*2.2.2.1.4 Dendrimers*

*Immune Response Activation and Immunomodulation*

from nuclease activation [64, 65].

better provocation [23, 50, 61, 67, 68].

*2.2.2.1.3 Polyethyleneimine (PEI)*

negatively charged colloids [73].

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

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

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

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

*2.2.2.1.2 Chitosan*

**136**

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 classification [25, 75–78].

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

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

### *2.2.2.1.5 Poly(lactic-co-glycolic acid) (PLGA)*

PLGA is a solid polymeric material approved by the Food and Drug Administration (FDA) for nanoparticle-based drug and gene delivery systems. Their biocompatibility, biodegradability, reliability and high stability characteristics during storage provide advantages in delivery systems [20, 80–82]. PLGA nanoparticles can easily pass through vessels in vivo without damaging the tissues surrounding the tumor and thus accumulate with the mechanism of "enhanced permeability retention" (EPR) in solid tumors. However, PLGA particles are less efficient in encapsulating nucleic acids because hydrophobic properties of PLGA are not compatible with anionic, hydrophilic properties of nucleic acids. In addition, difficult preparation conditions and pH decreases during PLGA hydrolysis inactivate nucleic acid loading and prevent polyplex formation. In order to overcome these drawbacks, effective gene transfer systems can be formed with different formulations made with different molecular interactions [21, 48, 52, 61, 83–86].

For example, PLGA nanoparticles can be processed with materials such as PEI to increase positive charge distribution and provide a stronger penetration of nucleic acid. After penetration of the nucleic acids, PEI, PEG cross-linking material and cell penetration peptides can be used for effective encapsulation and stabilization of the nano-carrier system [48, 81, 85]. It is also one of the systems to produce PLGAbased adjuvants in sizes of 200–300 nm by such means as cationic hydrophilic properties by condensing PLGA with cationic polymers such as polyethylenoxide (PEO) and polyethylene glycol methacrylate (PEGMA), emulsifying solvent diffusion method without shear stress [48, 81, 85].

In one study, DNA-loaded PLGA particles were fabricated by a double emulsion water in oil in water (w/o/w) method, in which energy is introduced to the system typically by either sonication or homogenization, and they were provided with submicron size (generally 0.1–10 μm). Then, conjugation of PLL to PLGA was achieved through the coupling agent at different percentages to create pDNA/ PLGA/PLL (poly-l-lysine) complex. This system achieved effective gene transport by acquiring cationic adjuvant property. [87]. In another study, it has been reported that the PLGA particle, which is condensed with the cationic lipid DOTAP, provides efficient pDNA encapsulation by forming a cationic adjuvant system [88].

#### *2.2.2.1.6 Polyethylene glycol (PEG)*

Polyethylene glycol (PEG) is a highly hydrophilic, non-immunogenic, semicrystalline, linear polyether diol used as a non-ionic polymer consisting of ethylene oxide monomers. PEG, a polymer approved by the Food and Drug Administration (FDA), is non-toxic at low density and does not damage active proteins or cells. PEG is excreted completely through the kidneys (<30 kDa PEGs) or stool (>20 kDa PEGs) [89]. In addition, functionalities by conjugation of different terminal groups such as amino, carboxyl and sulfhydryl groups can be increased. It is soluble in aqueous solutions and in most organic solvents like methanol and dichloromethane [89].

The physical properties of the PEG material vary with the molecular weight. With an increase in the molecular weight, viscosity of PEG increases, while the water solubility decreases. Furthermore, the high solubility of PEG in organic solvents provides a great advantage in preparing solid dispersions. PEG provides stability to coating particles. The flexibility of the polymer chain, which allows the polymer units to rotate freely, ensures that the PEG protects the particles. Thanks to its high hydrophilic property, it creates a protective shield around the particulate. Nowadays, PEG is used not only to increase stability and circulation time of particles in vivo, but also to target particles to the desired areas [90].

**139**

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

In vivo transfection experiments with DC-Chol/DOPE liposomes with 1% PEG coating showed that the PEG coating increases the stability and longevity of the adjuvant, while it decreases the pH sensitivity and thus decreases the transfection rate. This pH sensitivity is important for the vaccination strategies carried out in the treatment of an existing tumor tissue [55]. However, there is no indirect disadvantage of PEG coating on the transfection rate, as there is no intention to improve the present tumor tissue, but there is no need for pH sensitivities for adjuvants in vaccine studies designed to protect against tumor formation [23, 54, 56, 61].

The pHEMA [poly(2-hydroxyethyl methacrylate)] polymer is the polymerized non-toxic form of HEMA (hydroxyethyl methacrylate) which is a toxic monomer. Hydrogels are hydrophilic and are capable of holding water up to thousands of times more than their own dry mass. For this purpose, pHEMA that is virtually uncharged is a three-dimensional hydrophobic polymer that can swell in water or biological fluids, and it can be used with a large number of pathways [91, 92]. Because of its high water content such as those in body cells, it is used in ureters, cardiovascular implants, contact lenses, tissue restorative surgical materials and many dental applications [93, 94]. pHEMA is also used in the pharmaceutical industry and in tissue engineering because of its biocompatibility and similar physical properties as living tissues [93]. In addition, the pHEMA polymer has been developed by virtue of its high biocompatibility properties and successful complexes formed by a wide variety of cationic compounds. It is also used in DNA purification, RNA adsorption and drug and enzyme transport [91, 95, 96]. These approaches shed light on the creation of new adjuvant systems for the transport of genetic material using pHEMA [91, 93, 97]. In our previous studies, we purposed to develop new pHEMA-based adjuvant systems to increase the immune effectiveness and protectivity of the DNA vaccine. Within this scope, cationic pHEMA-His/PEG, pHEMA-Chitosan/PEG, pHEMA-PEI/ PEG and pHEMA-DOTAP/PEG particles were developed. As a result, all pHEMAbased adjuvant systems, which can be produced in nano-sizes and in the desired properties, have been shown to increase in vitro transfection efficiency compared to naked DNA by using them in different pDNA/adjuvant formulation ratios. When compared to Lipofectamine 2000 agent, pHEMA-PEI and pHEMA-DOTAP adjuvant

formulations are promising candidates for gene transfection agents [98].

Advances in adjuvant systems have led to the development of biodegradable, environmentally responsive and biocompatible vaccine carriers (e.g., droplet-based microfluidic devices). An ideal adjuvant system should effectively interact with both the pDNA and cellular membrane and should not elicit an immune response or cytotoxicity. Characterization studies of pDNA vaccine-loaded delivery systems are carried out by size and zeta potential measurements, transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), gel retardation assay with agarose gel electrophoresis, PicoGreen assays,

Cellular uptake adjuvant/nucleic acid formulations mainly depend on type, size, shape as well as composition, surface chemistry and/or the carrier charge. These are key factors, which affect carrier/cell interactions and the transfection

*2.2.2.2 Characterization of adjuvant systems*

robustness assays and FT-IR [26, 31, 99–102].

**2.3 Cellular uptake of delivery systems**

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

*2.2.2.1.7 Poly(2-hydroxyethyl methacrylate) (pHEMA)*

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

In vivo transfection experiments with DC-Chol/DOPE liposomes with 1% PEG coating showed that the PEG coating increases the stability and longevity of the adjuvant, while it decreases the pH sensitivity and thus decreases the transfection rate. This pH sensitivity is important for the vaccination strategies carried out in the treatment of an existing tumor tissue [55]. However, there is no indirect disadvantage of PEG coating on the transfection rate, as there is no intention to improve the present tumor tissue, but there is no need for pH sensitivities for adjuvants in vaccine studies designed to protect against tumor formation [23, 54, 56, 61].

## *2.2.2.1.7 Poly(2-hydroxyethyl methacrylate) (pHEMA)*

*Immune Response Activation and Immunomodulation*

*2.2.2.1.5 Poly(lactic-co-glycolic acid) (PLGA)*

sion method without shear stress [48, 81, 85].

*2.2.2.1.6 Polyethylene glycol (PEG)*

PLGA is a solid polymeric material approved by the Food and Drug Administration (FDA) for nanoparticle-based drug and gene delivery systems. Their biocompatibility, biodegradability, reliability and high stability characteristics during storage provide advantages in delivery systems [20, 80–82]. PLGA nanoparticles can easily pass through vessels in vivo without damaging the tissues surrounding the tumor and thus accumulate with the mechanism of "enhanced permeability retention" (EPR) in solid tumors. However, PLGA particles are less efficient in encapsulating nucleic acids because hydrophobic properties of PLGA are not compatible with anionic, hydrophilic properties of nucleic acids. In addition, difficult preparation conditions and pH decreases during PLGA hydrolysis inactivate nucleic acid loading and prevent polyplex formation. In order to overcome these drawbacks, effective gene transfer systems can be formed with different formulations made with different molecular interactions [21, 48, 52, 61, 83–86].

For example, PLGA nanoparticles can be processed with materials such as PEI to increase positive charge distribution and provide a stronger penetration of nucleic acid. After penetration of the nucleic acids, PEI, PEG cross-linking material and cell penetration peptides can be used for effective encapsulation and stabilization of the nano-carrier system [48, 81, 85]. It is also one of the systems to produce PLGAbased adjuvants in sizes of 200–300 nm by such means as cationic hydrophilic properties by condensing PLGA with cationic polymers such as polyethylenoxide (PEO) and polyethylene glycol methacrylate (PEGMA), emulsifying solvent diffu-

In one study, DNA-loaded PLGA particles were fabricated by a double emulsion water in oil in water (w/o/w) method, in which energy is introduced to the system typically by either sonication or homogenization, and they were provided with submicron size (generally 0.1–10 μm). Then, conjugation of PLL to PLGA was achieved through the coupling agent at different percentages to create pDNA/ PLGA/PLL (poly-l-lysine) complex. This system achieved effective gene transport by acquiring cationic adjuvant property. [87]. In another study, it has been reported that the PLGA particle, which is condensed with the cationic lipid DOTAP, provides

efficient pDNA encapsulation by forming a cationic adjuvant system [88].

ticles in vivo, but also to target particles to the desired areas [90].

Polyethylene glycol (PEG) is a highly hydrophilic, non-immunogenic, semicrystalline, linear polyether diol used as a non-ionic polymer consisting of ethylene oxide monomers. PEG, a polymer approved by the Food and Drug Administration (FDA), is non-toxic at low density and does not damage active proteins or cells. PEG is excreted completely through the kidneys (<30 kDa PEGs) or stool (>20 kDa PEGs) [89]. In addition, functionalities by conjugation of different terminal groups such as amino, carboxyl and sulfhydryl groups can be increased. It is soluble in aqueous solutions and in most organic solvents like methanol and dichloromethane [89]. The physical properties of the PEG material vary with the molecular weight. With an increase in the molecular weight, viscosity of PEG increases, while the water solubility decreases. Furthermore, the high solubility of PEG in organic solvents provides a great advantage in preparing solid dispersions. PEG provides stability to coating particles. The flexibility of the polymer chain, which allows the polymer units to rotate freely, ensures that the PEG protects the particles. Thanks to its high hydrophilic property, it creates a protective shield around the particulate. Nowadays, PEG is used not only to increase stability and circulation time of par-

**138**

The pHEMA [poly(2-hydroxyethyl methacrylate)] polymer is the polymerized non-toxic form of HEMA (hydroxyethyl methacrylate) which is a toxic monomer. Hydrogels are hydrophilic and are capable of holding water up to thousands of times more than their own dry mass. For this purpose, pHEMA that is virtually uncharged is a three-dimensional hydrophobic polymer that can swell in water or biological fluids, and it can be used with a large number of pathways [91, 92]. Because of its high water content such as those in body cells, it is used in ureters, cardiovascular implants, contact lenses, tissue restorative surgical materials and many dental applications [93, 94]. pHEMA is also used in the pharmaceutical industry and in tissue engineering because of its biocompatibility and similar physical properties as living tissues [93]. In addition, the pHEMA polymer has been developed by virtue of its high biocompatibility properties and successful complexes formed by a wide variety of cationic compounds. It is also used in DNA purification, RNA adsorption and drug and enzyme transport [91, 95, 96]. These approaches shed light on the creation of new adjuvant systems for the transport of genetic material using pHEMA [91, 93, 97].

In our previous studies, we purposed to develop new pHEMA-based adjuvant systems to increase the immune effectiveness and protectivity of the DNA vaccine. Within this scope, cationic pHEMA-His/PEG, pHEMA-Chitosan/PEG, pHEMA-PEI/ PEG and pHEMA-DOTAP/PEG particles were developed. As a result, all pHEMAbased adjuvant systems, which can be produced in nano-sizes and in the desired properties, have been shown to increase in vitro transfection efficiency compared to naked DNA by using them in different pDNA/adjuvant formulation ratios. When compared to Lipofectamine 2000 agent, pHEMA-PEI and pHEMA-DOTAP adjuvant formulations are promising candidates for gene transfection agents [98].

#### *2.2.2.2 Characterization of adjuvant systems*

Advances in adjuvant systems have led to the development of biodegradable, environmentally responsive and biocompatible vaccine carriers (e.g., droplet-based microfluidic devices). An ideal adjuvant system should effectively interact with both the pDNA and cellular membrane and should not elicit an immune response or cytotoxicity. Characterization studies of pDNA vaccine-loaded delivery systems are carried out by size and zeta potential measurements, transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), gel retardation assay with agarose gel electrophoresis, PicoGreen assays, robustness assays and FT-IR [26, 31, 99–102].

#### **2.3 Cellular uptake of delivery systems**

Cellular uptake adjuvant/nucleic acid formulations mainly depend on type, size, shape as well as composition, surface chemistry and/or the carrier charge. These are key factors, which affect carrier/cell interactions and the transfection efficiency. Cellular uptake of nucleic acid-loaded delivery systems and their localization in 2D (monolayer culture) and 3D (multicellular tumor spheroids) in vitro cell culture models and also in vivo models are studied by multi-labeling 3D confocal fluorescence microscopy, flow cytometry, overlaid bright field fluorescence microscopy based on GFP expressions, luciferase assays and fluorescence images [26, 27, 100–102].
