messenger rna ( mrna ) has achieved great success in an increasing number of biological applications .
apropos , the notion of nonviral genetic vaccination is also increasingly associated with mrna instead of dna . given a mature drug and gene delivery field , mrna nanoparticle delivery science is often deferred or closely compared with dna and sirna systems [ 1 , 2 ] . however , as various reports have shown , unique properties of mrna delivery exist [ 3 , 4 ] and continue to be a relevant research focus today . mrna delivery science has made significant progress since the first demonstration of cell based mrna tumor vaccine delivery via rna loaded dcs .
they include the optimization of the mrna molecular structure [ 6 , 7 ] , direct in vivo administration of mrna [ 8 , 9 ] , delivery routes [ 3 , 4 ] , evaluation of rationally designed gene carriers [ 1014 ] , and , recently , self - replicating rna . along this developmental trajectory
, dc - targeted nanoparticle gene delivery systems may be an imminent next step forward for nonviral tumor vaccine delivery . in this brief report ,
this will be followed by a brief discussion on three promising dc receptors that are suitable for targeted delivery of mrna nanoparticles for tumor vaccination .
ligands targeting surface receptors on dcs are molecules grafted onto surfaces of formulated nanoparticles , recognizable by dc - specific uptake mechanisms , and endow nanoparticles with the ability to be taken up exclusively by them .
this has the benefit of reducing effective doses of vaccine required through nonspecific uptake by other cell types . in the case of vaccines , which typically contains proinflammatory adjuvant molecules
since a wide variety of nanoparticle delivery systems exist , different ligand conjugation strategies have been developed . in this section
, we will discuss three conjugation strategies that are most often applied to gene delivery systems .
first , nanoparticles with solid cores such as poly(lactic - co - glycolic acid ) ( plga ) and inorganic nanoparticles ( e.g. , gold nanospheres , calcium phosphate ) possess excellent colloidal stability such that ligands can be covalently conjugated directly onto particles surfaces without aggregation . in plga systems , nanoparticles are formulated by emulsion techniques [ 1618 ] using plga - peg - cooh copolymer , which can be synthesized by grafting peg - cooh onto the ends of plga .
the resultant mrna infused plga nanoparticles bearing surface carboxylate groups ( cooh ) can be further functionalized with any ligands bearing amine groups ( e.g. , peptides , antibodies , nanobodies , and aptamers ) via n - hydroxysuccinimide ( nhs ) chemistry , which proceeds with good efficiencies under physiological conditions if nhs bearing ligands are applied in excess   ( figure 1(a ) , top )
. however , this conjugation strategy will require the colloidal nanoparticles to remain stable through every step of the conjugation process ( surface chemistry modifications , purification and lyophilization ) .
ligand conjugated nanoparticles are normally purified from the reaction mixture via centrifugation , and hence this strategy is compatible with formulations bearing a solid core because they can withstand compression without aggregation .
however , dialysis is not compatible with plga ( as well as other polyesters , e.g. , poly--amino esters ) as ester bonds in these polyesters undergo hydrolysis .
conversely , formulations that are chemically inert ( e.g. , gold nanoparticles , immunoliposomes , and polyamide - based nanoparticles ) but aggregate upon centrifugation can be purified by dialysis ( figure 1(b ) ) .
a similar approach uses functionalized amphiphilic surfactants commonly used to stabilize the plga nanoparticles in colloidal suspension ( figure 1(a ) , bottom ) .
these surfactants , which bear reactive chemical moieties ( e.g. , cooh , nh2 , and oh ) , are optimally incorporated on particle surfaces and amenable for subsequent conjugation with targeting ligands bearing compatible linkers . in particular , avidin - fatty acid surfactants have been applied to stabilize plga nanoparticles [ 22 , 23 ] .
the resulting nanoparticles can be subsequently functionalized with biotinylated ligands such as antibodies , which are easily available , to render user defined dc surface receptor targets such as dec-205 and dc - sign [ 22 , 24 , 25 ] .
however , notwithstanding the immunological consequences of antibodies , the sheer size of antibodies may result in low surface coverage due to steric hindrance .
this can be mitigated with more advanced ligands such as single chain fragment variable ( scfv ) [ 26 , 27 ] or aptamers , making this an attractive conjugation method .
second , targeting moieties can instead be incorporated as part of the carrier molecule ( polymer or lipid ) .
the ligand conjugated carrier is directly used to formulate the nanoparticles via coacervation between positively charged gene carriers and negatively charged mrna , and hence no additional step is needed to affix the ligands .
this strategy is typically applicable for electrostatically neutral , low molecular weight ligands to ensure that they do not interfere with the carrier molecule during nanoparticle formulation ( figure 1(c ) ) .
mannan / mannose , a sugar that interacts with c - type lectin / lectin - like receptors , is the most commonly applied dc - targeting ligand incorporated into nanoparticles using this approach .
a large number of mannosylated lipids and polymers have been developed hitherto for the purpose of vaccination [ 2936 ] .
for liposomal systems , mannose are grafted onto the head groups of lipids [ 2931 ] , while , for polymeric systems , they are normally covalently attached along the backbone of polymeric carriers [ 3236 ] . most of these systems are tested for delivery of different vaccine molecules including peptides , dna , and sirna with a consistent improvement in uptake efficiencies over nonmannosylated nanoparticles , which translates to an improved immunization outcome .
notably , midoux group elegantly demonstrated , as a proof - of - concept , that mrna - loaded mannosylated lipophosphoramides target dcs in vivo and translate into a better survival outcome based on a b16-f10 prophylactic tumor model [ 31 , 37 ] .
third , another tried and tested strategy for ligand conjugation primarily in liposomal systems exploits the use of hydrophobic interaction ( figure 1(d ) ) .
it is well known that liposomes / lipopolyplexes are not thermodynamically stable colloids that aggregate slowly over time [ 3840 ] .
aggregation is a fusion process when hydrophobic interactions between the lipid tails are stronger than the repulsive forces on the surfaces of the liposomes .
factors determining this balance include temperature , ionic concentration of the buffer , and amphiphilic property ( surface charge of the lipids versus length and number of the lipid tails ) . exploiting effects of temperature on lipid fusion , liposomes or lipopolyplexes encapsulated with mrna or other payloads can be incubated with ligand - micelles ( e.g. , dspe - peg-2000-x , where x = ligand ) at a temperature of 55c for at least 15 minutes .
due to increased hydrophobic interaction at a higher temperature , ligand conjugated lipids from these micelles can be transferred to the liposomes , effectively decorating them with the desired targeting ligands .
these ligand conjugated micelles can be prepared by reacting thiol ( sh- ) or amine ( nh2- ) bearing ligands with dspe - peg - nhs or dspe - peg - maleimide ( dspe : 1,2-distearoyl - sn - glycero-3-phosphoethanolamine ) available commercially with different peg molecular weight .
this so - called  postinsertion  strategy is a facile approach to functionalizing liposomes with any desired ligands . unlike plga system , dspe - peg - ligand
can be prepared separately and conveniently incorporated into formulated liposomes on demand [ 4143 ] .
the amount of peg coverage over a 100  nm liposome needed to prevent aggregation in serum is determined to be > 8 mole% ( based on total lipid content ) in the liposome formulation [ 44 , 45 ] .
a caveat to postinsertion strategy is that if the amphiphilicity of the micelles is significantly affected by an excessively hydrophilic head ( e.g. , highly charged aptamer , long peg chain ) , postinsertion method may fail because the increased hydrophobic interaction induced at a higher temperature may not be sufficient to trigger micelle fusion with the liposomes / lipopolyplexes .
when particles are administered into the body , unless the injected site is already the lymph node ( e.g. , intranodal administration ) or has a high density of antigen presenting cells ( e.g. , intradermal or intranasal administration ) , nanoparticles need to be passively transported from the site of administration to the lymph nodes via the body 's circulatory system such as the lymphatics or the systemic circulation [ 28 , 4648 ] . during passive transport from the site administration to the lymphoid tissues , nanoparticles may be taken up nonspecifically by bystander cells based on a range of physiochemical factors such as size , surface charge , and chemical structure of surface molecules .
targeting ligands may reduce such occurrences due to incompatible surface chemistries while increasing uptake efficiencies of nanoparticles when reaching the target site [ 4951 ] .
while generally it means selective delivery of the vaccine to dcs bearing specific surface receptors , direct outcome of receptor binding depends on what receptors are being targeted . targeting ligands can , amongst other functions , help increase the uptake by binding to receptors designed to endocytose larger particles , mitigate repulsive forces , or improve surface compatibility between the particles and the cell membrane .
since intracellular fate of the particles taken up by endocytosis   is determined largely by the mechanism through which they are being taken up , targeting ligands may help direct endosomes into specific intracellular trafficking pathways that are less degradative so that gene delivery efficiencies are increased .
dcs , unlike other somatic cells , possess unique endocytic receptors catered to antigen uptake and processing .
these receptors are special because they not only trigger particle uptake , but also mediate cross presentation and the development of the immune response . although cross presentation in dcs influenced the development of subunit nanoparticle vaccines , its impact on genetic vaccination is less conclusive .
the genetic vaccination delivery model has been described as a process where both bystander and antigen presenting cells are transfected [ 53 , 54 ] . according to this model , as illustrated in figure 2 , antigen presentation occurs through direct transfection of dcs and also through indirect transfer by transfected bystander cells . when the mrna nanoparticles are targeted to dcs directly , those that escape the endosomes will have a higher chance of being expressed . in dcs ,
endosome escape not only depends on the efficiency of the gene carrier , but also depends on the trafficking mechanisms . for example , cross presentation mechanisms in dcs can disrupt lysosome trafficking pathways via mediation of endosomal ph leading to higher delivery efficiencies .
but , on the other hand , intracellular trafficking pathways of nonprofessional antigen presenting cells often terminate at the lysosomes . when mrna nanoparticles are delivered without specific dc - targeting ligands , they will also transfect bystander cells .
the latter provide an alternative source of antigens by secreting them ( if the antigens are secretory in nature or designed with a secretory signal ) into the extracellular space for capture by dcs . finally , according to the consensus genetic vaccination
, the other indirect delivery mechanism occurs when transfected bystander cells become apoptotic due to significant stress caused by viral or tumor infection .
sufficient literature exists to suggest that indirect delivery mechanisms via bystander cells does not play a significant role in targeted delivery systems since targeted genetic nanoparticle vaccines consistently improve immunization outcomes [ 3234 , 5658 ] .
dc - specific receptors that not only increase uptake but also enhance transfection via less degradative intracellular trafficking pathways will be attractive for mrna nanoparticle tumor vaccination .
while a long list of dc receptors has been discovered to possess immune modulating function , only a few may benefit mrna delivery beyond uptake enhancements because they are also targeted towards less degradative intracellular trafficking pathways .
they are type i c - type lectins such as cd205 ( dec-205 ) and cd206 ( macrophage mannose receptor ) and type ii c - type lectins such as cd370 ( clec9a / dngr-1 ) .
it is a type i c - type lectin - like molecule consisting of a single polypeptide chain that functions as recycling endocytic receptor and caters for a wide range of cargos that include , notwithstanding lectin - like molecules , apoptotic cells , necrotic cells , and cpg .
dec-205 is an attractive target receptor because antigens delivered via this receptor are presented on both mhc - i and mhc - ii molecules .
furthermore , engagement of dec-205 does not lead to proinflammatory response , making it an attractive receptor target for tolerance immunization .
the anti - dec-205 ligand is one of the most developed ligands in immunotherapy . while ligands targeting most of the other dc - specific receptors continue to manifest in antibody molecules , anti - dec-205 ligands in form of scfv [ 26 , 27 ] and aptamer  
for example , being a cognate endocytic receptor for apoptotic cells , dec-205 will efficiently uptake both nano- and microparticles it comes into close contact with .
hence , given mrna nanoparticles tendency to aggregate in vivo ( increased particle sizes ) , administered dose will have higher bioavailability when targeted towards dec-205 .
in addition , cross presenting properties of dec-205 , thought to be results of  leaky endosomes  or less degradative endocytic pathway , will facilitate endosome escape of mrna nanoparticles into the cytoplasm and avoid the lysosomes .
the mannose receptor , another type i c - type lectin receptor with a well - established role in tissue homeostasis , recognizes sulfated carbohydrates , collagen , and oligosaccharides through its cysteine - rich domain [ 68 , 69 ] , fibronectin domain , and c - type lectin domains [ 71 , 72 ] , respectively .
the mannose receptors have been well - known endocytic receptors for decades in part because they are extensively studied as scavenging receptors in macrophages , which were initially thought to be the major antigen presenting cells before dcs were discovered .
the ligand for this receptor is mannose residue grafted on the gene carrier [ 2936 ] as previously described .
its role in antigen presentation was conclusively determined through the use of dcs derived from mannose receptor negative transgenic mice .
this study confirmed that dcs ' mannose receptors not only serve as uptake receptors [ 7477 ] , but also mediate cross presentation of soluble mannosylated antigens [ 7880 ] . since payload taken up via mannose receptor stably accumulates in the early endosome and is excluded from lysosomes for up to 6 hours [ 78 , 79 ] , this intracellular trafficking pathway is expected to be less degradative and highly attractive for mrna nanoparticle delivery .
dngr-1 or cd370 ) is a recently discovered endocytic receptor that is implicated in the clearance of damaged   and dead [ 82 , 83 ] cells .
this receptor , currently targeted via antibody , is restricted to a very small population of blood bdca3 dcs   ( in humans ) and its equivalent in mice models is cd8 dcs . due to its endocytic nature , antigen delivery properties of clec9a are rapidly investigated [ 85 , 86 ] .
recent reports show that clec9a are effective in cross presenting antigens for cell mediated immunity [ 83 , 87 ] and can be as effective t cell activators compared to langerin and dec-205 .
similar to other receptors capable of cross presenting soluble antigens , nanoparticles targeted to clec9a are expected to enter a less degradative intracellular trafficking pathway , leading to higher transfection efficiency .
restricted expression of clec9a to blood dcs may limit it as a practical receptor compared to the mannose receptor and dec-205 for targeted delivery to conventional dcs .
as a late bloomer , development of mrna therapeutics benefits from a plethora of related knowledge on similar delivery systems . advancing from passive targeting strategies employed for most mrna nanoparticle tumor vaccine to date , active targeting of mrna nanoparticles to dcs will further improve current therapeutic outcome for the treatment of cancer .
practicable conjugation strategies as well as target receptors reviewed in this paper will provide a convenient reference to facilitate future development of targeted mrna nanoparticle vaccine .
 the use of nanoparticles encapsulating messenger rna ( mrna ) as a vaccine has recently attracted much attention because of encouraging results achieved in many nonviral genetic antitumor vaccination studies . 
 notably , in all of these studies , mrna nanoparticles are passively targeted to dendritic cells ( dcs ) through careful selection of vaccination sites . 
 hence , dc - targeted mrna nanoparticle vaccines may be an imminent next step forward . in this brief report 
 , we will discuss established conjugation strategies that have been successfully applied to both polymeric and liposomal gene delivery systems . we will also briefly describe promising dc surface receptors amenable for targeting mrna nanoparticles . 
 practicable conjugation strategies and receptors reviewed in this paper will provide a convenient reference to facilitate future development of targeted mrna nanoparticle vaccine . 