**2. Physiological barriers for siRNA**

The site of action of the SiRNA therapeutics is cytosol. The obstacles to siRNA administration vary depending on the targeted organs, disease, and routes of administration. In general, siRNA therapeutics are preferred as local or systemic injection. The local transport of siRNA has less obstruction compared to systemic administration. The first of the unique features of the local administration is the need to an easy formulation that requires easier production and application and secondly provides application of lower doses that limit intracellular (time-concentration-dependent) immune responses [14].

Therefore, the application is limited to easily accessible tissues such as the skin, eye, or mucosa, but successful results have been achieved. However, for the local administration of siRNA, the nervous system, lung, digestive, vagina, and inner coronary walls were selected as target regions [15].

For example, intranasal inhalation of the siRNA against the respiratory syncytial virus was carried out with TransIT-TKO commercial transfection agent and was found significantly effective in reducing viral infection [16].

In a recent study by Wu and colleagues, they developed novel PEGylated lipoplex-entrapped alginate scaffold system for vaginal epithelium-targeted siRNAs for the treatment of HIV virus infections. They indicated in the study the potential of the biodegradable PLAS system for the sustained delivery of siRNA/oligonucleotides to vaginal epithelium [17].

The main advantage of the systemic method is rapid action and biodistribution but also a wide range of applications. In addition, intravenous or intraperitoneal (IP) injection therapy is successfully applied for many diseases in the clinic. However, it causes significant difficulties for siRNA in systemic transmission. The transition steps of the siRNA from the application site to the target action site are shown in **Figure 1**.

As described above, after intravenous injection (1), siRNA is distributed to organs through the blood stream (3) and also undergoes elimination [7].

The siRNA molecules have unwanted physicochemical properties due to their negative charge, large molecular weight, size, and instability. After systemic administration, the naked siRNA administered without vectors is rapidly degraded by serum endonucleases (4), excreted by the kidney (7), or taken up by the macrophages (APC cells) (5) of the mononuclear phagocyte system (MPS) [7, 18].

**81**

**Figure 1.**

*Applications of Lipidic and Polymeric Nanoparticles for siRNA Delivery*

of the RNA backbone and by the use of nano-sized carriers (2) [7, 60].

particles could be very effective especially in in vivo experiments.

The result is a short plasma half-life below <10 minutes, so it cannot have enough time to reach the target and/or perform its functions, and therefore the gene silencing is ineffective. These problems are partially addressed by chemical modifications

Nanoparticle studies showed that the nanoparticles below 6 nm were rapidly excreted by renal excretion and nanoparticles in the range of 150–300 nm were taken by the cells of the MPS in the liver and spleen. It is stated that nanoparticles between 30 and 150 nm can hold in the bone marrow, kidney, etc. In addition, it has been reported that red blood cells (RBCs) affect the transport and distribution of microparticles in human microvasculature (diameter <100). In fact, due to collisions with RBCs, microparticles are pushed toward the artery wall where RBCs are virtually absent depending on the shape and size by a radial movement called the "waterfall effect." This event is also called margination, and they have stated that the extravasation cannot occur unless vectors are exposed to margination [14].

In a study done by D'Apolito et al. [20], they found that 3-μm particles had better marginalization than 1 μm. For this reason, it is seen that the size of the prepared

*An illustration of a nanocarrier, barriers at systemic circulatory, mechanisms of uptake of siRNA from cell (modified from the 26th literature). In the following text, the numbers in the figure are mentioned.*

*DOI: http://dx.doi.org/10.5772/intechopen.86920*

#### *Applications of Lipidic and Polymeric Nanoparticles for siRNA Delivery DOI: http://dx.doi.org/10.5772/intechopen.86920*

*Antisense Therapy*

polymeric nanoparticle.

**2. Physiological barriers for siRNA**

tration-dependent) immune responses [14].

otides to vaginal epithelium [17].

coronary walls were selected as target regions [15].

found significantly effective in reducing viral infection [16].

Taking advantage of these excellent properties of nanoparticles, various delivery systems for siRNA have recently used the clinical trial phase, and these methods are

As the latest development in this issues, the US Food and Drug Administration approved the Onpattro (patisiran) infusion on 10 August 2018 for the treatment of peripheral nerve disease (polyneuropathy) caused by hereditary transthyretin-mediated amyloidosis (hATTR) in adult patients. It is also the first FDA approval of a new class of drugs called small interfering ribonucleic acid (siRNA) treatment. FDA anticipates that this is the beginning of a new and exciting generation of therapeutics [11]. Patisiran is covered in a lipid nanoparticle (LNP) that carries the drug to the liver, but this carrier molecule can trigger its own immune response. Therefore, steroid, acetaminophen, and antihistamines should be used to reduce the likelihood of immune reaction in patients before taking patisiran administered by intravenous (IV) infusion. Further research is underway to design a drug delivery system that can reach the target tissue without causing an immune response. **Table 1** presents examples of clinical trials and current status of siRNAs prepared with lipidic and

The site of action of the SiRNA therapeutics is cytosol. The obstacles to siRNA

Therefore, the application is limited to easily accessible tissues such as the skin, eye, or mucosa, but successful results have been achieved. However, for the local administration of siRNA, the nervous system, lung, digestive, vagina, and inner

For example, intranasal inhalation of the siRNA against the respiratory syncytial

virus was carried out with TransIT-TKO commercial transfection agent and was

In a recent study by Wu and colleagues, they developed novel PEGylated lipoplex-entrapped alginate scaffold system for vaginal epithelium-targeted siRNAs for the treatment of HIV virus infections. They indicated in the study the potential of the biodegradable PLAS system for the sustained delivery of siRNA/oligonucle-

The main advantage of the systemic method is rapid action and biodistribution but also a wide range of applications. In addition, intravenous or intraperitoneal (IP) injection therapy is successfully applied for many diseases in the clinic. However, it causes significant difficulties for siRNA in systemic transmission. The transition steps of the siRNA from the application site to the target action site are shown in **Figure 1**. As described above, after intravenous injection (1), siRNA is distributed to

The siRNA molecules have unwanted physicochemical properties due to their

organs through the blood stream (3) and also undergoes elimination [7].

negative charge, large molecular weight, size, and instability. After systemic administration, the naked siRNA administered without vectors is rapidly degraded by serum endonucleases (4), excreted by the kidney (7), or taken up by the macrophages (APC cells) (5) of the mononuclear phagocyte system (MPS) [7, 18].

administration vary depending on the targeted organs, disease, and routes of administration. In general, siRNA therapeutics are preferred as local or systemic injection. The local transport of siRNA has less obstruction compared to systemic administration. The first of the unique features of the local administration is the need to an easy formulation that requires easier production and application and secondly provides application of lower doses that limit intracellular (time-concen-

followed as a very effective and promising treatment for various disease.

**80**

The result is a short plasma half-life below <10 minutes, so it cannot have enough time to reach the target and/or perform its functions, and therefore the gene silencing is ineffective. These problems are partially addressed by chemical modifications of the RNA backbone and by the use of nano-sized carriers (2) [7, 60].

Nanoparticle studies showed that the nanoparticles below 6 nm were rapidly excreted by renal excretion and nanoparticles in the range of 150–300 nm were taken by the cells of the MPS in the liver and spleen. It is stated that nanoparticles between 30 and 150 nm can hold in the bone marrow, kidney, etc. In addition, it has been reported that red blood cells (RBCs) affect the transport and distribution of microparticles in human microvasculature (diameter <100). In fact, due to collisions with RBCs, microparticles are pushed toward the artery wall where RBCs are virtually absent depending on the shape and size by a radial movement called the "waterfall effect." This event is also called margination, and they have stated that the extravasation cannot occur unless vectors are exposed to margination [14].

In a study done by D'Apolito et al. [20], they found that 3-μm particles had better marginalization than 1 μm. For this reason, it is seen that the size of the prepared particles could be very effective especially in in vivo experiments.

#### **Figure 1.**

*An illustration of a nanocarrier, barriers at systemic circulatory, mechanisms of uptake of siRNA from cell (modified from the 26th literature). In the following text, the numbers in the figure are mentioned.*

#### *Antisense Therapy*

The vectors carrying siRNA enter the tissue interstitium following extravasation. After entering the tissue interstitium, siRNA is moved to the target cells across the interstitial space. Transport along the vessel walls can occur via diffusion, convection through the capillary pores, and transcytosis. The diffusion is directed by the concentration gradient.

One of the unique properties of tumor microvessels is leakage (6) from endothelial discontinuity. The pore size of tumor microvessels ranges from 100 to 780 nm in diameter. In contrast, microvessels in most normal tissues have less leakage. For example; the tight junctions between endothelial cells are generally <2 nm, and the pore size in the capillary venules is <6 nm, the endothelium of the renal glomeruli is 40–60 nm, and the sinusoidal endothelium in the liver and spleen has large pore sizes of about 150 nm [19].

Leakage in tumor vessels increases siRNA/carrier extravasation, since the naked siRNA cannot enter the direct cell membrane due to its high molecular weight, large size, and negatively charged phosphate skeleton from the anionic cell membrane; the carrier systems are gaining importance here, again [21, 22].

There is an evidence that endocytosis (8) plays an important role, although not all of the entry mechanisms are well understood. The most common endocytosis used by nanocarriers is clathrin-mediated endocytosis through endocytic route receptor [23].

Rejman et al. [24] showed that the size and nature of the carrier vector affect the internalization mechanism. Nanoparticles of about 1 μm size are taken up via macropinocytosis, and ~120 nm size are taken up via clathrin-mediated endocytosis, whereas nanoparticles ~90 nm are taken up via clathrin/caveolae-independent endocytosis and caveolin-mediated endocytosis [25, 26].

Besides surface properties and size of the nanoparticles, surface modifications of the nanoparticles are also important in siRNA transport. The most important of these surface modifications is the coating of nanoparticles with PEG. The PEGylation is performed to keep the particles in the systemic circulation longer, and the positive results were obtained from until today. However, in addition to this positive effect, it has been reported that the increased PEGylation from 1–2 to 5% mol reduced the transfection efficiency by neutralizing the positive surface load required for siRNA involvement instead of increasing the transfection [27].

After reaching the target cell, the siRNA is subjected to endocytosis internalization, a process involving encapsulated siRNA in endocytic vesicles fused with endosomes. Endocytotic vesicles are initially associated with early endosomes and then mature late endosomes before fusion with lysosomes in the cell. Lysosomes are then formed. After internalization into the cell, the siRNA should escape from the fragmentation in the endosomes and be released from the carrier to the cytosol in order to be loaded to RNA-induced silencing complex (RISC) [28].

Sardo et al. found that endosomal escape increased with another modification with pH-sensitive polymers. In this study, they prepared a siRNA delivery system based on inulin (Inu), a plenty and natural polysaccharide. Inu was functionalized via the conjugation with diethylenetriamine (DETA) residues to form the complex Inu-DETA. The results of the study showed that while homogenous diffusion of siRNA was performed in JHH6 cytoplasm via micropinocytosis and clathrinmediated endocytosis, it was found that it did not allow caveola-mediated passage and no siRNA activity [29].
