Biopolymer‐based Carriers for DNA Vaccine Design

Abstract Over the last 30 years, genetically engineered DNA has been tested as novel vaccination strategy against various diseases, including human immunodeficiency virus (HIV), hepatitis B, several parasites, and cancers. However, the clinical breakthrough of the technique is confined by the low transfection efficacy and immunogenicity of the employed vaccines. Therefore, carrier materials were designed to prevent the rapid degradation and systemic clearance of DNA in the body. In this context, biopolymers are a particularly promising DNA vaccine carrier platform due to their beneficial biochemical and physical characteristics, including biocompatibility, stability, and low toxicity. This article reviews the applications, fabrication, and modification of biopolymers as carrier medium for genetic vaccines.


DNA vaccines
Thed evelopment of vaccines has been one of the most significant advances of modern medicine and has led to improved public health and life expectancy.I th as been reported that two to three million lives per year are saved worldwide due to vaccination. [1] Theprocess of immunisation was first observed in China and the Middle East in the 12 th century.B yu sing the skin or pustule liquid of patients with smallpox, disease resistance in another patient was achieved. [2,3] However,the first vaccine was developed in 1798 by Edward Jenner who reported that injecting humans with cowpox led to the protection against smallpox. [4] Conventional vaccines are predominantly composed of inactivated (killed) pathogens or pathogen subunits,f or example toxins,p olysaccharides or proteins,a nd live-attenuated (weakened) viruses. [5] Antigens from these pathogens are recognised by the immune system as being foreign, which results in the induction of an immune response,t he production of antibodies,a nd the establishment of immunological memory.S uccessful traditional vaccines have been developed against numerous bacterial and viral pathogens and have been most effective for disease control. [6] Unlike conventional vaccines,DNA vaccines are bacterial plasmids designed to carry aspecific encoding gene,which is responsible for expression of the desired antigen in the host and leads to the induction of an immune response. [7,8] Instead of using proteins from pathogens to stimulate the immune system, DNAvaccines deliver an instruction for the protein to be produced in the body (Figure 1). Major benefits of DNA vaccines,compared to conventional vaccines,are high specificity and the possibility of introducing additional sequences to the plasmid, for example,adjuvants,which can potentiate the immunostimulatory effect of the expressed antigen. [9] In addition, the expressed immunising antigen is exposed to Over the last 30 years,genetically engineered DNAhas been tested as novel vaccination strategy against various diseases,i ncluding human immunodeficiency virus (HIV), hepatitis B, several parasites,a nd cancers.H owever,the clinical breakthrough of the technique is confined by the lowt ransfection efficacy and immunogenicity of the employed vaccines.Therefore,c arrier materials were designed to prevent the rapid degradation and systemic clearance of DNAi nt he body.I nt his context, biopolymers are aparticularly promising DNA vaccine carrier platform due to their beneficial biochemical and physical characteristics,i ncluding biocompatibility,s tability,and low toxicity.T his article reviews the applications,f abrication, and modification of biopolymers as carrier medium for genetic vaccines. the same species-specific post-translational modifications (e.g. phosphorylation or glycosylation) as the natural viral infection. There is no need for live vectors or complex biochemical production strategies.T he interest in DNA vaccines was further boosted by promising animal studies, and generally,c heaper production as well as facile transport and storage. So far, DNAvaccines have been approved for veterinary use against West Nile Virus in horses, [10] canine melanoma, [11] infectious haematopoietic necrosis in farm-raised Atlantic salmon, [12] and as gene therapy for growth hormone-releasing hormone in pigs. [13] Thef irst human trial of DNA-based vaccines was conducted in 1998 and targeted human immunodeficiencyvirus type 1(HIV). [14] Currently,there are many ongoing human clinical trials that target various types of cancer, autoimmune diseases,and infectious diseases,such as human papilloma virus (HPV), HIV,hepatitis Band coronavirus (Table 1). [5,15] Results of these first clinical trials reported DNAvaccines to be safe and well tolerated, but showed low immunogenicity, which was attributed to insufficient protein expression levels. [5,11] This low efficacycan be overcome by optimisation of the plasmid-encoded antigen to increase antigen expression per cell or by increasing the transfection rate through polymer and lipid formulations,aswell as enhancement of the immune response by addition of molecular adjuvants. [9] Notably,m RNAv accines have recently emerged as another nucleic acid vaccination strategy,a sr eviewed by Pardi et al. [16] and Maruggi et al. [17]  concerns in vivo,w ere overcome using sophisticated nanotechnology.T he mRNAi sencapsulated in lipid nanoparticle formulations to protect it against rapid nuclease degradation in vivo and deliver it efficiently to the cytoplasm. [18] In contrast to DNAv accines,m RNAv accines do not require entry to the nucleus and efficient transcription, thereby excluding the risk of insertional mutagenesis.However,DNA vaccines are known to be generally more stable than mRNA.
Herein, we will give an overview of biopolymer-based formulations with emphasis on nanostructured biopolymers as carriers for DNAvaccines and introduce modifications that have been shown to enhance vaccine efficiency.D ue to the comparable mechanism of action of DNAv accines and mRNAvaccines,t he design considerations presented in this Review may also be useful for mRNAv accine delivery systems.

DNA Vaccines:M ode of Action
Thea dministration of DNAv accines leads to the transfection of cells at the injection site.Upon internalisation and translocation to the nucleus,r esident cells,s uch as keratinocytes in the skin and myocytesi nm uscle tissue,e xpress the vaccine-encoded antigen and eventually excrete it through apoptosis or exosomes. [19] APCs,such as dendritic cells (DCs), circulate searching for pathogenic structures,d etect and internalise the exogenous antigen and introduce it to their endolysosomal degradation pathway.T he DCs then progress to the lymph nodes,w here they present peptide fragments stemming from the antigen to CD4 + Tcells.The activation of CD4 + Tcells is achieved through the association of the major histocompatibility class II (MHCII) complex on APCs and the T-cell receptor on CD4 + cells and supported by further interaction through co-stimulatory ligand-receptor binding.MHC complexes are aset of genes coding for MHC cell surface glycoproteins,which present pathogen fragments to Tcells and thus help the immune system to recognise at hreat. [20] CD4 + Tcells play am ajor role in orchestrating the immune response by contributing to Bcell priming and the activation of cytotoxic CD8 + Tcells. [21,22] Furthermore, exogenous antigens can be cross-presented from transfected apoptotic somatic cells to immature CD8 + Tcells through the MHCI pathway,w hich results in the activation of cytotoxic Tlymphocytes (CTL), triggering as trong cellular immune response. [23] In addition to this indirect route,DCs can also be directly transfected by DNAv accines.T he direct route involves the endocytosis and expression of the DNAvaccine in DCs,which results in the parallel activation of CD8 + cytotoxic Tcells and distinct CD4 + Thelper cells through the binding with MHCI and MHCII, respectively,a sw ell as with the specific co-stimulating receptors. [24] Upon activation, immature CD8 + and CD4 + Tcells begin to proliferate due to the autocrine production of IL-2, aTcell growth and differentiation factor ( Figure 2). CD8 + Tcells clonally expand upon the interaction with IL-2 to increase the effective amount of cytotoxic, antigen-specific Tcells.C D4 + Tcells,h owever, start to differentiate upon binding to IL-2 and become Thelper cells (Th0). These helper cells produce IL-2, IL-4, and interferon gamma (IFN-g)a nd can further proliferate into Th1and Th2effector Tcells.T h1 cells are responsible for activating the cellular immune system by further supporting the proliferation of cytotoxic CD8 + Tcells. [25] In contrast, Th2c ells stimulate the humoral immune response by inducing Bcell-mediated antigen production. In this way,DNA vaccines can trigger both ahumoral and cellular immune response,w hich is as ignificant advantage over commonly used vaccines. [26] While the humoral immune response targets extracellular pathogens,c ellular immunity is responsible for annihilating intracellular pathogens,such as cells infected with viruses or bacteria.
Theproliferation into Th1orT h2 cells can be modulated by changing the mode of administration. While intramuscular administration was shown to mostly induce Th1-type immune response,i ntradermal treatment results in as trong humoral, Figure 2. Mode of action of DNA vaccines. Extracellulara ntigens, stemmingfrom keratinocytes or myocytes, are presented to naive CD4 + Tcells through the MHCII pathway (I), which leads to clonal expansion and differentiation to Th0. While the interactiono fTh0 with IL-12 results in the formation of Th1, the binding to IL-4 promotes the stimulation of Bcells, which eventually form antigen-specific antibodies and memory Bcells. Extracellularantigens can also be introducedtothe MHCI pathway through cross-presentation (II). In addition, APCs can be directly transfected with pDNA. After cellular uptake (III), APCs express pDNA (IV) and present the resulting antigen through the MHCI pathway (V) to naive CD8 + Tcells, which clonally expand upon the interactionw ith IL-2 to form CTL and memory Tcells, and to naive CD4 + cells through the MHCII pathway (VI).
or Th2-mediated, immune response. [21] Furthermore,t his multifaceted mechanism leads the formation of long-living memory Band Tcells to form an integral part of the adaptive immune system, which allows for af aster and stronger immune response after repeated infection with ap reviously encountered antigen. [27] 1.2. Design of DNA Sequences pDNAs equences used in vaccine design are typically comprised of an expression (or transcription) unit and ap roduction unit ( Figure 3). Thet ranscription unit consists of av iral-hybrid or eukaryotic promoter region (I), an intron (II), as equence that encodes the antigen of interest(III), and apolyadenylic acid (polyA) signal (IV).
Thep romoter sequence (Figure 3, I) provides as trong binding site for RNAp olymerase and thus controls the transcription of the plasmid into mRNA, which is eventually translated into the desired antigen of interest. Them ost commonly used promoter stems from the human cytomegalovirus (CMV), as it drives high antigen expression levels in various cell lines. [28] However,v iral promoters are quickly deactivated through gene silencing and often show only transient gene expression. Therefore,v iral/mammalian hybrid-promoters,s uch as ac ombination of CMV and the human elongation factor 1a promoter,c an be employed to delay the deactivation effects caused by gene silencing in vitro and in vivo. [29] Ty pically,a ni ntron sequence (II) is introduced between the promoter (I) and the antigen of interest (III) region on the plasmid. Introns are non-coding regions in ag ene and were found to increase the antigen expression efficacy significantly. [30] Thei ntron is followed by the antigen of interest (III) region, which codes for the desired immuno-genic protein. Thef inal element of the transcription unit is ap olyA signal (IV), which stabilises the mRNAt ranscripts and eases the export from the nucleus. [31,32] Thep roduction unit consists of an origin of replication (V) sequence and antibiotic resistance genes (VI). The origin of replication is needed to amplify the plasmid in the host cell, and typically consists of ab acterial region, responsible for successful replication and selection in abacterial host, and an eukaryotic region, which allows for the expression in mammalian cells. [33] Thea ntibiotic resistance genes facilitate the antibiotic-selected plasmid production in bacterial cultures.H owever, legislators strongly discourage the use of antibiotic resistance genes in in vivo trials to prevent potential antibiotic resistance of the patient and the integration into the human genome. [34]

Delivery of DNA Vaccines
To date,n oD NA vaccine has been approved for use in humans.The major challenge for DNAvaccines still resides in insufficient gene expression and low immune system activation. In order to overcome this,research has been focusing on DNAsequence design, vaccine formulation, and the mode of delivery. [35] In terms of delivery,c onventional needle-based delivery approaches can be classified into intradermal, subcutaneous, intravenous,a nd intramuscular injection with varying depth of skin penetration. [36] Intradermal delivery is considered to be more effective than intramuscular or subcutaneous injection due to the dense DC network present in the dermis. [37] Intravenous application is performed with the goal to deliver DNAplasmids to APCs in secondary lymphoid organs,although with varying effectiveness. [36] Alternatively,DNA vaccines can be delivered via mucosal barriers by,f or example,o ral, nasal, or pulmonary uptake, which induces local immunity at mucosal sites as well as whole-body immunity. [38] Mucosal immunisation is regarded to be effective against viruses,a sh ost infection is primarily induced by entering through mucosal surfaces. [38,39] Moreover, these methods are particularly user-friendly,a st hey do not require needles,s pecialised equipment, or skilled operators. [39] However,a dministration of naked DNAt hrough these routes lacks major features desirable for robust immunisation, such as in vivo stability,s pecific targeting, high cellular uptake,a nd immune system modulation. Hence,delivery via the skin has been enhanced by use of electroporation, microneedles,o rn eedle-free delivery systems such as gene guns or biojectors,a ll of which require specialised equipment. [36,37] Delivery efficacy can additionally be increased by combining the pDNAw ith suitable carriers,w hich not only improves the plasmid stability,b ut can also enhance gene expression and immune response.Biopolymers have recently emerged as suitable carrier materials and have significantly increased the potential of DNAvaccine formulations. The plasmid can be divided in ap roduction and transcription section. The production unit is responsible for successful replication in the host and consists of an origin of replication and antibioticresistance gene region. In contrast, the transcription section consists of ap romoter region, commonly separated from the antigen of interest sequence by an intron. The sequence is completed by ap olyA signal to stabilise mRNA and facilitate nuclear export.

Biopolymers as DNA Vaccine Carriers
Liposomes,p olymers,v irosomes,c ell-penetrating peptides (CPPs), and live bacteria have been successfully used as DNAc arriers (Figure 4). [35,37,40] Amongst these,p olymers show high physicochemical versatility and low toxicity, provide protection from enzymes that can interfere with the structural integrity of the vaccine,a nd enable,c ost-effective production. [41] Furthermore,t hey are more rigid and stable than liposomes and do not pose risks regarding anti-vector immunity as in the case of virosomes and live bacteria. [37,42] An additional advantage of polymers is the ability to design various nanostructures with tuneable size and different surface properties,w hich have been particularly useful for the design of smart drug delivery systems.Infact, anumber of polymer nanoformulations have already been approved for clinical use. [43,44] Among polymers for DNAv accine design, biopolymers ( Table 2) are preferred over synthetic polymers due to their biocompatibility,favourable cellular interactions, biodegradability,a nd often facile production, for example with the help of bacteria or using enzymes. [45] Biopolymers are polymers which are naturally formed by living organisms. [46] In abroader sense,they can be defined as synthetic polymers made from monomer units obtained from living organisms, [47] such as crustaceans,s eaweed, and corn. [48][49][50] Thef ollowing sections will give an overview of the most significant advances that emerged from the development of biopolymer carriers and their use in DNAvaccine formulation.

Design of Biopolymer Vaccine Carriers
Biopolymeric DNAvaccine systems contain at minimum ac ore scaffold and the pDNA. Theb iopolymer core can be modified with functional moieties or co-polymers to enhance the vaccine delivery ( Figure 5). Thep DNAc an either be adsorbed on the surface of the nanoparticles (NPs) [101,102] or incorporated within the core. [48,[103][104][105] Further modification of these basic designs can be made by addition of polymeric shells, [51,105] the deposition of NPs in ap olymer matrix, [105][106][107] and preparation of compounded NPs composed of two different material domains within the particle. [108,109] Moreover, metal-based NPs,s uch as gold [110] and iron oxide NPs, [111,112] liposomes, [113][114][115] and ar ange of hydrogels have been used in combination with biopolymers to introduce new functionalities. [99,116,117] When designing DNAvaccine carrier materials,different biological barriers need to be taken into account. On an extracellular level, these include the rapid clearance of foreign genetic material from the bloodstream, deactivation through serum proteins,a nd degradation of DNAt hrough DNases.Once the target cell is reached, carrier-bound DNA needs to be internalised through the membrane via phago-, pino-, or endocytosis.F inally,u pon internalisation, the vaccine is required to escape phago-or endosomal vesicles and travel across the cytoplasm to reach the nucleus,w here DNAn eeds to dissociate from the carrier to enable the expression of the encoded antigen. [118] In short, the DNAc arrier not only needs to ensure the integrity of pDNA, but also needs to be equipped with functional groups that enable stability in biological fluids, prolonged circulation to reach target cells,o vercoming of extracellular and intracellular barriers,a nd safe delivery to the nucleus.This might seem like aformidable challenge,but significant advances have been made in the past decade that took us closer to the rational design of successful DNA carriers.T he next sections will give an overview of the most important strategies applied to increase the efficacy of DNA vaccines using biopolymer carriers.  . Biopolymer-based DNA vaccine delivery designs. Nanostructured biopolymers, which can include additional elements such as functionalm oieties or co-polymers. Commonly employed designs include NPs with both surface-adsorbed and incorporated pDNA. Nanocarriers can be further enclosed by polymer layers or incorporated in compounded formulationso rpolymer matrices. Advanced systems include NPs with metallic cores, the use of liposomes,orhydrogels.

Angewandte Chemie
Reviews negatively charged or neutral polymers,s ince electrostatic binding is no longer the driving force for DNAcondensation ( Figure 7). This approach led to efficient immobilisation of pDNAt o5 b-cholanic acid-modified chitosan, [170] and 1,2distearoyl-sn-glycero-3-phosphoethanolamine-conjugated HA, [171] as well as for the incorporation of DNAw ithin the hydrophobic core of PLGA particles. [98,172,173] In addition to the use of positive charge and hydrophobic interactions,hydrogen bonding can be exploited to condense DNA. [174] Fore xample,g uanidinium moieties in argininecontaining materials were shown to form hydrogen bonds with phosphate groups in the backbone of DNAa nd were successfully used for DNAcondensation. Furthermore,these groups interact with the cell surface,w hich significantly improves the cellular uptake. [174,175] Another strategy to enable condensation is the use of coordinating multivalent metals cations that can bind to phosphate groups or nucleobases. [176,177] Zinc ions have been employed to aid the interaction between phosphate groups of DNAa nd two different biopolymeric species,h istidineconjugated PLL [178] and dipicolylamine-modified HA, [179] but there is definite scope for use of other ions,particularly those that might be helpful adjuvants,i ncluding nickel, beryllium, cobalt, and palladium. [180] In addition to being an excellent coordinating ion, incorporation of zinc ions via histidine has been shown to increase endosomal release increasing the rate of transfection. [178]

Improved Stability and Solubility of DNA Biopolymer Systems
Stability and solubility of DNAvaccine formulations are not only essential to ensure the efficacy and safety of the vaccine post administration, [181] but also to enable long-term storage without compromising the product quality. [182] Notably,s tability considerations need to be taken into account when deciding on the route of administration (e.g. oral, parenteral) and the dosage form (e.g.dry powder,suspension) of DNAvaccines.
One of the stability issues commonly encountered with nanoformulations and the production of NP carriers is aggregation, which is especially important for suspension formulations. [183] NPs are characterised by high surface-areato-volume ratios,r esulting in ah igh surface energy.C onsequently,t hese particles tend to form thermodynamically favourable aggregates with lower overall surface energy, particularly in biological media. [183,184] To prevent aggregation, asurface coating with hydrophilic polymers,s uch as polyethylene glycol (PEG), can be introduced to the nanoformulation. PEG has been shown to prevent particle interaction and increase their circulation time in blood, thus enhancing the probability of reaching the targeted tissue.P EG surface modification has been demonstrated for PLGA-, [98] PDA-, [185] gelatine-, [186,187] polyspermine-, [141] and PLA-based [188] DNAc arriers.
Shielding against aggregation of positively charged delivery systems can also be achieved using the negatively charged biopolymer chondroitin sulfate. [67] Coating PLL, [67] dendrigraft PLL, [129] poly-arginine, [67] and protamine [135] NPs with chondroitin sulfate has shown to reduce agglutination with erythrocytes and cytotoxicity associated with highly positively charged delivery systems. [157] Furthermore,H ashimoto and co-workers showed that functionalisation of chitosan with hydrophilic lactose residues supressed self-aggregation as well as aggregation of chitosan NPs with serum proteins. [189] Proteins abundant in blood, such as HSA [78,143,190] and bovine serum albumin (BSA), [149] known for their role in the transport of various biologically important molecules,ranging from hormones to fatty acids,h ave also been employed as nanocarriers for gene delivery.A stheya re naturally present in the blood circulation, unfavourable interactions with other serum proteins are minimised, ensuring low levels of aggregation and favourable pharmacokinetic profiles. [190] In terms of chemical stability,p DNAn eeds to be wellprotected from degrading DNase enzymes.O ne way to achieve this is to pack the pDNAw ithin the NP core to block the enzyme access. [96,191] However, it was shown that enzyme degradation can also be minimised by adsorption of pDNAonthe surface of the carrier. [77,82] This is thought to be due to the deformation of pDNAupon binding to nanosized spheres [192] as well as to steric hindrance posed by the carrier core. [193]  Considering different routes of DNAv accine delivery, administrating genetic vaccines via the oral route presents an appealing alternative to intradermal or intramuscular injection due to its non-invasive and convenient nature,a nd because of low sterility concerns. [194] In addition, it was shown that oral administration is particularly interesting for local therapy of GI diseases,s uch as dental caries, [195] colorectal cancer, [196] and inflammatory bowel disease,t hat is,C rohns disease and ulcerative colitis. [197] However,o ral delivery systems are faced with especially challenging chemical stability issues.U pon oral administration, the nanoformulation encounters the highly acidic stomach environment, which is crowded with gastric enzymes that can degrade the carrier material, thus impeding DNAdelivery.This can be overcome by careful design of scaffold materials and DNA-interacting species.F or example,i th as been shown that alginate nanospheres can efficiently protect DNAwhen passing the GI tract and facilitate GI epithelial uptake through superior mucoadhesive and mucopenetrating properties. [198,199] Furthermore, tertiary systems comprised of alginate,c hitosan, and pDNA were more efficient for in vivo oral delivery than chitosan/ pDNAN Ps alone. [200,201] This is probably due to the dissociation of alginate-chitosan crosslinks at pH 1.5, which results in the formation of aprotective layer of insoluble alginate on the carrier surface. [201] Furthermore,t he introduction of polycaprolactone and poly(2-hydroxyethylmethacrylate) to gelatine and chitosan NPs,r espectively,p roved to be ag ood strategy to protect DNAc argo from the harsh conditions of the GI tract. [202,203] Theprotein zein can be used as oral DNAvaccine carrier as well. Its amphiphilic character,o wing to its hydrophobic amino acid content of over 50 %and high glutamine content, facilitates aggregation into NPs with hydrophobic cores and hydrophilic outer shells.Inacidic environments,such as in the stomach, zein nanocarriers are insoluble,e xtremely resistant to the low pH, and have been shown to protect their?c argo from enzymatic degradation. [48,87] It has been demonstrated that immunisation of mice with chitosan carriers modified with an outer shell of zein leads to higher antibody titers compared to mice vaccinated with particles lacking zein. [88] Finally,i ti sd esirable to develop DNA-carrier systems stable during storage and transport without extensive cooling, which can be energy demanding and not suitable for low income countries.D esigning carriers that enable storage and transport at room temperature is asignificant advantage over existing strategies,a nd has been successfully demonstrated for HA and chitosan-containing NPs,w hich remained stable both in lyophilised and liquid form over 12 months at ambient conditions. [204] Closely related to the stability and key to successful DNA vaccine formulations is the solubility of the carrier material. Many biopolymers used for the design of DNAvaccines are intrinsically soluble in aqueous media. However,s ome commonly used biopolymers,s uch as chitosan, show only limited solubility at physiological pH. This can be overcome by increasing the hydrophilicity of the polymer backbone through chemical modification, such as the deacetylation of residual acetylated amines, [205,206] introduction of carboxymethyl moieties to the C6 hydroxy group, [56,68,207,208] trime-thylation, [55, 165-167, 207,208] or addition of larger hydrophilic groups such as N-[(2-hydroxy-3-trimethylammonium)propyl]chloride [56,159] and polymeric methacrylates. [209]

Improved Cell Uptake
In order to increase the immune response and therapeutic activity of DNAvaccines,efficient delivery and release of the genetic cargo within the targeted cell is crucial. Cellular uptake is one of the most significant steps to ensure the biological activity of vaccines,a nd it depends on the interaction between the cell membrane and the carriers, which can result in several endocytotic pathways as shown in Figure 8. [210] Thei nternalisation depends on the cell type as cells bear different types and numbers of membrane proteins and lipids,aswell as on the physiochemical properties of the carrier. [211] We will not go into details on cellular uptake and release of nucleic acids,s ince these have been reviewed by Degros et al., [212] Medina-Kauwe et al., [213] and Zhou et al. [214] In the following sections,w ew ill discuss properties of biopolymers that have been reported to promote different routes of DNAvaccine uptake.

Non-Specific Cell Uptake
Non-specific,a dsorptive endocytosis is promoted by cationic polymers to ah igher degree than by negatively charged or neutral molecules. [152] Endocytosis is enabled by adsorption of the positively charged DNA-polymer systems with negatively charged proteoglycans on the outer cell membrane surface. [174,215] In addition to the strategies for the introduction of positive charge reviewed in Section 2.2, non-specific cellular uptake can be facilitated through the use of CPPs (Figure 9, left). [78,90] CPPs are short, positively charged peptides that can translocate across the cell membrane without impairing the cellular integrity and were first discovered in viral proteins. Although the mechanism of action is complex and beyond the scope of this Review,their positive charge plays an important role to facilitate the adsorption onto the negatively charged cell membrane leading to the uptake through several endocytic pathways. [210,216] Ac ommonly used CPP is HIV-1t ransactivator of transduction sequence (Tat), which was found to enhance gene transfection efficiency.T he introduction of both Tata nd arginine-glycine-aspartate (RGD) peptide was shown to significantly increase the efficiency of HSA nanovectors. [78] It was also demonstrated that the degree of cross-linking to the polymer backbone had ap ronounced influence on the properties of the carrier.B esides positively charged polymers and CPPs,calcium ions (Ca 2+ )can be used to aid cell internalisation as well as endosomal escape.C a 2+ ions mediate several cell processes,i ncluding induction of endocytosis.Itwas demonstrated that the favourable gradient for transport of these ions into the cell (Ca 2+ concentration is 10 4 times higher in the extracellular space compared to the cytoplasm) plays an important role in gene delivery. [217] Ca 2+modified alginate-sulfate NPs were investigated as DNA Angewandte Chemie Reviews delivery systems and mechanistic studies indicated the possibility of clathrin-mediated endocytosis. [218] However,itwas reported that large amounts of positively charged polymers,e specially if bearing high charge density, can cause cytotoxicity [219] and promote nonspecific interaction with negatively charged serum proteins and subsequent clearance by the reticuloendothelial system. [220] That indicates the importance to control the amount of positive charge and charge density.
One approach to reduce positive charge without reducing non-specific cellular uptake includes introduction of hydrophobic moieties such as phenylalanine [102] or oleoyl groups [68] to the positively charged polymer backbone.A lthough this was shown to decrease the toxicity and enhance cellular uptake through hydrophobic interactions with the cell membrane, [221] careful introduction of such modifications is needed in order to avoid significant decrease in solubility and the stability of the system.

Target-Specific Cell Uptake
Targeting ligands can enhance accumulation of the carrier system in as pecific tissue or cell type,m inimise non-specific uptake,and facilitate internalisation. Small molecules,such as folate, [141,148,163,222,223] alendronate, [224] lactose, [189] mannose, [113,[225][226][227] and TLR7 agonist, [208] as well as peptides, [207,228,229] oligonucleotides, [187] and polymers [68,129,142,172,173,222] have been introduced to biopolymerbased nanovectors in order to improve targeting of specific receptors (Figure 9, right). In addition to the choice of ligand, Figure 9. Design strategies to enhance cell uptake of DNA vaccine carriers. Cationic polymers, CPPs, and hydrophobic substituents can promote non-specific cellular uptake. Targeting lectin-binding receptors (mannose receptor or DEC-205) and toll-like receptors (TLR2 or TLR7) can facilitate accumulationi nA PCs. Targeting tumour-specific receptors( folate, CD44) can increase uptake by cancer cells. Figure 8. Internalisation of nanocarriers.Internalisation can occur through an umber of endocytic pathways:pinocytosis (clathrin-mediated endocytosis, caveolae-mediated endocytosis, clathrin/caveolin-independent endocytosis), phagocytosis, and macropinocytosis. Once internalised, the cargo is entrapped in endosomes and eventually ends up in the lysosome. The pDNA should evade endolysosomal degradation and enter the cytoplasm by endosomal escape to enable pDNA translocation into the nucleus for transcription.

Angewandte Chemie
Reviews control over surface density is essential to achieve high targeting efficiencya nd internalisation.
Specific delivery and enhanced uptake of DNAvaccines into APCs has been associated with effective immunisation. Lectin-binding receptors,s uch as mannose receptors CD206 and DEC-205, are abundantly expressed on the surface of APCs,i ncluding macrophages and DCs, [225][226][227]230] and introduction of mannose enables preferential uptake of APCs. [113,[225][226][227] TheD EC-205 receptor is particularly interesting,a sit initiates MHCI and MHCII pathways by antigen endocytosis, leading to the stimulation of both CD4 + and CD8 + Tcells. [231] To enable DEC-205 targeting,S uresh et al. fused anti-DEC-205 antibody to pDNA-loaded chitosan and designed aD NA vaccine against severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein. [228] This carrier has demonstrated that targeted delivery to nasal DCs is apotent strategy to achieve enhanced immunogenicity of alow-dose DNAvaccine.
In addition to lectin-binding receptors,T LRs have been explored for macrophage targeting.M odification of chitosan NPs with TLR agonists,T LR-7 [208] and TLR-2, [207] has significantly increased IL-8 levels in THP-1 macrophages compared to bare chitosan NPs.
As mentioned in the introduction, DNAvaccines represent ap romising strategy to induce as pecific long-term immune response.T his is particularly interesting for the induction of an immunological memory and systemic immune response in cancer treatment. [232] Besides targeting receptors on APCs,different cancer types can be treated by induction of specific CTLs.N amely,f olate and CD44 receptors are significantly overexpressed receptors on several tumour cells compared to normal cells.T herefore,i ntroducing folic acid to chitosan, [223] polyspermine, [141] chondroitin sulfate-PEI, [222] and dextran [148,163] led to enhanced immunisation in hepatocellular,lung, and ovarian cancer,respectively.Biopolymer scaffolds,such as endogenous polysaccharides,HA, and chondroitin sulfate,specifically bind to CD44 receptors.

Designing Carriers that Enable Endosomal Escape
Am ajor challenge drug and gene delivery systems are facing is their entrapment in endosomes.E ndosomes are organelles responsible for intracellular sorting and contain numerous enzymes.Ifthe genetic material remains entrapped in the endosome,itisdegraded by lysosomal proteases,which ultimately results in low transfection efficacy. In order to escape endolysosomal degradation, several mechanisms are involved in this process,s uch as pore formation in the endosomal membrane,the pH buffering effect, and the fusion into the lipid bilayer. [233] Throughout the maturation of the endosomes,t he pH decreases from physiological pH 7.4 to pH % 6.5 in the early endosome,pH% 6.0 in the late endosome and pH % 5.0 in the lysosome due to the activity of membrane-bound ATPase pumps (proton pumps), which pump protons across the endosome and lysosome membrane into the vesicle interior through ATPh ydrolysis. [234] Thep resence of cationic poly-mers,s uch as PEI, [91,142,144,147,152,235] polyamidoamine, [151] succinyl tetraethylene pentamine, [164] spermine, [89,139-141, 161, 162, 236] and imidazole-containing molecules,s uch as histidine, [83,90] can all lead to endosomal rupture via the "proton-sponge" effect. This effect is associated to the large buffering ability of these molecules due to proton binding,w hich leads to more protons being pumped. Consequently,this results in accumulation of chloride ions and water, osmotic swelling,a nd ultimately rupture of endosomes.E ven though this mechanism has been extensively used to explain the endosomal rupture,i th as been heavily debated. [234] Nevertheless,m olecules with high buffering capacity have been shown to play an important role in enabling endosomal escape and have been employed to enable the release of genetic material. For example,C heng et al. [153,154] have designed dextran nanocarriers containing histidine-rich peptides as ap romising material for safe and efficient gene therapy.I nt heir study, dextran was grafted with arginine-histidine peptides (R x H y ), and low cytotoxicity as well as high gene expression were achieved by using low molecular weight dextran carriers with ah igh degree of substitution. Thep resence and ratio of histidine was important both for the DNAc ondensation and the control of endosomal escape,while arginine residues were primarily used for DNAc ondensation and enhanced cell uptake.T his and other studies [64,83,90,139] demonstrated the advantage of imidazole substituents and indicated ap ossible route to designing efficient DNAvaccine carriers.
In addition to functional groups with high buffering capacity,i tw as also shown that endosomal escape can be enhanced in the presence of molecules able to penetrate lysosomes (lysosomotropic agents), such as chloroquine, [237,238] cationic lipids, [239] and membrane-disrupting peptides. [159,240] Thel atter are particularly interesting as they are mainly derived from viral and bacterial vectors,w hich developed efficient strategies to escape endosomes. [233] For example,membrane-disrupting peptides,s uch as haemagglutinin subunit 2(HA2), [240] afusogenic peptide from influenza virus,a nd LLO,acholesterol-dependent toxin produced by Listeria monocytogenes, [241] have been used to modify pDNA delivery systems and enhance delivery into the cytosol of the target cells.A lthough their mode of action is different, they both result in endosomal escape.W hile HA2 undergoes conformational change during acidification, which enables fusion trough the endosomal membrane,LLO is active at low pH but is degraded in the cytosol and enables endosomal escape through the formation of pores in lipid bilayers. [80] 2.6. Improved pDNA Release and Delivery to the Nucleus Dissociation of the pDNAfrom the nanocarrier is one of the most important steps in achieving higher transfection efficacies.Efficient release can be achieved either by masking the strong electrostatic interaction of pDNAa nd the carrier or by introduction of astimuli-responsive degradation system.
Masking of the positive charge is probably the simpler, but less controllable route and can be achieved by introducing as econd polymer to the nanocarrier.F or example,i tw as reported that incorporating polyanionic polymers,s uch as alginate [200,201,242] and poly(g-glutamic acid), [108,109] or an egatively charged protein like a-casein, [243] into chitosan nanostructures can reduce the strength of the interaction between DNAa nd the particles,f acilitating release and increasing transfection. However, it should be noted that this could also lead to aloss of DNAasitistransported to the cell of interest.
Studies have also demonstrated that the net positive charge can be decreased by grafting hydrophobic molecules such as l-phenylalanine [102] or by addition of flexible bulky groups,s uch as pullulan. [64] Additionally,X ua nd co-workers integrated ad endritic lipopeptide,acharge-reversible polymer, and an APC-targeting material into aD NA vaccine delivery system through layer-by-layer assembly. [51] They used poly(allylamine hydrochloride)-citraconic anhydride,acommonly used charge-reversible polymer,w hich can be hydrolysed in mild acidic environments in endosomes,r esulting in the destabilisation of the carriers and release of pDNA. Compared to the traditionally used cationic polymer,P EI 25k , the carrier system based on the charge-reversible polymer resulted in lower toxicity and eight-fold higher transfection efficiency.
However,am ore controllable route to pDNAr elease is the use of stimuli-responsive linkers.InDNA vaccine systems, stimuli-responsive disintegration of the carrier is mainly achieved by incorporation of disulfide bonds and pH-sensitive bonds.
Thed isulfide bond (-SÀS-) can be cleaved into thiols (-SH) in the presence of intercellular glutathione. [244] The concentration of glutathione is around 100-1000 times higher in several intracellular compartments compared to the extracellular environment (2-10 mm compared to 2-20 mm), which contributes to the efficient cleavage of disulfide modified polymers. [245] This strategy was employed to facilitate unpacking of pDNAf rom polymer carriers such as dextran, [148,163] HA, [69] polyarginine, [90] and LLO, [79,80] which were modified with positively charged groups via disulfide bonds.
In addition to disulfide bonds,a cid-degradable ketal esters, [89] diacrylate cross-linkers, [141] and bis-amide bonds [139] were used to improve the release of DNAfrom polyspermine gene delivery systems in acidic environment.
Furthermore,L iu and co-workers have reported the dissociation of pDNAf rom dextran-quantum dots [246] enabled by cleaving the C=Nb ond in Schiff bases in acidic conditions (pH < 6.5). [247] In another study,W ang and co-workers have designed aP DA-PEI nanovector bound to PEG-phenylboronic acid via ap H-responsive boronate-ester bond. [185] They have shown that the complex remained stable at physiological pH but could be cleaved after internalisation in endosomes. Additionally,n ear-infrared (NIR)-light irradiation and good photothermal conversion of the carrier system has resulted in quick endosomal release.Inresponse to the acidic pH within cancer cells and the NIR light irradiation, the nanovector was able to overcome multiple barriers and result in efficient gene delivery.
Once released, pDNAn eeds to enter the nucleus to be transcribed for successful DNAv accination. To aid the nuclear transport of the genetic material, carriers can be additionally modified with an uclear localisation signal (NLS). NLS is ashort peptide with high content of positively charged lysines or arginines,derived from eukaryotic nuclear proteins and viral proteins,w hich can efficiently mediate intranuclear transport. [248] Guan et al. introduced NLS derived from simian virus 40 (SV40) large Tantigen to ac ationic HSA-DNAs ystem, significantly increasing its gene expression efficacy in vitro and in vivo. [190] Similarly,p rotamine,a na rginine-rich protein commonly used for DNAcondensation and membrane translocation, has been shown to aid the nucleus uptake when attached to biodegradable anionic polymers such as chondroitin sulfate [135] or hydrophobic moieties such as cholesterol. [83] This increase in nuclear transport is attributed to the presence of NLS-like regions consisting of four to six arginine repeats.

DNA Vaccine Delivery Materials with Immunostimulatory Effects
As outlined in Section 1.1.,i mmunostimulants,s uch as IFN-g,IL-2, or IL-4, play an important role during an immune response,i.a. through promoting Tcell differentiation. This is the reason why vaccine formulations often contain adjuvants. Adjuvants are organic or inorganic vaccine additives,f or example,a luminum salts in hepatitis vaccines or monophosphoryl lipids in shingles vaccines,and are employed to trigger astronger immune response,for example through stimulating the secretion of immunostimulants. [249] Adjuvants have also been employed to boost the immunogenicity of DNAv accines.F or example,J iang et al. reported that modifying chitosan NPs with methacrylatebased polymers resulted not only in asignificant stabilisation of DNA, but also led to higher antibody levels and IFN-g secretion compared to the administration of naked DNA. [209] Further to these findings,Y ue and co-workers reported that the introduction of CpG motifs to chitosan-NP-carriers increased T-cell proliferation, the production and release of IFN-g,I L-2, as well as IL-4. [250] TheC pG motif is aw ellstudied adjuvant in gene delivery and consists of as hort, synthetic single-stranded DNAs equence,w hich contains mostly cytosine and guanine building blocks.I nn ature,t he unmethylated CpG motif can be found in bacterial genomes, but it is very rare in the vertebrate genome.T hus,w hen brought into the organism of av ertebrate,t he foreign CpG DNAi sr ecognised as an invading species,w hich results in as trong immune response.I nanutshell, CpG represents ap athogen-associated molecular pattern (PAMP), which is recognised by aspecific pattern recognition system in TLR-9 of APCs. [251] Interestingly,n ot only the addition of adjuvants but also the size of the carrier complex plays ar ole in provoking an immune response.This phenomenon was observed in various inorganic and biopolymeric delivery systems.H owever,n o universal, linear size-immune-response relationship was derived, indicating that other factors,i ncluding shape,c hemical composition, surface modification, and charge of the carrier,c ontribute,t oo. [252] While Yuea nd co-workers found that smaller CpG-modified chitosan NPs triggered astronger immune response than larger particles, [250] Kim et al. explored the potential of monodispersed polypyrrole NPs and discovered that medium-sized NPs (60 nm in diameter) caused as tronger immune response than their smaller and larger counterparts. [253] These studies point towards the need for ar ational design approach that aims to address different factors related to stability,s olubility,t ransport, and immune system response of DNAvaccines.

Conclusion and Future Directions
DNAv accines have been developed as as uitable alternative to conventional vaccines.D espite not being approved for human use yet, numerous DNAvaccine formulations have been proposed and used in clinical trials to tackle,i .a.,v iral and bacterial infections,p arasites,a sw ell as cancers.T here are several advantages of DNAvaccines that should prompt more studies in the field. DNAvaccines are cheaper to make and easier to formulate than conventional vaccines.T hey are based on the expression of antigens,w hich can be presented to immune cells,m aking them versatile and adaptable to ar ange of diseases.H owever,p ressing challenges remain, including the difficulty of administering genetic material precisely and safely as well as the low immunogenicity of the first formulations.S ome of these obstacles have been overcome by using DNAn anocarriers,b ased on polymers,l ipids, and inorganic materials.Biopolymer-based nanomaterials are particularly promising carrier candidates due to their intrinsic biocompatibility,b iodegradability,s ustainable availability, and manifold possibilities to modulate their physicochemical and biological properties by adjusting the size,c hemical composition, or surface functionalisation. These materials can be further modified to improve their solubility and stability, ability to immobilise DNA, cellular uptake,i ntracellular release,aswell as immunogenicity.
Thepromising initial advances of biopolymer-based DNA vaccines,p articularly for the treatment of cancers,a re reassuring, but the major challenge of the field, the insufficient immune response in humans through administered DNA, abides.H ence,t he field will need to focus on translating the promising results of preclinical studies,c ommonly conducted in small mammals and non-human primates,t o efficient human therapies.T his is often very difficult due to the significantly different structure of the immune system in humans and animals.F urthermore,w ee xpect that particularly for complex diseases,like cancer, clinicians will embrace an approach based on the combination of therapies,s uch as checkpoint inhibition, cell therapy,a nd DNAv accines to address the issue of low immunogenicity in the future. Recently,c ombinational approaches have also been employed in dealing with coronavirus infections,w herein antivirals and anti-inflammatory agents are being used to counter viral replication and respiratory distress,respectively. Administration of vaccines alongside these conventional therapies will effectually counter the virus that enters the body.
Adevelopment we will need to see in the next few years is am ore efficient identification of biomarkers,w hich will enable development of diagnostic strategies and provide us with the information on patients most likely to benefit from use of the DNAv accine approaches.Amore nuanced and holistic understanding of immunological markers and their correlation to vaccine efficacyw ill inform us about polymer design and its subsequent delivery strategy.
Finally,s cale up of vaccine-based delivery systems with focus on reproducibility and stability is another crucial aspect to consider both during the carrier material design and implementation in the clinic. We believe that in this context, biopolymers will emerge as sustainable and scalable material of choice for the formulation of DNAand mRNAvaccines,as their monomers are readily available and their production is cost-effective.T he next few years will see more research in the field of biopolymer-based DNAvaccines to increase their in vivo efficacyand ultimately enable their application in the clinic.