c IgG2a and total IgG titers.Another study reported that the immunogenicity of a recombinant Plasmodium vivax protein vaccine could be enhanced by subcutaneously co-delivering the vaccine with GLA in an oil-in-water emulsion formulation[42].The intradermal administration of GLA within oil-in-water emulsions to human skin explants was found to enhance the capacity of skin DCs to activate both CD4+T cells and the emigration of Langerhans cells within skin tissues[43].Oil-in-water emulsions containing GLA and recombinant hemagglutinin are currently in phase 2 clinical trials for the prevention of seasonal inf l uenza[44].The adjuvanticity of the hemagglutinin emulsion prepared with GLA was found to increase the titers of hemagglutinin-specif i c antibodies relative to the formulation prepared without GLA,following intramuscular administration to healthy adults.

The successfulcommercialization ofsqualene-based emulsions in marketed vaccines suggests that emulsion adjuvant approaches warrant further exploration.Formulation stability during storage and the development of biocompatible oilcomponentsmaybecrucialconcernsforthedevelopmentof new emulsion formulations for vaccine delivery.

2.3. Synthetic polymer-based nanodelivery systems

Polymeric nanocarriers(Fig.1C)have been examined as vaccine delivery systems to take advantage of their ability to offer the controlled release of antigens or adjuvants.Poly(lactic-coglycolic acid)(PLGA)is the most widely used biodegradable synthetic polymer nanocarrier with a relatively long history of biomedical use.The PLGA particle size has been varied and surface modif i cations have been introduced into vaccine formulations for use in oral,mucosal,and systemic delivery.

The sizes,surface modif i cation,and release prof i les of PLGA particles were shown to affect the immunogenicity of entrapped antigens.The average size of an aerosolized PLGA or poly(L-lactic acid)-based nanoparticle pulmonary hepatitis B virus vaccine formulation was suggested to inf l uence the immunogenicity of the antigen[45].Nanoparticles larger than 500 nm induced the generation of antigen-specif i c secretary IgA more effectively than smaller nanoparticles(＜500 nm)in rats.

The surfaces of the PLGA polymeric nanoparticles were modif i ed with lipids to enable the co-delivery of antigens and adjuvants[46].In a recent study the OVA antigen was covalently anchored to a pegylated phospholipid bilayer coating formedonthePLGAnanoparticlesurfaces,andalipidadjuvant, suchasMPLor α-galactosylceramide,wasincorporatedintothe lipid bilayer.Subcutaneous co-delivery of OVA with MPL in the surface lipid coating of PLGA nanoparticles was shown to enhance the antigen-specif i c IgG titer values more than 10 folds.Other groups have conjugated a candidate P.vivax malaria antigen,VMP001,to the surfaces of lipid-coated PLGA particles,and MPL has been incorporated into the lipid membranes[47].In the study PLGA particle surfaces were coated with lipids composed of DOPC,DOPG,and 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[maleimide].

The prolonged release of antigens delivered by PLGA nanoparticles was shown to enhance the immune response to the model OVA antigen[8].PLGA nanoparticles provided an OVA release prof i le that extended over 10 weeks and was much slower than the release prof i le obtained from a liposomal formulation.In line with the sustained release prof i le, an OVA-PLGA particulate vaccine formulation induced a higher antibody titer compared to the liposome formulation 8 weeks after subcutaneous administration in mice.

The synthetic PLGA vaccine SEL-068(Selecta Bioscience, Inc.,USA)is now in phase I clinical trials for the prevention of nicotine addiction and relapse(http://www.selectabio.com). SEL-068 was designed to facilitate smoking cessation and is the f i rst nano-vaccine to enter clinical trials.The SEL-068 formulation incorporates a universal peptide antigen for eliciting T helper cell response and an immunostimulating TLR agonist into the PLGA polymer matrix.Nicotine,a B cell antigen,is covalently linked to the nanoparticle surfaces.No dose-limiting systemic toxicities were observed in a repeatdose GLP safety and eff i cacy study in cynomolgus monkeys.

Other biodegradable polymers,including poly(ε-caprolactone)and polyanhydrides,have been tested for their utility in vaccine delivery applications.The H1N1 hemagglutinin antigen was incorporated into chitosan-coated polycaprolactone nanoparticles.Intranasal delivery of the chitosan-polycaprolactone nanocarriers containing H1N1 hemagglutinin was shown to produce serum IgG levels and secretory IgA levels in nasal and lung lavage that were higher than those produced following intranasal delivery of the soluble antigen to Balb/c mice[48].

Polyanhydride nanocarriers have been tested as vaccine delivery systems to take advantage of their biodegradable and safety prof i les.Polyanhydride nanoparticles containing Yersinia pestis antigens were examined for intranasal vaccination [49].The recombinant protein F1-V was co-loaded with the adjuvant MPL to induce the generation of F1-V-specif i c antibodies detected 23 weeks post-vaccination.Polyanhydride microparticles were shown to be taken up by DC cells and to induce antigen-specif i c proliferation of both CD4+and CD8+ T cells[50].

Numerous polymeric nanoparticles have been designed to physically entrap antigens within polymer matrices.Electrostatic polyplexes have also been tested for their utility in delivering subunit antigens[51]or plasmid DNA vaccines [52,53].Hepatitis B surface antigen was loaded onto cationicL-poly lactic acid microspheres via the formationof electrostatic complexes[51].A single subcutaneous immunization with HBsAg complexed onto the surfaces of cationic microspheres was found to induce comparable levels of serum IgG responses and higher cellular immune responses as compared to two injections of HBsAg with adjuvant alum in a mouse model.

Cationic polymers have been used to form complexes with negatively charged plasmid DNA vaccines.Deacylated cationic polyethyleneimine was used to form complexes with the human immunodef i ciency virus(HIV)CN54gp140 antigen [52].Pulmonary administration of the polyplexes elicited an immune response and protected the immunized mice against viral challenge.PEGylated poly[2-(N,N-dimethylamino)ethyl methacrylate]was used to deliver HIV gag DNA vaccine[53]. Intranasal administration of the polyplexes was observed to enhance the gag-specif i c serum IgG levels and IFN-γ secreting splenocytes in mice as compared to the naked DNA vaccinetreated group.

Synthetic polymer-based vaccine delivery systems can provide a sustained release prof i le for vaccine antigens over prolonged periods of time.The release prof i le may be tuned by controlling the molecular weights of the synthetic polymers and the encapsulation conditions.Relatively few biodegradable and biocompatible synthetic polymeric delivery systems have been tested to date,and the safety prof i les of the biodegradation products have not been extensively studied.Progress in the f i eld of biodegradable polymer development is anticipated to accelerate polymer-based vaccine delivery studies.

2.4. Natural biopolymer-based nanodelivery systems

Natural biopolymers,including chitosan and pullulan,have been studied as vaccine or adjuvant delivery systems.Chitosan is biocompatible,nontoxic,biodegradable,and provides a cationic charge that may facilitate endocytosis.In the context of vaccine delivery,chitosan displays valuable properties, including mucoadhesivenessand anadjuvanticity thatacts by promoting the maturation of DCs.

The mucoadhesiveness of chitosan has been exploited in mucosalvaccinedeliveryformulations.A recentstudy showed that an N-trimethyl chitosan nanocarrier carrying a model antigen OVA provided prolonged residence in nasal mucosa compared to PLGA nanoparticles bearing the same antigen[54].Whereas intramuscularly administered N-trimethyl chitosan-based vaccine formulations provided immune responses that were comparable to those generated by the PLGA-based vaccine,intranasally administered N-trimethyl chitosan-based vaccine formulations induced higher antigen-specif i c secretory IgA levels compared to PLGA carriers.These results suggest the importance of mucoadhesiveness in vaccine carriers for enhancing the induction of mucosal immunity.

Chitosan nanoparticles were studied as mucosal vaccine delivery systems[55].Chitosan nanoparticles that encapsulated the hemagglutinin-split inf l uenza virus were prepared by ionically crosslinking the chitosan polymers in the presence of sodium tripolyphosphate.Two intranasal vaccination doses of the hemagglutinin-split inf l uenza virus entrapped in chitosan nanoparticles induced higher systemic and mucosalantibody responses compared to the responses generated by a vaccine prepared from the hemagglutinin-split inf l uenza virus alone in mice.

The intracellular fate of the HBsAg encapsulated in chitosan nanoparticles has been examined[56].The cellular fates of the HBsAg and chitosan nanoparticles were followed by f l uorescently labeling the HBsAg and chitosan with Cy3 and Cy5,respectively.The HBsAg-containing chitosan nanoparticles were found to be endocytosed by bone marrowderived DC,and the HBsAg was found to dissociate from the nanoparticles 6 h after cellular uptake.The dissociation of HBsAg from the chitosan nanoparticles was thought to result from the chitosan matrix degradation in the lysosome.

Cholesteryl-conjugated pullulan polymer-based nanoscale hydrogels were studied as potential vaccine vehicles[57].A non-toxic subunit fragment of the Clostridium botulinum type-A neurotoxin BoHc/A was loaded into the nanogel and administered intranasally.The viscosity of the nanogel promoted the retention of BoHc/A in the nasal mucosal layer. After release from the nanogel,BoHc/A was taken up by the mucosal DCs.The intranasal nanogel formulations provided high titers of the antigen-neutralizing serum IgG and antigenspecif i c sIgA in the absence of additional mucosal adjuvants.

Synthetic and natural polymer hybrid vaccine delivery systems have been investigated.For example,chitosancoated polycaprolactone nanoparticles were developed for the intranasal delivery of the recombinant inf l uenza A virus (A/California/07/2009)H1N1hemagglutinin protein[48].About 66.5%of entrapped antigens were released from the nanoparticles over 63 days.A single intranasal immunization with the antigen-loaded chitosan-coated polycaprolactone nanoparticles resulted in a total IgG response that exceeded the response achieved after a booster intramuscular administration of the free subunit antigen solution in Balb/c mice.

2.5. Carbon-based nanodelivery systems

Carbon-based nanomaterialsand carbon nanotubesin particular were recently investigated as antigen delivery systems[58].Carbon-based nanodelivery systems are insoluble, non-degradable,and mimicbacteriain size and shape.Carbon nanotubes are not intrinsically immunogenic,display a low toxicity,are capable of carrying multiple antigens,and are taken up rapidly by antigen-presenting cells.Such features support the feasibility of using carbon nanotubes as antigen carriers.

Zeinali and colleagues examined the utility of singlewalled carbon nanotubes(SWCNTs)in vaccine delivery systems[59].A purif i ed tuberculin protein derivative was loaded onto a carboxyl group-functionalized SWCNT through covalent binding.Following subcutaneous administration,the antigen-SWCNT conjugate was found to stimulate the production by Th1 cells of cytokines,such as IFN-γ and IL-12,at levels comparable to those generated through administration of the conventional tuberculosis BCG vaccine.

A recent study reported the importance of carbon nanotube type on the induction of immunogenicity[60].In the study,a commercially available fungicide azoxystrobin was loaded onto four types of carbon nanotube(short and singlewalled,short and multi-walled,long and single-walled,and long and multi-walled).The resulting immuneresponseswere thencompared.The shortand multi-walled carbonnanotubes 0.5 μm in length and 50 nm in diameter were found to provide the highest anti-azoxystrobin IgG antibody titers in rabbits.

Carbon magnetic nanoparticles were constructed to permit the tracing of carbon-based vehicles[61].Magnetic resonance imaging data showed that magnetic nanoparticles 20-80 nm in diameter were rapidly distributed to the spleen,followed by the kidneys and the inguinal lymph nodes.The preferential targeting of carbon magnetic nanoparticles to DCs was used to enrich DCs ex vivo using an external magnet.The biotinylated model antigen hen egg lysozyme protein and the biotinylated TLR ligand CpG were attached to avidin-coated carbon magnetic nanoparticles to improve the induction of T cell activation and antigen-specif i c IFN-γ responses compared to the free protein antigen.

The nanostructures of carbon-based materials were reported to affect the antigen presentation process[62].Graphene oxide nanosheets(Fig.1D)differed from C60 fullerenes in itsability topromoteantigenpresentationto theT cells.C60 fullerenes stimulated MHC class I antigen presentation of OVA,whereas graphene oxide suppressed antigen presentation by DCs.Moreover,unlike the C60 fullerenes,graphene oxide reduced the immunoproteasomes in DCs.

Although most carbon-based vaccine delivery systems have been tested through systemic administration,a recent studyexamined thefeasibilityofusingcarbon nanoparticles for oral vaccine delivery[63].Carbon nanoparticles 470 nm in diameter bearing 40-60 nm pores were synthesized using silica as a template.Instead of loading the antigens via surface adsorption,the model antigen,bovine serum albumin,was encapsulated within the meso-and macropores of the nanoparticles.The rigid structure of the nanoparticles resisted destruction in the oral digestive tract and appeared to protect the antigens from the harsh environments of the alimentary canal after oral delivery.In this study,the IgG and cytokine immune responses elicited by the oral bovine serum albumin entrapped in the porous nanoparticles were comparable to those elicited by parenteral administration of bovine serum albumin with Freund's adjuvant.

Carbon-based nanomaterial vaccine delivery systems are relatively new and less well studied than other systems. Several investigations have indicated the feasibility of carbonbased systems for systemic or oral antigen delivery.Future developmentsshould demonstratewhether thelack of biodegradability will promote or reduce the safety of a carbonbased nano delivery system.

3. Current&future developments

Nanotechnology-based vaccine delivery systems have been developed to enhance the immunogenicity of a vaccine antigen by modulating antigen delivery to the immune cells. Nanocarrier formulations of vaccines offer the advantages of co-delivery of antigen and immunomodulator[64].Numerous studies have reported enhanced immunogenicity of nanocarrier-based vaccines upon co-delivery with an immunomodulator.Single immunization of OVA and MPL co-formulated with the PLGA nanocarrier induced much higher systemic and mucosal immune responses after oral delivery than OVA alone[65].Micelle formulations that include a PEG-modif i ed cationic polypeptide can co-deliver OVA and TLR3 agonists while increasing vaccine-induced antibody production by more than a factor of 70[66].The subcutaneous vaccination of pH-responsive micelle nanoparticles containing amphiphilic diblock copolymers conjugated to OVA and CpG oligonucleotides displayed remarkably higher CD8+T cell responses compared with the free form or a physical mixture[67].

In addition to the co-delivery of antigens and immunomodulators,modifying the surfaces of nanocarriers can contribute to the delivery of antigens specif i cally to relevant immune cells.Imiquimod(TLR7 agonist)-entrapped PLGA nanocarriers were coated with a chitosan derivative(N,N,N-trimethylated chitosan)to improve the protective response generated by mucosal immunization[68].Another chitosan derivative,glycol chitosan,was decorated onto the surfaces of PLGA nanoparticles for use in a nasal vaccination[69].Glycol chitosan-coated PLGA nanoparticles showed a lower clearance rate and a higher local uptake in the nasal cavity compared with chitosan-coated PLGA nanocarriers.

Multifunctional nanovaccines can signif i cantly increase the immune response generated by the target-specif i c,effective,and stable delivery of an antigen.However,the use of many-component nanovaccines with complex structures can increase production costs and complicate the manufacturing process.Many nanovaccines have been manufactured in small batches for research use[70].Batch-to-batch variations with respect to particle size,shape,and quality are critical problems in the area of nanoparticle synthesis,and these problems must be addressed during scale-up for clinical trial testing[71,72].Surface-modif i ed nanoparticulate vaccines require complicated synthesis procedures that can require complex purif i cation processes and high expenses.Scale-up processes tend to be time-consuming for pharmaceutical applications.Self-assembled nanovaccine technologies would be benef i cial for reducing obstacles to the development of industrial-scale manufacturing protocols.

4. Conclusions

Nanotechnology platforms present promising strategies for improving the immunogenicity of a vaccine antigen.Nanocarriers that are useful for the formulation and delivery of antigens and adjuvants offer many advantages over alternative adjuvant approaches,including improved stability,sustained release kinetics,lower immunotoxicity,and targeting to specif i c and selective immune cells.In addition,nanoparticles eff i ciently deliver antigens due to their nanoscale size,solid form,ease of surface modif i cation,and ability to co-deliver antigens along with adjuvants.Although the applications of nanotechnologies for nanocarrier-based vaccine delivery are currently in a nascent investigational stage,and only a few products are being tested in clinical trials,these vaccine delivery systems may potentially be used more broadly for the prevention and treatment of infectious disease.

Acknowledgment

This work was supported by grants from the Korean Health Technology R&D project,Ministry of Health and Welfare (Grant No.A092010),and from Ministry of Science,ICT and Future Planning(No.2014023227),and from Business for Cooperative R&D between Industry,Academy,and Research Institute funded Korea Small and Medium Business Administration(No.C0010962).

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*Corresponding authors.Tel:+82 2 880 2493;fax:+82 2 882 2493.

E-mail addresses:shimg@snu.ac.kr(G.Shim),ohyk@snu.ac.kr(Y.-K.Oh).

Peer review under responsibility of Shenyang Pharmaceutical University.

http://dx.doi.org/10.1016/j.ajps.2014.06.002

1818-0876/? 2014 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/3.0/).