**2.5 Disulfide linkage**

carboxylic groups on the PEGylated liposomes to form a nanoparticulate system able to target the infarcted heart. The system was effective in *in vitro* testing against cardiac cells [37]. Due to their stability, the rate of hydrolysis of amide bonds is lower when compared to ester bonds. This slower rate of hydrolysis affects the release of drugs, thus affecting the activity of the conjugated drugs [31]. The amide

Hydrazones are formed by the action of hydrazine on ketones or aldehydes functional groups [38]. Their basic structure is R1R2C]NNH2 which is formed when oxygen in ketones and aldehydes is replaced with the dNNH2 functional group. pHsensitivity attributes of hydrazones bond have been used in the formation of stimuli responsive nano/micro-drug delivery system. At a lower pH, the bond decomposes efficiently while at basic pH, hydrazones are usually stable [39]. The instability of hydrazone bonds in acidic mean molecules can be cleaved in acidic intracellular environment of endosomes or lysosomes, tumor tissues, and bacterial infection sites. Hydrazone bonds have been successfully used to covalently load drugs into delivery systems resulting to pH-responsive nano/micro-dosage forms that can effectively target a disease that alters physiological pH to acidic (**Figure 4**) [38, 40, 41].

Thioether bond is formed from the reaction between the thiol group containing SH group and first carbon of maleimide that is attached to the drug carrier [42]. Conjugation via thioether bonds is favored technique as the bond is formed under mild conditions, at room temperature, and in aqueous solution [26]. Thioether linkage makes it possible to link peptides to a delivery system or drugs to peptide (**Figure 5**). Several drug delivery systems have been reported to employ thioether linkage as a means of covalently loading drug on to them. mAb trastuzumab and nanoparticle doped with doxorubicin were successfully loaded in a drug delivery system via thioether linkage [43]. DOX was conjugated to a drug delivery system via thioether bond through poly(ethylene glycol) polymer having two linkers of maleimide and n-hydroxysuccinimide (nhs). The conjugates showed better cancer uptake when compared to free DOX. The better uptake was attributed to better affinity of the system to the HER2 receptor of breast cancer cells. When compared to the free drug, the conjugated delivery system had a longer blood circulation with less toxic effects when compared to the free drug [28]. Similar results were reported by Park et al. who formulated immunoliposomes conjugated with monoclonal antibodies (mAB) [44]. Additionally, another liposomal covalent system has also been reported by Kirpotin et al.; in the system, a free thiol group was used to conjugate antibodies to the nanocarriers. The carrier showed increased cellular uptake resulting in better tumor reduction [45]. Thioether linkage has also been applied successfully on carbon nanotubes functionalized by folic acid. The system employed in targeted delivery of DOX against cancer [46]. From literature reports, thioether-

conjugation of drugs to drug delivery systems is summarized in **Table 1**.

*Nano- and Microencapsulation - Techniques and Applications*

**2.3 Hydrazones conjugates**

**2.4 Thioether linkage**

**Figure 5.**

**142**

*Illustration showing maleimide thiol covalent linkage to drug delivery systems.*

Two thiol groups' conjugation results in the formation of a disulfide bond. One group originates from a nanocarrier and the other from a ligand [47]. Disulfide drug conjugates have shown to be stable in the extracellular environments but easily broken down in intracellular reductive intracellular environment. There is an increasing number of drug formulations that incorporate disulfide bonds being reported, for nano/micro-drug delivery system. Disulfide bonds are being designed to exploit differences in the reduction potential at disease location and the whole body at large [18]. Formulating environmentally responsive drug delivery systems has been made possible due to the desirable attribute of disulfide bonds. Lu et al. using a disulfide-bridged created mesoporous silica nanoparticle covalently loaded with folic acid (FA) and decorated bull serum albumin (BSA) for improved tissue biocompatibility and effective dual pH/glutathione (GSH) response drug releasing drug delivery system. Disulfides have also been employed as cleavable linkers in drug conjugates or to formulate stimuli-responsive carriers, and this has resulted in disulfides linker-based mAb drug covalent linkages (ADCs) in clinical trials [48].
