Keywords

14.1 Introduction

Targeted gene delivery is the way of delivering genes to cells, tissues, and organs through local or systemic blood circulation. This enables the interaction of genes directly on the sites of the intended diseases and produces therapeutic benefits. By enhancing therapeutic molecular activity at certain regions while lowering toxic side effects at normal sites, this selective delivery keeps the systemic effect to a lower level. This type of delivery is used in the therapy known as gene therapy (Zhou et al. 2017; Han et al. 2016; Kuang et al. 2017; Liu et al. 2016; Wang et al. 2016; Chen et al. 2017; Saha et al. 2017; Kemp et al. 2016). Gene therapy has earned considerable interest over the last 20 years as a favorable future treatment of choice for major diseases like cancer, AIDS, cardiovascular or neuronal disorders, as well as hereditary single gene abnormalities. For gene therapy to be effective, the therapeutic gene must be delivered to the infected cells of the patient (Mulligan 1993; Verma et al. 2000; Wirth et al. 2013). By injecting a gene into a patient’s cell, gene therapy may allow medical practitioners to treat disorders without the use of medications or surgery. Numerous techniques of therapy are being investigated by some scientists and medical professionals, including (1) substituting a disease-causing mutated gene with a healthy gene, (2) “knocking out” or inactivating a mutated gene, and (3) inserting new genes into the cells to help defend against the ailments. The interaction between the gene delivery system and the target cell must be thoroughly understood to develop an efficient gene delivery system. A plasmid-based gene expression system that controls a gene’s activity within the target cell, a gene that encodes a particular therapeutic protein, and a gene delivery system that regulates the transfer of the gene expression plasmid to a particular site inside the body include the three elements that form the gene delivery systems (Han et al. 2000; Mahato et al. 1999) (Fig. 14.1).

Fig. 14.1
A flowchart of the steps involved in gene therapy. It includes the insertion of a gene of interest into a vector, injection of the vector-containing gene into tissue of interest, and release of genetic material to cause gene expression, mutant gene inactivation, and mutant gene correction.

Schematic representation of gene therapy

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Since the first human gene therapy study was launched in 1990 (Blaese et al. 1995; Basarkar and Singh 2007), there has been massive interest in delivery materials for better gene delivery. The availability of reliable and secure carrier for the delivery of genes plays a vital role in gene delivery systems.

14.2 Gene Delivery Materials

The materials used for gene delivery are categorized into three types:

A flowchart exhibits the materials used for gene delivery. They are viral vectors, non-viral vectors, and physical method.

14.2.1 Viral Vectors

These are the vectors in which viruses act as delivery systems that deliver genes to the target cells. Depending on their capacity to inject their DNA into host cells, these vectors deliver genes. These capitalize on a virus’s capacity to replicate its own genetic material. Because the shape of the virus prevents DNA liposome breakdown, hence viruses are an excellent method for delivering genes (Kamimura et al. 2011; Van Nies et al. 2018; Singh et al. 2017). Viral-based vectors were first developed as a transgene expression technique in the 1980s. In order to protect chimpanzees against hepatitis B, the vaccinia virus was utilized as a vaccine vector in 1984 (Moss et al. 1984). Seventy percent of all gene therapy clinical trials conducted globally as of 2015 involved viral vectors for gene delivery (Ako-Adounvo et al. 2017). The replication gene of viral vectors used for the delivery of genes is removed during genetic engineering and replaced with the therapeutic gene (gene of interest) (Thomas et al. 2003). This approach retained the virus’s capacity to infect host cells (Basarkar and Singh 2007).

The main characteristics to be considered for choosing a viral vector are as follows:

  1. 1.

    Vector should be safe to be handled.

  2. 2.

    Vector should show less toxic effects in infected cells.

  3. 3.

    Vector should be stable enough to ensure the repeatability of the work.

  4. 4.

    Vector should be capable of being altered for cell-type specificity.

  5. 5.

    Vector should be able to integrate marker genes for facilitating identification.

  6. 6.

    Vector should be capable of carrying significant foreign genes.

  7. 7.

    Vector should be effective in the transducing and transfecting process (Ako-Adounvo et al. 2017).

Viral vectors can be classified into three categories.

A flowchart of the classification of viral vectors. The viral vectors are categorized into D N A and R N A-based viral vectors for gene delivery, and oncolytic viral vectors based gene delivery.

14.2.1.1 DNA-Based Viral Vectors for Gene Delivery

DNA-based viral vectors that deliver genes generally last longer and are incorporated into the genes. Viral vectors are used in DNA-based gene delivery systems to transfer genetic material to the host cells. The genetic components are effectively delivered to the host cell by the viral vectors (Wivel and Wilson 1998). Plasmids delivering transgenes for gene therapy are among the viral vectors based on the DNA for the delivery of genes (Crooke 1998). The group of materials known as DNA-based viral vectors has evolved as a promising option for gene carriers for gene therapy in a variety of diseases, including cardiovascular disorders, neurological diseases like Parkinson’s and Alzheimer’s disease, AIDS, and cancer (Stull and Szoka 1995; Patil et al. 2005a).

Some of the main viral vectors based on the DNA used for delivery of genes are as follows:

14.2.1.1.1 Adenovirus

In 1953, human adenoid tissue cultures yielded adenoviruses, which are linear double-stranded and non-enveloped DNA viruses (Rowe et al. 1953; Campos and Barry 2007; Majhen and Ambriović-Ristov 2006) (Fig. 14.2). Adenoviruses can be genetically modified to have a very large capacity for the introduction of transgenes due to their large viral genomes (36–38 kb). The high-capacity “gutless” Helper-dependent adenovirus, for instance, may deliver 37 kb of the transgene (Kamimura et al. 2011; Volpers and Kochanek 2004). Although adenoviral vector systems have a high transgenic capacity, the host immunological response and subsequent transitory expression of gene severely restrict their usage as delivery systems (Kamimura et al. 2011).

Fig. 14.2
A schematic representation of the icosahedral adenovirus cell. It consists of a hexagonal outer covering made of capsid proteins. Filaments of fiber protein extend outward from the capsid. The capsid encloses a D N A genome, which is the genetic material of the organism.

Structure of adenovirus

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14.2.1.1.2 Poxvirus

Poxvirus is a desirable option for immune-based cancer treatment because of the absence of viral integration into the host cellular genome, the maximum size (25 kb) of the gene insert, and it elicits high immune activation. One of the main examples of poxvirus is the vaccinia virus.

14.2.1.1.3 Vaccinia Virus

All cells are infected by the vaccinia virus, but even after multiple injections, the tumor immune response is not completely suppressed by the host immunological response to the vector (Fig. 14.3). The administration of vaccinia in immunocompromised cancer patients is made possible by the availability of attenuated viruses, and the data suggests that this carrier improves tumor immune rejection. The major adverse effects were moderate flulike symptoms that lasted for 1–2 days and localized skin rashes/irritation at the injection site that typically lasted 4–5 days. The immune response at the cellular level and the clinical response were not correlated (Gardlík et al. 2005; Ayllón Barbellido et al. 2008).

Fig. 14.3
A schematic representation of vaccinia virus structure. The organism consists of an outer and an inner membrane, that encloses the core. The core includes viral enzymes and the D N A genome.

Structure of vaccinia virus

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14.2.1.1.4 Adeno-Associated Virus

These viruses are having properties like targeting non-proliferating cells, having discrete genome insertion sites, exhibiting little immunogenicity, and having no known toxicity (Fig. 14.4). Using an AAV vector, “suicide” gene therapy was demonstrated to be effective in oral cancer cells. Additionally, antisense or ribozyme genes have been effectively transferred using AAV vectors in preclinical models of cancer. It has been shown that AAV vectors can successfully transduce CD34+ blood cells, the brain, and the liver.

Fig. 14.4
A schematic representation of the adenovirus. The organism consists of an icosahedral structure that encloses the D N A genome.

Structure of adeno-associated virus

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There are numerous limitations in using the adeno-associated virus as a viral vector.

  1. 1.

    Some cells need to be infected at very high multiplicities.

  2. 2.

    The AAV genome is compact, with only space for an additional 4.8 kb of DNA.

  3. 3.

    Since effective packing cells have not yet been devised, the manufacturing of viral particles is still extremely labor-intensive (Gardlík et al. 2005; Kay et al. 1997; Biçeroğlu and Memiş 2005; Mulherkar 2001; Xi and Grandis 2003; Zhou et al. 2004).

14.2.1.1.5 Herpes Simplex Virus

It is a virus that is large in size with a broad range of actions that show continued gene expression from protracted infection (Fig. 14.5). The HSV poses slight chances of insertional mutagenesis since it stays outside the nucleus (episomal). HSV type 1 (HSV-1) strains form the basis for the majority of herpes virus vectors. This double-stranded DNA virus has various unique characteristics, such as the capacity to stay dormant in tissues and to revive at an infection site. HSV-1 replicates once it has infected a cell, leading to cell and infection of neighboring cells. Furthermore, HSV-1 is a widespread human infection that infrequently results in serious illnesses. HSV vectors can quickly and effectively transmit genes while accommodating significant amounts of foreign DNA.

Fig. 14.5
A schematic representation of the herpes virus. The organism consists of an oval outer covering with spikes of glycoprotein. The outer covering encloses a hexagonal capsid that consists of the D N A genome. The space between the outermost layer and the capsid is the tegument.

Structure of herpes simplex virus

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Herpes simplex virus has some limitations, such as the following:

  1. 1.

    Poor transfection efficiency.

  2. 2.

    Large genome size.

  3. 3.

    The function of HSV is confined due to its high affinity toward neuronal cells. However, some researchers are taking advantage of this limitation to target the neurons (Ayllón Barbellido et al. 2008; Gardlik et al. 2011; Roman et al. 2011).

14.2.1.2 RNA-Based Viral Vectors for Gene Delivery

Infectious RNA transcripts can now be directly translated by RNA-based viral vectors for the delivery of genes. Gene delivery with RNA is typically temporary and not permanent. Oncoretro-viral vectors, human foamy virus, and lentiviral vectors are RNA-based viral vectors for delivery of genes used in the treatment of genetic diseases. The advanced system provides negative-strand RNA templates with RNA-dependent polymerase complexes (Mogler and Kamrud 2015). Patients who received transplantation for AIDS-related lymphoma have used lentiviral vector-altered CD34(+) cells as RNA-based gene delivery methods for HIV (DiGiusto et al. 2010).

14.2.1.2.1 Retrovirus

One of the main RNA-based viral vectors for the delivery of genes is a retrovirus. Retroviruses are RNA-based viruses that are single-stranded that have genomes that can accommodate transgenes up to 10 kb in size (Barquinero et al. 2004; Daniel and Smith 2008) and can contain genes up to 7–11 kb in size (Kamimura et al. 2011; Barquinero et al. 2004). The nuclear pores of proliferating cells do not act as barriers to retroviral vectors, which are very successful in dividing cells (Nayerossadat et al. 2012; Bushman 2007). Additionally, retroviruses are highly useful for interventions that favor permanent gene transfer (Anson 2004; Mancheño-Corvo and Martín-Duque 2006). However, in addition to their potential for pathogenicity and immunogenicity, retroviral vectors also carry the risk of mutation because of how efficiently they incorporate into the DNA of the host cell (Basarkar and Singh 2007; Anson 2004). Additionally, target cells may become randomly infected when retroviral vector systems are used (Yi et al. 2011).

The limitations of retrovirus are as follows:

  1. 1.

    Less vector titer

  2. 2.

    Lesser transfection efficiency exhibited in in vitro studies

  3. 3.

    Particle instability

  4. 4.

    Difficult to transduce nondividing postmitotic cells

It is important to remember that each viral vector system has specific advantages and disadvantages, requiring a distinct study to determine the applications for which each is most appropriate (Mancheño-Corvo and Martín-Duque 2006; Siemens et al. 2003).

14.2.1.3 Oncolytic Viral Vectors for Gene Delivery

These viruses are used as a novel type of treatment for diseases related to cancer. Lately, they have emphasized oncology in an attempt to improve the effectiveness of their treatment interventions (Howells et al. 2017). The benefits and drawbacks of the many modifications made to them to improve infectivity and therapeutic safety for the interaction of tumor cells and oncolytic viruses (OVs) have been discussed. Through the development of T cells expressing IL-18R or IL-12R2, oncolytic adenoviruses co-expressing IL-18 and IL-12 enhance tumor-specific immunity (Choi et al. 2011). The ultimate objective is to develop a virus that can successfully multiply inside the host, find a specific target, and kill cancerous cells. Adenovirus-induced decorin expression triggers p53 activation and mitochondrial apoptosis, which kill cancer cells (Yoon et al. 2017). With the support of gene therapy, oncolytic adenovirus vectors provide a promising treatment option for cancer (Choi et al. 2015). IFN- and TNF-producing T cells that express IL-23 and p35 are activated to provide antitumor immunity. According to studies, cytokine immune-gene therapy is one of the most effective treatments for cancer (Choi et al. 2013; Hernandez-Gea et al. 2013; El-Aneed 2004; Baban et al. 2010).

14.2.2 Nonviral Vectors

In substitution for typical viral-based vectors, the nonviral vector strategy was developed. Lipids, polymers, and peptides are nonviral vector approaches explored for gene delivery (Godbey and Mikos 2001; Zhi et al. 2013; Eliyahu et al. 2005).

A flowchart depicts the three approaches for non-viral vectors for gene delivery. They are lipid, polymer, and peptide-based gene delivery.

14.2.2.1 Lipid-Based Gene Delivery

Currently, the nonviral vector that has received the most emphasis is lipid-based gene delivery, which has shown promise for controlling cellular gene expression in both research and therapeutic contexts. They self-build with DNA having a negative charge to produce a cationic-charged complex, which leads to the production of a complex of plasmid DNA and cationic lipids (Wasungu and Hoekstra 2006). They are cationic at normal pH and are made up of a neutral lipid or cholesterol and cationic lipid (de Ilarduya et al. 2010). DNA is delivered into the cytoplasm when lipoplex comes in contact with cellular plasma membrane and internalize into the cell through endocytosis. This causes the lipoplex to become unstable. However, the delivery of other medicinal macromolecules has been the predominant use for anionic liposomes (Mayhew and Papajadjopoulos 1983).

They are classified into two types:

A flowchart exhibits the two types of lipid-based gene delivery. They are cationic lipid-based drug delivery and anionic lipid-based gene delivery.
14.2.2.1.1 Cationic Lipid-Based Gene Delivery

Researchers have been working on designing and examining cationic lipid formulations for effective gene transfer since 1983; [1,2-bis(oleoyloxy)-3-(trimethylammonio)propane] (DOTAP) (Leventis and Silvius 1990), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (Felgner et al. 1987), 3β[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) (Gao and Huang 1991), and dioctadecylamido glycylspermine (DOGS) (Behr et al. 1989) are the common agents for cationic lipids. To facilitate endolysosomal escape, cationic lipids are typically combined with the neutral lipid dioleoylphosphatidylethanolamine (DOPE) (Farhood et al. 1995).

14.2.2.1.1.1 N-[1-(2, 3-Dioleyloxy)Propyl]-N,N,N-Trimethylammonium Chloride (DOTMA)

N-[1-(2, 3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, or DOTMA, was used as a lipofectin. DOTMA was combined in a 1:1 ratio with a neutral lipid called dioleoylphosphatidylethanolamine (DOPE) to enhance the transfection efficacy of lipofectin (Fig. 14.6). One of the early developed, extensively studied, and widely available cationic lipids for the delivery of genes was DOTMA. In an attempt to increase transfection efficiency and decrease toxicity, various research groups prepared modified DOTMA by altering its key functional components, such as linker, its head group, hydrocarbon chains, and linkage bonds (Vaheri and Pagano 1965; Ren et al. 2000). It was discovered that the cytotoxicity linked to the synthesized monovalent lipids depended on the density of the plated cells and the lipids’ structural properties (Vaheri and Pagano 1965).

Fig. 14.6
Chemical structure of DOTMA molecule. The molecule is ionic and consists of N plus and C l minus entities. The N atom is single-bonded to 2 methyl groups and 2 alkene chains with an ether substitution in each.

N-[1-(2, 3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (Vaidya et al. 2022)

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14.2.2.1.1.2 2,3-Dioleyloxy-N[2(Sperminecarbaxamido)Ethyl]-N,N-Dimethyl-1-Propaminium Trifluoroacetate (DOSPA)

Cationic lipid developed as a derivative of DOTMA is 2,3-dioleyloxy-N[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate, or DOSPA. DOSPA and DOTMA are almost similar in structure (Fig. 14.7). The main distinction between them is that DOSPA contains a spermine functional group that is linked to the hydrophobic chains by a peptide bond, enabling more effective DNA packing (Jain et al. 1989).

Fig. 14.7
Chemical structure of DOSPA molecule. It consists of a long chain with ammonium 4 N plus entities. The terminal N atom is single-bonded to a branched alkene chain.

2,3-Dioleyloxy-N[2(sperminecarbaxamido)ethyl]-N,N-dimethyl-1-propaminium trifluoroacetate (Li et al. 2015)

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14.2.2.1.1.3 N-[1-(2,3-Dioleyloxy)-Propyl]-N,N,N-Trimethylammonium Chloride (DOTAP)

N-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTAP) (Felgner et al. 1987) is the most well-researched lipid which has been used to in vivo genetically modify a number of animal organs (Zhu et al. 1993) (Fig. 14.8). The key distinction among DOTMA and DOTAP is that DOTAP has ester bonds instead of ether bonds connecting the backbones, which can be hydrolyzed to help break down lipids and lessen toxicity. Using a cationic lipid complex containing cholesterol and DOTAP, high tumor selectivity can be achieved as per the experimental studies. When compared to liver tissue, this approach yields a minimum of ten times more expression for every milligram of tumor tissue (Wang et al. 2013).

Fig. 14.8
Chemical structure of DOTAP molecule. The molecule is ionic, with the N plus moiety containing an organic part that is electrostatically bonded to the C l minus ion. The N plus is single-bonded to an ether-substituted alkene chain that has 2 C ketone groups.

N-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTAP)

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14.2.2.1.1.4 3[N-(N′,N′-Dimethylaminoethane)-Carbamoyl]Cholesterol (DC-Chol)

3[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol was discovered in 1991 (Gao and Huang 1991) (Fig. 14.9). Unlike DOTAP and DOTMA, DC-Chol has a tertiary amine group that may assist to prevent lipoplex aggregation and promote increased expression of transgene (Ajmani and Hughes 1999).

Fig. 14.9
Chemical structure of D C Chol molecule. It is a cation with a positive charge present on the N atom. The molecule also consists of 3 six-membered rings, one five-membered ring, and a hydrocarbon tail.

3[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) (Vaidya et al. 2022)

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14.2.2.1.1.5 Di-Octadecyl-Amido-Glycyl-Spermine (DOGS)

DOGS, also known as di-octadecyl-amido-glycyl-spermine, is marketed as transfectam and has similar characteristics to DOSPA (Fig. 14.10). Both DOGS and DOSPA have 2 18-carbon alkyl chains in addition to a spermine group. These two are different from one another since DOSPA contains a quaternary amine. Contrarily, DOGS lacks quaternary amines; instead, it has saturated chains and is connected to the head group by a peptide linkage. Numerous cell lines have been transfected using DOGS. The delivery of the chloramphenicol acetyltransferase (CAT) reporter plasmid without any cytotoxic effects was demonstrated using DOGS (Behr et al. 1989).

Fig. 14.10
Chemical structure of D O G S molecule. The molecule consists of 2 N H 2 plus and 2 N H 3 plus ions, along with 2 other N atoms. The terminal N atom is single-bonded to two 18-membered alkane chains.

Di-octadecyl-amido-glycyl-spermine (DOGS) (Zhi et al. 2018)

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14.2.2.1.2 Anionic Lipid-Based Gene Delivery

Plasmid DNA, anionic lipids, and cations constitute the components of anionic lipoplexes (Srinivasan and Burgess 2009). Gene delivery using anionic lipids is often not very desirable or effective. Phospholipids like phosphatidylglycerol, phosphatidylserine, and phosphatidic acid, which are naturally present in biological membranes, are often employed as anionic lipids in gene delivery. As a nonviral means of delivery, DNA encapsulation into anionic and neutral liposomes has also been investigated. Owing to electrostatic repulsive interactions that exist among an anionic head group of the lipids and the phosphate backbone of DNA, an anionic liposome cannot effectively attach to anionic DNA. To use anionic lipids for cell-specific targeting, DNA must be enclosed. However, the DNA matrix’s size and shape restrict the uses of its encapsulated form (Ledley 1995).

A few number of cell types, including hippocampal neurons and CHO cells, have been recorded for gene delivery using a variety of anionic liposomes (Ledley 1995; Patil et al. 2004, 2005b). Nevertheless, despite extremely positive and hopeful outcomes, our total understanding of anionic lipofection is still restricted due to numerous challenges, few of which will definitely test our scientific acumen. The absence of reproducibility is one of the primary causes. Furthermore, because of their systematic administration, it is linked to undesirable side effects from nonspecific immune system cytotoxicity that results in a variety of unfavorable consequences.

14.2.2.2 Polymer-Based Gene Delivery

In an attempt to overcome the immunogenic and carcinogenic issues related with viral vectors, carriers based on the polymer for the delivery of genes have been designed as their alternative. Due to its tremendous scope for the design of safe and reliable vectors, it has garnered a lot of interest over the past two decades (Kang et al. 2012).

Researchers studying gene delivery methods have investigated the possible advantages of polymeric vehicles for genes in cationic biopolymers including chitosan derivatives and liposomes (Ginn et al. 2018; Hulin-Curtis et al. 2016). Polymeric materials can conceal the DNA of negative charges and compress the big genes into smaller tiny molecules when used with cationic polymers for gene delivery. The polyplex is the complex of cationic polymer-based nucleic acid. One important targeting delivery system for gene therapy is the gene complex. The majority of studies concentrate on the impact of ligands that are attached to the DNA complex by covalent bonding. The targeted ligands can combine with the cationic polymers. Poly(l-lactide) is a polymer that can be a potential alternative that is frequently used to link the targeted ligands (Stone 2010; Manno et al. 2006; Kabanov and Kabanov 1995; Buwalda et al. 2012). Under a wide range of circumstances, compaction occurs during cationic polymer condensation of plasmid DNA. Condensing agents mainly utilized are multivalent cationic polymers (Bloomfield 1991, 1996).

One of the main classes of cationic polymers used as condensing agent is polyamidoamine (PAMAM) dendrimer.

Polyamidoamine Dendrimers (PAMAM Dendrimers)

A group of highly branched cationic polymers known as polyamidoamine dendrimers may condense DNA and deliver it to a number of cell types with minimal cytotoxic effects (Kukowska-Latallo et al. 1996). These are circular polymers that are highly branched and are frequently used in transfer of genes using nonviral vectors.

The most widely utilized dendrimers for the delivery of genes using nonviral vectors are the 6-generation Starburst™ PAMAM dendrimers, either in fragmented or intact form. The first polyamidoamine (PAMAM) dendrimers were developed by Tomalia et al. (1985) (Fig. 14.11). The process used to form this type of starburst dendrimer involves adding methyl acrylate by Michael addition to an original core (such as ethylenediamine or ammonia) that has a number of branching points at its center. Next, the resulting esters are amidated with ethylenediamine. Later investigations made use of PAMAM-OH dendrimers with hydroxyl ends (Fig. 14.11b). PAMAMOH that had been neutralized on the surface showed advantages, including less toxicity and lower transfection efficiency (Lee et al. 2003). l-Arginine was used to modify these dendrimers by forming degradable ester linkages (Fig. 14.11c). In fact, the l-arginine ester-grafted PAMAM-OH G4 had a higher transfection and reduced toxic effects than the earlier l-arginine amide-grafted PAMAM G4 (Nam et al. 2009).

Fig. 14.11
3 chemical structures. 1. Chemical structure of G 3 P A M A M. It has a branching structure made of amide groups. 2. Structure of hydroxyl-terminated P A M A M O H. 3. Structure of P A M A M O H modified by L-arginine ester.

Polyamidoamine dendrimers, (a) G3 PAMAM dendrimer (Tomalia et al. 1985). (b) Hydroxyl-terminated PAMAM-OH dendrimer (Lee et al. 2003). (c) l-Arginine ester-modified PAMAM-OH dendrimer (Nam et al. 2009)

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The primary benefit of the fractured dendrimer’s structural design is the presence of highly dense amine at the molecule’s periphery, which facilitates effective condensation of nucleic acids while releasing up the amino group present inside for a proton sponge throughout endolysosomal acidification, facilitating most effective endosomal escape. In comparison with the intact polymer, these dendrimers exhibit much increased (>50-fold) levels of expression of reporter gene. This finding’s cause is unknown, although it is possible that one of the key causes is that the polymer’s enhanced flexibility and improved capacity for complexing with DNA serve to promote gene expression (Tang et al. 1996). Researchers have looked at the tertiary structures that resemble noncondensing plasmid DNA in complexes with less hydrophobized stearyl-poly(l-lactide) (Kim et al. 1998a). The process of adding hydrophilic components, such as or hydrophobic stearyl chains, dextran, polyethylene glycol, hydrophobic stearyl chains, or hyaluronic acid, has however been facilitated by DNA condensation (Toncheva et al. 1998; Maruyama et al. 1997a; Katayose and Kataoka 1997; Kim et al. 1997).

Polymer-based gene delivery has been divided into three categories:

A flowchart presents the three types of polymer-based gene delivery. They are gene delivery based on polysaccharides, polyethyleneimine, and poly, L-lysine.
14.2.2.2.1 Polysaccharide-Based Gene Delivery Systems

On the basis of grafted oligoamine residues for natural polysaccharides, a range of biodegradable cationic polymers were identified for the delivery of genes (Azzam et al. 2002). The grafting concept enables 3D contact with anionic surfaces of double-stranded DNA chains by attaching side chain oligomers to hydrophilic polysaccharides, which are branched or linear in nature. The polysaccharide-based gene delivery technology suggests that the cationic polysaccharide’s structure has a key impact in the transfection activity for delivery of genes. The delivery of compounds such as plasmids and oligonucleotides through mucosa had been explored using colloidal polysaccharide particles (Janes et al. 2001). Nanoparticles made of natural polysaccharides have also been investigated as methods of delivering drugs and genes (Liu et al. 2008). The mechanism for manufacturing polysaccharide-based nanoparticles included electrolyte complexing, ionic and covalent cross-linking, and hydrophobic polysaccharide self-assembly. Natural polysaccharides like chitosan have been evaluated as vehicles for drugs or genes (Morris et al. 2010).

14.2.2.2.1.1 Chitosan

It is a biodegradable polysaccharide that is linear in nature comprising of N-acetyl-d-glucosamine and b-1,4-linked d-glucosamine residues. Owing to its non-allergenicity, biodegradability, biocompatibility, mucoadhesive property, and great binding with DNA, it is one of the highly reported naturally produced cationic gene polymers used in nonviral gene transfer. In a series of experiments using both experimental animals and humans, chitosan displayed little cytotoxicity and increased transfection effectiveness (Rao and Sharma 1997; Aspden et al. 1997).

14.2.2.2.2 Gene Delivery Systems Based on Polyethyleneimine

In most cases, polyamines that turn cationic under physiologic conditions are the cationic polymers used for gene complexation. Due to their great complex stability, polyethyleneimines (PEIs), which were initially proposed by Boussif et al. (1995), are one of the most extensively researched and regarded as the reference of nonviral vectors for gene delivery. Compared to other polycations like PLL, PEIs provide a transfection that is significantly more effective and resistant to nuclease degradation. It might be because PEIs form more compact and effective complexations and have higher charge densities. Amino groups present in the PEIS have the ability to increase their ability to act as buffers. These result in the rupture of lysosome and then permeate into the cytoplasm through the effect of the proton sponge (Benjaminsen et al. 2013; Luu et al. 2012). The other functional molecules can receive some buffering capacity enhancement from PEI. PEI conjugated poly(cystamine bis(acrylamide)-diaminohexane) [poly(CBA-DAH)] was designed to improve transfection efficiency and reduce weight ratio. Multiple disulfides constitute poly(CBA-DAH), which are broken down in the cytoplasm by an intracellular reducing agent like glutathione (GSH) (Doss et al. 2013; Hong et al. 2006; Oupický and Li 2014; Wen et al. 2011). The triple peptide that constitutes GSH was formed in the cytosol from precursor amino acids (Chakravarthi et al. 2006).

The improved buffering ability of other compounds including imidazole, histidine, and PEI was also demonstrated in other investigations (Bello Roufaï and Midoux 2001; Pack et al. 2000; Pires et al. 2011; Yang et al. 2008; Zhang et al. 2015). Additionally, water-soluble, branching poly(ethylenimine)-cholesterol lipo-polymers for gene transport were produced (Wang et al. 2002). Gene delivery uses altered linear polyethylenimine-cholesterol conjugates for DNA complexation (Furgeson et al. 2003). A gene delivery approach using polyethylenimine-cholesterol/DNA complexes which are linear in nature had been investigated for tumor effectiveness and biodistribution (Furgeson et al. 2004). A biodegradable gene carrier made of polyethyleneimine with acid-labile links had been developed and evaluated (Kim et al. 2005). Gene delivery technologies consisting of reducible poly(amidoethylenimine)s have been developed and evaluated (Christensen et al. 2006, 2007; Jeong et al. 2007, 2010).

14.2.2.2.3 Poly(l-Lysine)-Based Gene Delivery Systems

The main cationic polymer used for the transfer of genes is poly(l-lysine) (PLL). PLL falls under the category of cationic-charged polymers at neutral pH. A hydrophilic cationic amino group is mainly found in PLL. PLL has the capacity to join with DNA to build a polyelectrolyte complex. In physiologic conditions, PLL (pKa 10.5) is protonated, which ionically interacts with DNA’s negatively charged phosphate groups and builds a polyelectrolyte complex of nanoparticulate range (Laemmli 1975). To facilitate the co-adsorption of plasmid DNA, PLL-based replica particles are cross-linked using a homo-bifunctional linker have been developed for the delivery of genes (Zhang et al. 2010). For the in vivo delivery of genes to the liver, poly-l-lysine/DNA polyplexes have been stabilized (Kwoh et al. 1999). There have been reports on the development and evaluation of poly-l-lysine-based carriers for the delivery of genes (Choi et al. 2000). Modified poly(l-lysine) such as N-terminal modified poly(l-lysine) antibody conjugate is utilized as a vehicle for delivering specific genes in endothelial cells present in the lungs of the mouse (Trubetskoy et al. 1992). Terplex DNA delivery systems and DNA nanoparticle carriers with grafted poly(l-lysine) polysaccharide copolymers were initially developed and recognized as a carrier of genes (Kim et al. 1998a; Maruyama et al. 1997b). For gene delivery systems, terplex delivery systems and polyethylene glycol grafted poly(l-lysine) were also designed (Kim et al. 1998b; Choi et al. 1998a,b, 1999). In the area of biomedical applications, clinical evaluations are now being conducted (Park et al. 2006; Meel et al. 2016; Bodles-Brakhop et al. 2009; Pulkkanen and Yla-Herttuala 2005; Rainov 2000; Young et al. 2006; Yoshida et al. 2004; Weichselbaum et al. 2002; Amer 2014; Jayant et al. 2016; Nam et al. 2015).

14.2.2.3 Peptide-Based Gene Delivery

Genomic carriers based on peptides provide a number of advantageous properties. With the aid of the naturally occurring amino acids ornithine, arginine, and lysine, which provide positive charges, electrostatic interaction can condense the pDNA (Plank et al. 1999) and perform a variety of other functions, including endosomal escape and receptor-targeted delivery. Oligolysine peptides are a substitute for heterogeneous polylysine peptides that include lysines of a fixed length (l-lysine). It is also possible to carry out site-specific adjustments because of the specific structure. According to studies, pDNA can be compacted by oligolysine molecules with 13 or more lysine monomers (Wadhwa et al. 1997), and a peptide with 18 lysines can form stable polyplexes with pDNA that are guarded against breakdown (Adami et al. 1998).

Cross-linking has been shown to significantly enhance the stability of DNA complexes. In order to establish bioreversible disulfide linkages through oxidation, cysteine has been added to the peptide sequence. They studied modifications of the Trp-Lys20 peptide by replacing one to four of the lysines with cysteines. The best transfection efficiency was demonstrated by a peptide containing two terminal cysteines (McKenzie et al. 2000a). Similar transfection effectiveness was attained using reduced lysine chains that contained only two terminal cysteines and four lysines (McKenzie et al. 2000b).

Fusogenic peptides, such as amphipathic peptides with large amounts of basic amino acids, like Tat (Fawell et al. 1994), melittin (Boeckle et al. 2006; Chen et al. 2006), KALA (Wyman et al. 1997), and others (Lehto et al. 2010; El Andaloussi et al. 2011), were used to mediate endosomal escape as an alternate choice to the mechanism of proton sponge (Zorko and Langel 2005). These peptides can damage endosomes by interacting with their lipid membrane. Numerous polymers, cationic peptides, or other gene delivery vehicles were combined with fusogenic peptides.

14.2.3 Physical Method of Gene Delivery

The physical method of transferring genes is not vector-mediated and does not require carriers.

It involves various methods such as electroporation, sonoporation, photoporation, magneto-fection, needle injection, hydroporation, and gene guns.

  • Electroporation: This method involves the use of electromagnetic pulse, which helps in the formation of pores in the membrane of the cell to enable the genetic materials into the cell.

  • Sonoporation: This technique involves the usage of sound waves for the creation of pores in the membrane of the cell for the movement of genetic materials into the cell.

  • Photoporation: The method of photoporation helps in the formation of pores in the cell membrane by using a laser pulse for entering the genetic materials into the cell.

  • Magneto-fection: It involves utilizing magnetic particles that have complexed with DNA and an external magnetic field to concentrate nucleic acid particles inside target cells.

  • Needle injection: This method involves the usage of needles to directly inject genetic materials into the cell.

  • Hydroporation: This is the technique that involves the use of the hydrodynamic capillary effect to alter the permeability of the cell membrane for the entry of genetic materials into the cell (Sung and Kim 2018).

14.3 Conclusion

In recent years, gene therapy has garnered significant interest and can be used as a promising treatment of choice for chronic conditions caused by genetic abnormalities. Gene therapy is a type of treatment in which genes are mainly used for the treatment of diseases. In gene therapy, components used for gene delivery are the essential components for the treatment of genetic diseases. The major aim of the research on gene delivery systems is to design proper vectors for the treatment of serious life-threatening diseases like cancer, Alzheimer’s, and AIDS. Several types of materials used in gene delivery systems are summarized and discussed here.

As all we know, viral vectors are playing a vital role in clinical trials of gene delivery such as adenovirus, herpes simplex virus, adeno-associated virus, retrovirus, etc. These vectors are efficient to carry the genome into target cells. On the other hand, nonviral vectors are also gaining attention in gene therapy. The materials utilized for nonviral vectors are lipids, peptides, and polymers. There are different categories of lipids such as anionic lipids and cationic lipids which are used for the delivery of genes. This lipid-based gene delivery acts as a promising approach to altering gene expression at a cellular level in therapeutic and research applications. Polymeric gene delivery systems have gained interest in basic research and clinical applications. These gene delivery systems have several merits such as cost-effectiveness, easy scale-up production, and easy modulation of DNA loading capacity. However, materials that are stable and monodisperse in size must be used for the effective delivery of genes. Hence the main advantages of gene delivery systems should be assessed carefully to reach the desired target by building an effective system with proper biodistribution to first-pass organs and rapid clearance of complexes for efficient gene delivery. In the future, our main research has to focus on DNA and RNA molecular techniques to become the main treatment in the biomedical field.