*2.2.2.1 Nanoparticulate adjuvant systems and DNA vaccine delivery*

Nanoparticles are matrix systems called nanospheres or nanocapsules according to the method of preparation, which vary in size from 1 to 1000 nm and are prepared with natural or synthetic materials. They are also the matrix systems in which the active substance nucleic acid is solubilized, adsorbed or electrostatically interacted with the surface [23, 45].

In vaccine development studies, nanotechnology is becoming increasingly important. Numerous nanoparticles of varying composition, size, shape and surface properties are used to both increase vaccine efficacy and target vaccination. With these properties, nanoparticles play an active role in in vitro/in vivo drug and gene delivery systems. Because nanoparticles are biodegradable and easily recognizable by antigen-presenting cells (APCs), they can be easily endocytosed and used as successful vaccine carriers in vaccine delivery systems [22, 25, 40].

Nanoparticle-mediated delivery of DNA vaccines has the advantages of increasing transfection efficiency and immunogenicity, inducing both cellular and antibody responses, and not requiring special equipment during administration; there are some disadvantages, such as, the long-term effects of nanoparticles in the body are not yet known [1]. As a DNA vaccine carrier, natural polymeric nanoparticles such as chitosan, alginate, pullulan and inulin (2–1000 nm); synthetic polymeric nanoparticles such as PLGA, dendrimer, PLA, PHB and PEI (2–1000 nm); inorganic nanoparticles such as gold, silica-based and carbon-based nanoparticles (2–1000 nm); nano-liposomes (100–400 nm) which are phospholipids that can be organized in nanoscale and non-infectious virus-like particles (VLP) (20–800 nm) generated by packaging the nucleic acid into biocompatible capsid proteins [1, 9, 22] are used. In recent years, the use of complex nanoparticles to overcome the disadvantages of enhancing the function of single-source nanoparticles has been discussed. Polyethylene/PLGA, polyg-glutamic acid/chitosan, polyethylene/chitosan, dendrimer/polyethylene glycol, polyg-glutamic acid/polyethyleneimine (PEI), chitosan/tripolyphosphate and polyethylene glycol/liposome nanoparticles are remarkable DNA transport systems [1, 22, 25, 40, 46, 47]. The overall efficiency of nanoparticle-based DNA delivery systems depends on four basic factors. These factors explain the necessity of nanoparticle-based adjuvant systems. Accurate plasmid DNA is integrated with the nanoparticle into the target cell correctly, the nanoparticle is removed from the endosomal vesicles and is transferred to the cytoplasm, then is transferred to the mitochondria or nucleus [23, 25].

In recent years, there has been a tremendous improvement in the area of gene delivery system with the advent of cationic polymers. These polymers bind with nucleic acid(s) to form complex structures known as polyplexes, which have increased transfection efficiency [10, 48]. Some of the examples of the polycationic polymers are chitosan, polyethyleneimine (PEI), poly(2-hydroxy ethyl methacrylate) (pHEMA), polyamidoamine (PAMAM) dendrimers, polylactic-co-glycolic acid (PLGA), polyethylene glycol (PEG) and poly-L-lysine (PLL) and the negatively charged phosphate group of pDNA, which have cationic properties. [10, 23, 42, 43, 49–51]. Many factors such as molecular weight (MW), surface charge, charge

**135**

*Current State of the Art in DNA Vaccine Delivery and Molecular Adjuvants: Bcl-xL…*

density, hydrophilicity and morphology significantly influence the gene transfection efficiency of cationic polymers. For this reason, in general, optimization of various forms of these cationic polymers with pDNA is required to increase transfection efficiency. The polyplex structure electrostatically formed with DNA packs it into smaller structure, pDNA, that interacts effectively with the negatively charged cell membrane and is endocytosed easily into the cell. In addition, cationic polymers are suitable for specific applications like conjugation of targeted ligands and various moieties, which provide them with specific characteristics [21, 22, 24, 25, 52].

Since their discovery in 1964, liposomes are extensively studied and used in the pharmaceutical and cosmetic industries. It is a generic name for single- or multilamellar vesicles in lipid-based nano- and micro-dimensions, whose surface charge can be changed by lipid formulation. Thanks to biocompatibility, biodegradability, low toxicity and low immunogenicity, liposomes have been used as potential nucleic acid carriers both in vitro and in vivo systems. In the composition of cationic liposomes, neutral lipids, cationic lipids and/or anionic lipids can be complexed in different ratios for creating ideal co-lipid. Neutral lipids (DOPE, DPPC, DOPC and Chol) are also often involved in the cationic liposome structure for more efficient transport systems, although sometimes only cationic lipids (DOTAP, DC-Chol, MMRIE, DODAP, DDTAP and DDA) are used [8, 36, 53]. The choice of ideal colipids is important during the formulation phase [54–56], since they significantly

There is a strong correlation between morphology and performance of lipoplex (liposome and DNA complex). There are several models that determine morphology such as the external model (DNA is attached to the cationic liposome surface), the internal model (DNA is surrounded by the cationic liposomes), the cationic liposome beads model on the DNA sequence and the spherical model. In addition to the liposomal composition, cationic lipid/nucleic acid (N/P) load ratio, preparation methods, ionic strength and temperature also affect lipoplex formulation and morphology. While high N/P ratios accept that the lipid/DNA complex is compatible with the global model with effective DNA concentration [22, 54, 57, 58], the low N/P ratio accepts the model of cationic liposome beads on the DNA sequence. In addition, high concentrations of cationic liposomes can cause cytotoxicity. The particle size of the lipoplexes is also influential on transfection efficiency. In general, large lipoplexes are more effective with in vitro transfection of nucleic acids because large pieces allow rapid sedimentation, maximum cell contact with the cell membrane and easier separation after endocytosis. Small particles, on the other hand, are much

more effective, safe and suitable for in vivo transport of nucleic acids [57, 59].

coating to protect surface charges [23, 48, 54, 56, 57, 61].

Various studies have shown that cationic liposomes (DC-Chol/DOPE) formed at various ratios of 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) and 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) are effective pDNA carriers in the preclinical as well clinical trials for plasmid transfection [55, 57, 60]. In another study, it was reported that the vaccine adjuvants with high immunogenicity were obtained with cationic liposomes formed at 50:50 ratio (50 mol%) with DC-Chol/DPPC (3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol/1,2-dipalmitoyl-sn-glycero-3-phosphocholine) [53]. In many studies, it has been reported that surface modifications to cationic liposomes (PEGpolyethylene glycol coating, etc.) increase transfection efficiency due to more stable plasmid retention, longer circulation time and lower immunological response. Studies have shown that transfection efficiency is increased by about 55% with PEG

*DOI: http://dx.doi.org/10.5772/intechopen.82203*

affect the overall performance of cationic liposomes.

*2.2.2.1.1 Liposomes*

*Current State of the Art in DNA Vaccine Delivery and Molecular Adjuvants: Bcl-xL… DOI: http://dx.doi.org/10.5772/intechopen.82203*

density, hydrophilicity and morphology significantly influence the gene transfection efficiency of cationic polymers. For this reason, in general, optimization of various forms of these cationic polymers with pDNA is required to increase transfection efficiency. The polyplex structure electrostatically formed with DNA packs it into smaller structure, pDNA, that interacts effectively with the negatively charged cell membrane and is endocytosed easily into the cell. In addition, cationic polymers are suitable for specific applications like conjugation of targeted ligands and various moieties, which provide them with specific characteristics [21, 22, 24, 25, 52].

#### *2.2.2.1.1 Liposomes*

*Immune Response Activation and Immunomodulation*

immune response pattern [33].

interacted with the surface [23, 45].

adjuvants depends on its ability to stimulate antigen-presenting cells (dendritic cells and macrophages) and T and B lymphocytes of the natural defense system [11]. There is a need for definitive information on the structure, stability, safety and immunogenicity of the adjuvant to be used in an effective vaccine development process [32, 33]. For this purpose, the choice of adjuvant depends on factors such as antigenic structure, immunization scheme, mode of administration and desired

Nanoparticles are matrix systems called nanospheres or nanocapsules according to the method of preparation, which vary in size from 1 to 1000 nm and are prepared with natural or synthetic materials. They are also the matrix systems in which the active substance nucleic acid is solubilized, adsorbed or electrostatically

In vaccine development studies, nanotechnology is becoming increasingly important. Numerous nanoparticles of varying composition, size, shape and surface properties are used to both increase vaccine efficacy and target vaccination. With these properties, nanoparticles play an active role in in vitro/in vivo drug and gene delivery systems. Because nanoparticles are biodegradable and easily recognizable by antigen-presenting cells (APCs), they can be easily endocytosed and used as

Nanoparticle-mediated delivery of DNA vaccines has the advantages of increasing transfection efficiency and immunogenicity, inducing both cellular and antibody responses, and not requiring special equipment during administration; there are some disadvantages, such as, the long-term effects of nanoparticles in the body are not yet known [1]. As a DNA vaccine carrier, natural polymeric nanoparticles such as chitosan, alginate, pullulan and inulin (2–1000 nm); synthetic polymeric nanoparticles such as PLGA, dendrimer, PLA, PHB and PEI (2–1000 nm); inorganic nanoparticles such as gold, silica-based and carbon-based nanoparticles (2–1000 nm); nano-liposomes (100–400 nm) which are phospholipids that can be organized in nanoscale and non-infectious virus-like particles (VLP) (20–800 nm) generated by packaging the nucleic acid into biocompatible capsid proteins [1, 9, 22] are used. In recent years, the use of complex nanoparticles to overcome the disadvantages of enhancing the function of single-source nanoparticles has been discussed. Polyethylene/PLGA, polyg-glutamic acid/chitosan, polyethylene/chitosan, dendrimer/polyethylene glycol, polyg-glutamic acid/polyethyleneimine (PEI), chitosan/tripolyphosphate and polyethylene glycol/liposome nanoparticles are remarkable DNA transport systems [1, 22, 25, 40, 46, 47]. The overall efficiency of nanoparticle-based DNA delivery systems depends on four basic factors. These factors explain the necessity of nanoparticle-based adjuvant systems. Accurate plasmid DNA is integrated with the nanoparticle into the target cell correctly, the nanoparticle is removed from the endosomal vesicles and is transferred to the cytoplasm,

*2.2.2.1 Nanoparticulate adjuvant systems and DNA vaccine delivery*

successful vaccine carriers in vaccine delivery systems [22, 25, 40].

then is transferred to the mitochondria or nucleus [23, 25].

In recent years, there has been a tremendous improvement in the area of gene delivery system with the advent of cationic polymers. These polymers bind with nucleic acid(s) to form complex structures known as polyplexes, which have increased transfection efficiency [10, 48]. Some of the examples of the polycationic polymers are chitosan, polyethyleneimine (PEI), poly(2-hydroxy ethyl methacrylate) (pHEMA), polyamidoamine (PAMAM) dendrimers, polylactic-co-glycolic acid (PLGA), polyethylene glycol (PEG) and poly-L-lysine (PLL) and the negatively charged phosphate group of pDNA, which have cationic properties. [10, 23, 42, 43, 49–51]. Many factors such as molecular weight (MW), surface charge, charge

**134**

Since their discovery in 1964, liposomes are extensively studied and used in the pharmaceutical and cosmetic industries. It is a generic name for single- or multilamellar vesicles in lipid-based nano- and micro-dimensions, whose surface charge can be changed by lipid formulation. Thanks to biocompatibility, biodegradability, low toxicity and low immunogenicity, liposomes have been used as potential nucleic acid carriers both in vitro and in vivo systems. In the composition of cationic liposomes, neutral lipids, cationic lipids and/or anionic lipids can be complexed in different ratios for creating ideal co-lipid. Neutral lipids (DOPE, DPPC, DOPC and Chol) are also often involved in the cationic liposome structure for more efficient transport systems, although sometimes only cationic lipids (DOTAP, DC-Chol, MMRIE, DODAP, DDTAP and DDA) are used [8, 36, 53]. The choice of ideal colipids is important during the formulation phase [54–56], since they significantly affect the overall performance of cationic liposomes.

There is a strong correlation between morphology and performance of lipoplex (liposome and DNA complex). There are several models that determine morphology such as the external model (DNA is attached to the cationic liposome surface), the internal model (DNA is surrounded by the cationic liposomes), the cationic liposome beads model on the DNA sequence and the spherical model. In addition to the liposomal composition, cationic lipid/nucleic acid (N/P) load ratio, preparation methods, ionic strength and temperature also affect lipoplex formulation and morphology. While high N/P ratios accept that the lipid/DNA complex is compatible with the global model with effective DNA concentration [22, 54, 57, 58], the low N/P ratio accepts the model of cationic liposome beads on the DNA sequence. In addition, high concentrations of cationic liposomes can cause cytotoxicity. The particle size of the lipoplexes is also influential on transfection efficiency. In general, large lipoplexes are more effective with in vitro transfection of nucleic acids because large pieces allow rapid sedimentation, maximum cell contact with the cell membrane and easier separation after endocytosis. Small particles, on the other hand, are much more effective, safe and suitable for in vivo transport of nucleic acids [57, 59].

Various studies have shown that cationic liposomes (DC-Chol/DOPE) formed at various ratios of 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) and 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) are effective pDNA carriers in the preclinical as well clinical trials for plasmid transfection [55, 57, 60]. In another study, it was reported that the vaccine adjuvants with high immunogenicity were obtained with cationic liposomes formed at 50:50 ratio (50 mol%) with DC-Chol/DPPC (3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol/1,2-dipalmitoyl-sn-glycero-3-phosphocholine) [53]. In many studies, it has been reported that surface modifications to cationic liposomes (PEGpolyethylene glycol coating, etc.) increase transfection efficiency due to more stable plasmid retention, longer circulation time and lower immunological response. Studies have shown that transfection efficiency is increased by about 55% with PEG coating to protect surface charges [23, 48, 54, 56, 57, 61].

#### *2.2.2.1.2 Chitosan*

Chitosan is the best-known working polymer among the therapeutic natural polymers. It consists of D-glucosamine and N-acetyl-D-glucosamine units linked by β-(1,4) glycosidic bonds. The chitosan is derived from the chitin molecule and the thermoacetal deacetylation process forms the bonds. The chitin is a biopolymer in abundance in nature and is the cell wall of most of the fungi and bacteria of the shellfish and the outer shell of insects [62]. One of the most important applications of chitosan is its application as non-viral vector in gene therapy. For this reason, chitosan has recently been used for gene delivery systems for therapeutic purposes [50, 63]. The first chitosan/DNA complex was made about 25 years ago, and was composed of plasmid and chitosan in size of 150–600 nm. Positive charge, which allows it to complex easily with negatively charged DNA, allows the formation of nanocapsules (80–500) at various sizes that protect DNA from nuclease activation [64, 65].

Chitosan nanoparticles are formed by a wide variety of methods, by the formation of different bonds, by different polymer conformations and by various internal and external molecular interactions. In addition to covalent cross-linking and desolvation methods, ionic gelation is one of the most useful methods used in the handling of chitosan nanoparticles and is especially considered for polyelectrolyte sodium tripolyphosphate (TPP) [42, 43, 66]. In chitosan-based adjuvants, DNA is involved in structure determination, electrostatic interaction, encapsulation and surface adsorption [42, 50]. Chitosan has excellent biocompatibility, acceptable biodegradability, high biosecurity, low cytotoxicity and low immunogenicity. However, limited application of gene transport due to poor solubility at physiological pH, insufficient positive charge and low transfection efficiency can be prevented by various surface modifications on chitosan nanoparticles [25]. For example, TPP is useful in preparing chitosan nanoparticles and enhances its non-toxic nature. Due to the self-regulation nature of polycations and polyanions, it leads to the formation of linking complexes between TPP groups and chitosan amino groups. In the process of the protonation of the chitosan at physiological pH, TPP is covalently bound to chitosan amino groups providing structural change and better provocation [23, 50, 61, 67, 68].

#### *2.2.2.1.3 Polyethyleneimine (PEI)*

Polyethyleneimine is a gene-carrying cationic polymer that has high transfection efficiency in vitro and in vivo with lower cost, but can exhibit high toxicity with concentration pulse [69–71]. PEI receives proton in aqueous solutions and has a high positive charge. Thus, in vitro and in vivo DNA and oligonucleotide transport are promising candidates as non-viral transport systems [71, 72]. PEI and its derivatives are commonly known as an effective dispersant and cationic flocculent used for negatively charged colloids [73].

Adjuvant systems with PEI involve the electrostatic interaction of DNA with the cationic polymer and the formation of the polycation/DNA complex [74]. The DNA/ polymer complex (N/P) occurs when the amine group of this polymer interacts with the phosphate group of DNA [73]. In many studies, it has been shown that PEI has the advantages of holding pDNA with electrostatic bonds, binding to the cell surface and its endocytosis, and releasing pDNA into the cell. It also enhances the entry of the gene from cytoplasm to the nucleus [70]. This polymer is used for DNA-based immunotherapy or DNA vaccine delivery to ensure that the immune response is activated to provide a strong immune response [74]. Factors such as molecular weight, branching grade, ionic strength of solution, zeta potential and particle size affect the

**137**

*Current State of the Art in DNA Vaccine Delivery and Molecular Adjuvants: Bcl-xL…*

efficacy/cytotoxicity of PEI. PEI is coated with PEG and various ligands to increase transfection efficiency and reduce cytotoxicity. Meanwhile, the PEI in the linear structure helps to create an effective transport system than the branched PEI [69].

Dendrimers are nanometer-sized (1–100 nm) particles with a unique architectural structure in the form of spherical macromolecules, consisting of a central core, a hyperbranched mantle and a corona containing a peripheral reactive group. Dendrimers can be fabricated by convergent or divergent synthesis. The high-level control system on dendritic architectural synthesis makes dendrimers almost perfect spherical nanostructures with predictable properties. Dendrimers can build up ionic interactions with DNA, creating complexes with high stability and resolution. Costly production is the only disadvantage. Structures such as polyamidoamine and polypropylenimine are included in the dendrimeric clas-

Of the cationic compounds used in gene delivery, polyamidoamine (PAMAM)

dendrimers have been regarded as the most suitable gene carrier, due to the presence of abundant amino groups on the electrostatically interacting surface with negatively charged nucleic acid material and low polydispersity. This association at the nanoscale (nucleic acid-dendrimer pair) is called dendriplex [79]. These particular supramolecular structures not only protect the genetic material from nuclease degradation but also interact with the negative surface of the cell membrane and activate entry into the cell through endocytosis. The high amine content of PAMAM dendrimers provides significant buffering capacity in the endosomal pH range. This extraordinary buffering capacity plays a powerful driving force in the liberation of dendriplex complexes prior to enzymatic degradation to lysosomal enzymes in endosomes. These properties make PAMAM dendrimers the carriers that are obliged to carry out future polycation-based gene delivery studies. On the other hand, long-term storage stability and high biocompatibility make PAMAM dendrimers almost necessary to use as gene carriers

The number of generations on the transfection efficiency is important. Dendrimer generations G0–G3, low-grade PAMAM dendrimers, exhibit low gene transfection efficiency and low cytotoxicity, while G4–G8 dendrimers show high transfection efficiency and high cytotoxicity. For this reason, dendrimer generations G4–G5, which have low cytotoxicity as well as high transfection efficiency in gene transfer, are preferred [25, 76]. In addition, adding different moieties enhances various features of dendrimers. For example, high amine content on the PAMAM surface allows conjugation of various materials to improve transfection efficiency and reduce target cytotoxicity [24, 25, 49, 61]. PEG conjugation provides positively charged protective sheath, which reduces cytotoxicity, undesired interactions with blood components, and facilitates binding of the ligand. Adding hydrophobic moieties favors hydrophilic-hydrophobic balance, reduced cytotoxicity and facilitation of packaging back into the vector. With glucocorticoid conjugation, nuclear targeting and parental dendrimers (dexamethasone and triamcinolone acetonide conjugates) provide hydrophilic-hydrophobic equilibrium modulation. By cyclodextrin conjugation, increase of endosomal escape and decrease of cytotoxicity as well as oligonucleotides against enzymatic digestion are protected. Finally, by amino acid, peptide and protein conjugation, it is also possible to increase cell penetration (arginine and TAT peptide conjugation), cellular uptake, endosomal escape, serum resistance (histidine conjugation), and nuclear localization, and also target specific

*DOI: http://dx.doi.org/10.5772/intechopen.82203*

*2.2.2.1.4 Dendrimers*

sification [25, 75–78].

in vivo [24, 25, 49, 51, 61, 75].

receptors [24, 25, 51, 61, 77, 78].

efficacy/cytotoxicity of PEI. PEI is coated with PEG and various ligands to increase transfection efficiency and reduce cytotoxicity. Meanwhile, the PEI in the linear structure helps to create an effective transport system than the branched PEI [69].
