**2.10 Hydrogels**

Hydrogels are three-dimensional networks made of polymeric chains that are hydrophilic in nature. The polymer can be natural or synthetic and should contain a large amount of water. Although natural polymers show good biocompatibility, they are not suitable for protein or peptide delivery due to their improper mechanical strength. Natural polymers can also lead to an autoimmune response. However, hydrogels with the synthetic polymer can be designed to avoid these problems. Hydrogels can control the release of the protein or peptide of interest in response to pH [100, 101]. Hydrogels can also enhance transportation of the protein or peptide of interest. The difference of pH at different parts of the GIT has been utilized to control the release of insulin encapsulated in the hydrogels at the intestine (**Figure 16**). Hydrogels also showed rapid release of the encapsulated insulin once in the intestine. Finally, the encapsulation process of the protein (e.g., insulin) inside the hydrogel is highly efficient. These properties of the hydrogels make them promising systems for protein or peptide delivery [102].

**Figure 16.** *Protein or peptide therapeutic (black spheres) encapsulated in a hydrogel.*

#### **2.11 Injectable nanocomposite cryogels**

Although hydrogels are promising systems for protein or peptide delivery, they have some issues regarding the rapid release of the encapsulated protein or peptide of interest. This rapid release leads to a short duration of action which may not be optimal for some therapies. Another issue associated with the hydrogels is the possible denaturation of the encapsulated protein or peptide. Injectable nanocomposites have been developed to overcome some of the issues related to hydrogels. Kinetics of the release of the therapeutic protein or peptide can be controlled by using suitable components of the system [103]. This delivery system can also be used for the sustained release of various therapeutic proteins and peptides.

#### **2.12 Cell-penetrating peptides**

Cell-penetrating peptides (CPP) are capable of transporting the attached molecule across the cell membrane. Therefore, one way of enhancing the permeability of therapeutic proteins or peptides across the cell membrane is to attach them to a CPP (**Table 1**) [104]. One way CPP can penetrate the cell membrane is via endocytosis. Another way could be the perturbation of the lipid bilayer of the cell membrane by the CPP. Although minor disturbances in the membrane structure were found, toxic effects of CPP on the cell membrane have not been reported. Insulin attached to TAT (**Figure 17**) showed better permeability across the cell membrane [105].

#### **2.13 Protein crystallization**

Crystallization of therapeutic proteins offers many advantages over the traditionally used protein solution or amorphous form of the protein. The protein in the crystalline form shows significantly higher stability than the amorphous form. This higher stability is advantageous for therapeutic proteins since the high stability of the proteins maintains their biological function in different environments [106]. Crystalline form


**Table 1.**

*Examples of protein and peptides delivered by CPPs.*

**Figure 17.** *Insulin attached to a CPP.*

of lipase enzyme is used as a therapeutic enzyme in pathological conditions related to abnormalities in the digestion of lipid. However, a major limitation of crystallization approach is that not all proteins can be crystallized. Also, in some cases, protein crystallization is not efficient. Nevertheless, protein crystallization remains a promising approach for developing protein or peptide delivery systems [107, 108].
