**Abstract**

Advancement in chemistry holds a great promise in improving drug encapsulation that leads to superior drug delivery efficiency and the therapeutic efficacy of nano/micro-delivery systems. Drugs are being designed to specifically access the infection sites via covalent conjugation to nano/micro-delivery systems. This chapter focuses on techniques for achieving covalent encapsulation of drugs in nano/micro-delivery systems, how conjugation is applied to selectively influence pharmacokinetic profile, intracellular, and extracellular uptake, specific targeting to disease sites, binding to specific receptors, and controlled/sustained release. In addition, the effect of conjugation on drug efficacy and biosafety of the micro/ nanoparticulate drug delivery systems are discussed.

**Keywords:** covalent conjugation, sustained release, smart responsive, targeted delivery

## **1. Introduction**

For drugs to execute their effective mechanism of action, they require to reach its targeted site of action so that they can exert their intended action [1]. While conventional dosage formulations of drugs can achieve desired therapeutic concentrations, they are unable to effectively maintain the desired therapeutic concentrations, conferring a limited half-life thereby leading to ineffective treatment by the drugs [2]. Therefore, the development of novel drug delivery systems with the ability to improve on this limitation of the conventional drug delivery systems is needed.

Micro- and nano-carrier systems are among the approaches that have been successfully utilized for encapsulation of various types of drugs such as peptides, proteins, and low-molecular weight drugs [3–5]. These systems have been found to overcome limitations of conventional dosages forms such as improving solubility [6], bioavailability, and biodistribution of drugs [7], and targeting disease sites [8], hence contributing to a high proportion of the active drug reaching the targeted site. In addition, drug carrier systems protect the loaded drugs from premature degradation in the biological environment, thus enhancing bioavailability and cellular uptake. For effective delivery of drugs to occur, they have to be successfully loaded onto drug delivery systems as payloads. Two techniques are employed in

encapsulating drugs onto drug delivery systems. They include noncovalent physical encapsulation and covalent linking of the drugs to drug delivery systems.

Physical encapsulation of drugs into a carrier system involves hydrophobic interactions, electrostatic ionic interactions, and physical entrapping of drugs in the carrier matrix [9]. While the physical encapsulation of drugs into a carrier system is a popular technique, certain disadvantages are associated with it. For example, ionic complexation precipitation and dose dumping may take place if the effective attractions between the drug and the delivery system are reduced due to charge fluctuations and short-range attractions between monomers [10]. For hydrophobic drugs entrapped in the core of micelles, dose dumping may occur if micelles undergo hemodilution below the critical micelle concentration [11]. Moreover, challenges have been encountered in entrapping hydrophilic drugs in carrier systems using physical encapsulation [12]. These challenges have resulted in physical encapsulation with low loading efficiencies. Due to this, drug delivery scientists are resorting to covalently linking drugs to nano/micro-drug delivery system as an alternative method to physical encapsulation. This chapter discusses the techniques of covalent drug encapsulation, the mechanism of drug release from the covalent linkages, efficacy, and biosafety of drugs due to covalent conjugation and how disease site targeting via covalent conjugation is achieved.

#### **2. Techniques of covalent drug encapsulation**

Covalent drug conjugation involves attaching the drug into drug delivery systems via a physiologically labile bond [13]. A greater control over drug release is achieved by the covalent attachment of drugs to the drug delivery systems. Targeting and release of the drug from such systems is achieved through hydrolysable or biodegradable linkages between the payload and the micro/ nanosystems [14]. Most commonly employed bio-hydrolysable bonds include amide, disulfide, ester, thiol, and carbamate bonds [15]. Conjugation of the drug to a delivery system may also include covalent linkers. The choice of a covalent linkers used is determined by its selectivity for drug release and the environment in which the drug should be released. Covalently conjugated drugs have exhibited the ability to release drugs by cleaving conjugated bonds under internal or external stimuli such as pH, redox potential, enzyme, light, and thermal energy [16].

hydrophilic drugs with fatty acid is a popular technique to formulate selfassembling particulate prodrugs. This conjugation has shown to improve cellular uptake of hydrophilic drugs [22]. While viruses are intracellular obligate microorganisms, most of the drugs employed for their treatment are DNA nucleosides analogs which are highly hydrophilic with poor cellular uptake. Improving cellular uptake of these drugs usually improves their activity [23]. Agarwal et al. reported that conjugation of Emtricitabine (FTC) with myristic acid resulted to an analog that had 35.2 times higher activity than the nonconjugated drug against multidrugresistant HIV viruses strain B-NNRTI and B-K65R [21]. These results indicated that antiretroviral ester conjugation with fatty acids could generate more potent analogs with a better resistance profile than its parent compound [24]. Similar results have been reported via esterification of fatty acids with lamivudine (3TC) [21] and

*Synthesis of the fatty acid ester-linked Emtricitabine. Adapted from [21]. DTMTr, 4,4*<sup>0</sup>

*chloride; HBTU, hexafluorophosphate benzotriazole tetramethyl uronium; DPEA, N,N-*

Amide linkages can be used to covalently attach drugs to Nano/microcarriers using an anchor functionalized with carboxylic acid end groups [26]. Among the covalent linkages, amide bonds are the most widely used linkages to conjugate drugs to drug delivery systems. The conjugation is usually catalyzed by 1-ethyl-3-

dicyclohexylcarbodiimide (DCC) chemistry [27, 28]. The process involves reacting a carboxylic group with EDC and N-hydroxysuccinimidyl (NHS) to form an acyl amino ester that is subsequently reacted with an amine to create the amide bond.


*-dimethoxytrityl*

(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) or N, N<sup>0</sup>

acyclovir [25].

**139**

**Figure 1.**

**Figure 2.**

**2.2 Amide and linked drug conjugates**

*diisopropylethylamine; DMF, dimethylformamide.*

*Biodegradable bonds employed in covalent drug conjugation.*

*Nano/Microparticles Encapsulation Via Covalent Drug Conjugation*

*DOI: http://dx.doi.org/10.5772/intechopen.93364*

The main advantages of covalent linking over physical encapsulation include the enhanced residence time of the drug in the body, slow release, improved biodistribution, and therapeutic efficacy, as well as reduced systemic toxicity [16, 17]. Covalent drug conjugation to micro and nanosystems is achieved via special bonds that are biodegradable or cleaved inside the body or a special environment at disease sites. Special linker moieties and functional groups of the drug dictate the success of conjugation on a nano/microsystem [18]. Based on the functional groups available on the delivery system and the drug being conjugated, several conjugation methods have been devised (**Figure 1**). The section below discusses the techniques of drug conjugations and their application in drug delivery.

#### **2.1 Ester-linked drug conjugates**

Ester bonds are widely used in conjugating drugs to drug delivery systems [19]. The ester bond is formed when a hydroxyl group and a carboxylic acid group react. Drugs with carboxylic groups can therefore be conjugated to hydroxyl groups of the drug delivery system and vice versa (**Figure 2**). Linkers or spacers such as a succinic anhydride may be employed to facilitate the conjugation [20]. Esterification of

*Nano/Microparticles Encapsulation Via Covalent Drug Conjugation DOI: http://dx.doi.org/10.5772/intechopen.93364*

**Figure 1.** *Biodegradable bonds employed in covalent drug conjugation.*

**Figure 2.**

encapsulating drugs onto drug delivery systems. They include noncovalent physical

Physical encapsulation of drugs into a carrier system involves hydrophobic interactions, electrostatic ionic interactions, and physical entrapping of drugs in the carrier matrix [9]. While the physical encapsulation of drugs into a carrier system is a popular technique, certain disadvantages are associated with it. For example, ionic complexation precipitation and dose dumping may take place if the effective attractions between the drug and the delivery system are reduced due to charge fluctuations and short-range attractions between monomers [10]. For hydrophobic drugs entrapped in the core of micelles, dose dumping may occur if micelles undergo hemodilution below the critical micelle concentration [11]. Moreover, challenges have been encountered in entrapping hydrophilic drugs in carrier systems using physical encapsulation [12]. These challenges have resulted in physical encapsulation with low loading efficiencies. Due to this, drug delivery scientists are resorting to covalently linking drugs to nano/micro-drug delivery system as an alternative method to physical encapsulation. This chapter discusses the techniques of covalent drug encapsulation, the mechanism of drug release from the covalent linkages, efficacy, and biosafety of drugs due to covalent conjugation and how

Covalent drug conjugation involves attaching the drug into drug delivery systems via a physiologically labile bond [13]. A greater control over drug release is achieved by the covalent attachment of drugs to the drug delivery systems. Targeting and release of the drug from such systems is achieved through hydrolysable or biodegradable linkages between the payload and the micro/ nanosystems [14]. Most commonly employed bio-hydrolysable bonds include amide, disulfide, ester, thiol, and carbamate bonds [15]. Conjugation of the drug to a delivery system may also include covalent linkers. The choice of a covalent linkers used is determined by its selectivity for drug release and the environment in which the drug should be released. Covalently conjugated drugs have exhibited the ability to release drugs by cleaving conjugated bonds under internal or external stimuli

The main advantages of covalent linking over physical encapsulation include the

Ester bonds are widely used in conjugating drugs to drug delivery systems [19]. The ester bond is formed when a hydroxyl group and a carboxylic acid group react. Drugs with carboxylic groups can therefore be conjugated to hydroxyl groups of the drug delivery system and vice versa (**Figure 2**). Linkers or spacers such as a succinic anhydride may be employed to facilitate the conjugation [20]. Esterification of

such as pH, redox potential, enzyme, light, and thermal energy [16].

enhanced residence time of the drug in the body, slow release, improved biodistribution, and therapeutic efficacy, as well as reduced systemic toxicity [16, 17]. Covalent drug conjugation to micro and nanosystems is achieved via special bonds that are biodegradable or cleaved inside the body or a special environment at disease sites. Special linker moieties and functional groups of the drug dictate the success of conjugation on a nano/microsystem [18]. Based on the functional groups available on the delivery system and the drug being conjugated, several conjugation methods have been devised (**Figure 1**). The section below discusses the techniques of drug conjugations and their application in drug delivery.

encapsulation and covalent linking of the drugs to drug delivery systems.

*Nano- and Microencapsulation - Techniques and Applications*

disease site targeting via covalent conjugation is achieved.

**2. Techniques of covalent drug encapsulation**

**2.1 Ester-linked drug conjugates**

**138**

*Synthesis of the fatty acid ester-linked Emtricitabine. Adapted from [21]. DTMTr, 4,4*<sup>0</sup> *-dimethoxytrityl chloride; HBTU, hexafluorophosphate benzotriazole tetramethyl uronium; DPEA, N,Ndiisopropylethylamine; DMF, dimethylformamide.*

hydrophilic drugs with fatty acid is a popular technique to formulate selfassembling particulate prodrugs. This conjugation has shown to improve cellular uptake of hydrophilic drugs [22]. While viruses are intracellular obligate microorganisms, most of the drugs employed for their treatment are DNA nucleosides analogs which are highly hydrophilic with poor cellular uptake. Improving cellular uptake of these drugs usually improves their activity [23]. Agarwal et al. reported that conjugation of Emtricitabine (FTC) with myristic acid resulted to an analog that had 35.2 times higher activity than the nonconjugated drug against multidrugresistant HIV viruses strain B-NNRTI and B-K65R [21]. These results indicated that antiretroviral ester conjugation with fatty acids could generate more potent analogs with a better resistance profile than its parent compound [24]. Similar results have been reported via esterification of fatty acids with lamivudine (3TC) [21] and acyclovir [25].

#### **2.2 Amide and linked drug conjugates**

Amide linkages can be used to covalently attach drugs to Nano/microcarriers using an anchor functionalized with carboxylic acid end groups [26]. Among the covalent linkages, amide bonds are the most widely used linkages to conjugate drugs to drug delivery systems. The conjugation is usually catalyzed by 1-ethyl-3- (3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) or N, N<sup>0</sup> dicyclohexylcarbodiimide (DCC) chemistry [27, 28]. The process involves reacting a carboxylic group with EDC and N-hydroxysuccinimidyl (NHS) to form an acyl amino ester that is subsequently reacted with an amine to create the amide bond.

EDC has good water solubility enabling its direct application in aqueous solutions without the addition of any organic compounds, thus making it suitable for the attachment of bioactive molecules to the carrier surface [29].

Several nano/micro-delivery systems with amide-linked drug conjugates have been widely reported with a great success. Such a system was reported by Yousefpour et al. (**Figure 3**) who conjugated doxorubicin and monoclonal antibody, trastuzumab to chitosan to form nanoparticles with high conjugation capacity, enhanced and selective uptake by human epidermal growth factor receptor 2 (Her2+) on cancer cells compared with the nonconjugated drug. Similar conjugation was reported by Kurtoglu et al. who conjugated Ibuprofen to PAMAM dendrimer and mPEG via amide linkages [30]. The drug conjugates showed better results when compared to bare drugs.

Monoclonal antibodies and derived therapeutics have also been linked with adverse effects and toxicity. The associated toxic effects of monoclonal antibodies

(mAb) have limited their therapeutic application. However, antibody/drug covalent (ADC) conjugation-based platform has enabled selective delivery of a potent cytotoxic payload to target diseased cells, resulting in improved efficacy, reduced systemic toxicity, and preferable pharmacokinetics (PK), pharmacodynamics (PD), and biodistribution compared to traditional chemotherapy [35]. The success of such conjugations includes FDA approved Adcetris® which is a drug conjugate of Dolastatin 10 and monomethyl auristatin E (MMAE). The link between Dolastatin 10 and MMAE is N-terminal amine via the amide bond linked to a self-immolating

**Drug Drug delivery system Main findings Reference**

Aggregates • Similar activity to bare drugs

Nanoparticles • Enhanced and selective uptake

*Drug loading via amide conjugation functionalized for varied clinical and research applications.*

Adriamycin Micelles • *In vivo* high anticancer activity

Nanofibers and spherical nanoaggregates

*Nano/Microparticles Encapsulation Via Covalent Drug Conjugation*

*DOI: http://dx.doi.org/10.5772/intechopen.93364*

Gemcitabine Self-assembled prodrug • High loading capacity

Self-assembled prodrugs • Greater antitumor efficacy than free DOX. • High drug loading • Sustained drug release

• Low side effects

• Low on side effects • Sustained release of the drug

• High loading capacity

loaded drug

• Synergism of the co-conjugated drugs

• Reduction of drug side effects in Her2+ breast and ovarian cancers.

• Increased biological half-life of the

• Better activity than the bare drug

[31]

[32]

[33]

[29]

[34]

Doxorubicin (DOX)

Camptothecin and Capecitabine

Penicillin V and Cephradine

DOX and trastuzumab

**Table 1.**

**Figure 4.**

**141**

Apart from ADCs, amide bonds have been employed to link mAb to other drug delivery surfaces such as liposomes. Liposomes with 1,2-distearoyl-sn-glycero-3 phosphoethanolamine-N-[carboxy(polyethylene glycol) (DSPE-PEG-COOH) linked with mAb have been reported to prepare immunoliposomes. The attachment of mAb to the liposomes was achieved via surface dCOOH on the surface of liposomes and dNH2 of the mAB. This conjugation enhanced significantly the blood residence time of the mAb [36]. Another amide conjugation between targeted ligand and liposome was reported where the peptide was covalently attached to the

spacer, p-aminobenzyloxycarbonyl (PABC).

*Illustration of linking drugs to the delivery system via hydrazine bond.*

#### **Figure 3.**

*Schematic description of the procedures for the amide conjugation between chitosan and doxorubicin to form a self-assembling nano-drug delivery system [29].*

*Nano/Microparticles Encapsulation Via Covalent Drug Conjugation DOI: http://dx.doi.org/10.5772/intechopen.93364*


#### **Table 1.**

EDC has good water solubility enabling its direct application in aqueous solutions without the addition of any organic compounds, thus making it suitable for the

Several nano/micro-delivery systems with amide-linked drug conjugates have

Yousefpour et al. (**Figure 3**) who conjugated doxorubicin and monoclonal antibody, trastuzumab to chitosan to form nanoparticles with high conjugation capacity, enhanced and selective uptake by human epidermal growth factor receptor 2 (Her2+) on cancer cells compared with the nonconjugated drug. Similar conjugation was reported by Kurtoglu et al. who conjugated Ibuprofen to PAMAM dendrimer and mPEG via amide linkages [30]. The drug conjugates showed better results when

Monoclonal antibodies and derived therapeutics have also been linked with adverse effects and toxicity. The associated toxic effects of monoclonal antibodies

*Schematic description of the procedures for the amide conjugation between chitosan and doxorubicin to form a*

been widely reported with a great success. Such a system was reported by

attachment of bioactive molecules to the carrier surface [29].

*Nano- and Microencapsulation - Techniques and Applications*

compared to bare drugs.

**Figure 3.**

**140**

*self-assembling nano-drug delivery system [29].*

*Drug loading via amide conjugation functionalized for varied clinical and research applications.*

*Illustration of linking drugs to the delivery system via hydrazine bond.*

(mAb) have limited their therapeutic application. However, antibody/drug covalent (ADC) conjugation-based platform has enabled selective delivery of a potent cytotoxic payload to target diseased cells, resulting in improved efficacy, reduced systemic toxicity, and preferable pharmacokinetics (PK), pharmacodynamics (PD), and biodistribution compared to traditional chemotherapy [35]. The success of such conjugations includes FDA approved Adcetris® which is a drug conjugate of Dolastatin 10 and monomethyl auristatin E (MMAE). The link between Dolastatin 10 and MMAE is N-terminal amine via the amide bond linked to a self-immolating spacer, p-aminobenzyloxycarbonyl (PABC).

Apart from ADCs, amide bonds have been employed to link mAb to other drug delivery surfaces such as liposomes. Liposomes with 1,2-distearoyl-sn-glycero-3 phosphoethanolamine-N-[carboxy(polyethylene glycol) (DSPE-PEG-COOH) linked with mAb have been reported to prepare immunoliposomes. The attachment of mAb to the liposomes was achieved via surface dCOOH on the surface of liposomes and dNH2 of the mAB. This conjugation enhanced significantly the blood residence time of the mAb [36]. Another amide conjugation between targeted ligand and liposome was reported where the peptide was covalently attached to the 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 conjugation of drugs to drug delivery systems is summarized in **Table 1**.

linked drug delivery systems can be effectively employed in targeted delivery of the

*Nano/Microparticles Encapsulation Via Covalent Drug Conjugation*

*DOI: http://dx.doi.org/10.5772/intechopen.93364*

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].

Other covalent linkages that include carbamate linkage, Schiff bases, and polycyclic linkages have also been employed to form covalent linkages between the drug and delivery systems. Reaction between a diene and dienophile results to cycloaddition via the Diels-Alder chemistry which forms bicyclic compounds. This chemistry can be utilized to form polycyclic linkages between the drug and the delivery system [49]. Whereas other hydrolyzable linkages include carbamate [50, 51], oximes [52], and Schiff bases [53, 54]. These types of linkages are specifically designed to make drug delivery systems have targeting ability due to the physio-

Roughly 70% of new drug discoveries have shown poor aqueous solubility, while approximately 40% of the marketed immediate-release drugs are practically insoluble [55]. Additionally, the drugs that are highly soluble have been found to have membrane penetration difficulties [56]. Covalent modification of therapeutic compounds is therefore a strategy that enhances efficacies of the conjugated drugs by solving physicochemical problems associated with the drugs [57]. When hydrophobic drug molecules are attached to hydrophilic material or when hydrophilic drugs are attached to hydrophobic biomaterial or delivery systems, an amphiphilic system is formed. The resulting amphiphilic system can self-assemble into stable core-shell aggregates such as vesicles, classical micelles, unimolecular micelles, and nanorods

When amphiphiles are dispersed in water, the hydrophilic component of the amphiphile preferentially interacts with the aqueous phase (shell) while the hydrophobic portion tends to reside in the air or in the nonpolar solvent (core) in order to form stable assemblies [59]. Self-assemblies of drug conjugates are usually governed by forces such as hydrogen bonds, Van der Waals interactions, hydrophobic interactions, and electrostatic interactions [2]. Self-assembled drug conjugates often

conjugated drugs.

**2.5 Disulfide linkage**

**2.6 Other covalent linkages**

[2, 58].

**143**

logical changes brought about by the diseases.

**3. Self-assembly of covalent conjugated drugs**

### **2.3 Hydrazones conjugates**

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].

#### **2.4 Thioether linkage**

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-

**Figure 5.** *Illustration showing maleimide thiol covalent linkage to drug delivery systems.*

linked drug delivery systems can be effectively employed in targeted delivery of the conjugated drugs.
