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

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 [2, 58].

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

responsible for the drug release from the drug delivery systems. It is important to note that amides are stable bonds, which do not hydrolyze at physiological pH and

the drug in a chemically unmodified form [71, 72] (**Table 2**).

*Nano/Microparticles Encapsulation Via Covalent Drug Conjugation*

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

increase the efficacy of the drug [74, 75].

applications have also been discussed [79].

**145**

**5. Efficacy and biosafety of drugs due to covalent conjugation**

Particulate drug delivery systems have recorded significant progress in the delivery of small molecular drugs; however, some challenges such as poor drug loading, formulation instability, premature drug leakage, and poor blood circulation are still encountered. This has led to the discovery of newer strategies that can be used to overcome these challenges. Over the years, various research groups have explored the efficacy and biosafety of drugs that were covalently conjugated to nano/micro-delivery systems [73]. These include systems developed from polymers, dendrimers, and peptides among others; usually, the drug is bond to the biomaterial via a linker. The efficacy of drugs covalently conjugated has been significantly improved in terms of drug loading capacity and stability amongst other benefits. Biomaterials such as polymers that are covalently bonded to drugs have shown to be useful drug carriers which help to hold drugs and are tunable to

The other advantages drug conjugation provides include increased solubility of the drugs that are insoluble in water, thus enhancing a controlled release of the drug as there will be increased permeability through lipophilic tissues. This will, in turn, lead to an increased effective concentration of the drug at the targeted site [75]. Additionally, drug conjugated covalently to biomaterials are shielded from degradation or deactivation as well as increases the circulation time of the drug [76]. One or more of these advantages offered by drug conjugation has been reported by various groups of researchers. For instance, the efficacy of different poly(ethylene glycol) (PEG)-based anticancer drug conjugates has been extensively explored [73, 77, 78]. The details of the importance of nano/micro-delivery systems such as lysosomes, polymeric micelles, and polymeric nanoparticles in drug delivery

In a study [80], hydrazine-based doxorubicin-polymer conjugates were synthesized into doxorubicin-loaded nanoparticles. It was reported that the bioactivity of the drug was largely retained *in vitro*, and there was a tremendous reduction in systemic toxicity of doxorubicin upon nanoparticle conjugation *in vivo* when compared to the physical formulation of the drug. In addition, the nanoparticles

prevented the drug from disassembling upon interaction with serum proteins in the blood [81, 82]. The *in vitro* study showed that the doxorubicin-nanoparticle conjugate accelerated the release of the drug in acidic conditions and killed the cancer cell. In the same vein, Modarassi and colleagues investigated the drug release

Disulfide bonds usually exploit differences in the reduction potential at different locations within and upon cells to release the conjugated drugs. Due to this, different delivery platforms have been designed to achieve different targeted delivery strategies. Redox enzymes reduce disulfide bonds on and inside the cells resulting in drug release [18]. Other bonds like hydrazones, Schiff bases hydrolysis is catalyzed by acid environment. In an acid environment, the bond between the drug delivery system and the drug is broken to release the drug. Apart from direct drug linkage to the delivery systems [70], suitable linkers that are self-immolating such as paminobenzyloxycarbonyl (PABC) can also be involved in the reaction. The purpose of the linkers is to situate the cleavable delivery system away from the drug to allow facile release. Upon cleavage, the linkers rapidly fragment, leading to the release of

body temperature.

**Figure 6.**

*Schematic representation of self-assembly of drug conjugates and subsequent release mechanism of the drug from the self-assembly. Adapted with permission from [60].*

result to effective therapies as they possess better physicochemical properties that lead to enhanced drug penetration for highly hydrophilic and charged drug molecules [61], enhanced solubility for highly insoluble drugs, and increased residence time for drugs that are easily eliminated via the kidney [62]. For instance, most of anticancer drugs are hydrophobic in nature, and therefore, to produce selfassembled nanostructures with better therapeutic and formulation aspects, hydrophilic molecules or polymers are usually attached to them via a degradable linker to induce amphiphilicity and self-assembly (**Figure 6**) [61]. Self-assembly widely occurs in nature and has been borrowed by science to formulate self-assembled nano/micro-drug delivery systems with better therapeutic outcomes than original drug molecules [63].

#### **4. Mechanism of drug release from drug conjugates**

Covalent linkages alter the absorption, distribution, metabolism, and elimination (ADME) properties of an active drug [64]. Before conjugation, it is paramount to have a complete understanding of the physicochemical, structural relationship activity of the drug candidates. It is also important to understand the ability of the attached groups to cleave, leaving and exposing the functional groups responsible for the activity of the drug [65]. Moreover, once the drug is cleaved from the delivery system, the delivery system should be inactive and nontoxic [66]. Most of the drawbacks from covalent linkages of drugs to the delivery systems are the inability of the drug to cleave from the delivery system. The inability of the drug to detach from the drug delivery system may lower the activity of the drug due to poor bioavailability [67]. Therefore, the chosen covalent linking technique should have the ability to easily cleave to enable the release of the drug.

Esterification is a common technique for conjugation because esterases are widely distributed in body tissues that easily cleave the ester bonds leaving the free drug to act. Esterase is a hydrolase enzyme that splits esters into an acid and alcohol in a chemical reaction with water called hydrolysis [68]. The easy cleaving of esters makes the use of ester linkages as an attractive technique. Breakage of amide bonds is via hydrolysis of the carbon-nitrogen bond, and this results in a carboxylic acid and either ammonia or an amine [69]. This cleavage of amides is

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

responsible for the drug release from the drug delivery systems. It is important to note that amides are stable bonds, which do not hydrolyze at physiological pH and body temperature.

Disulfide bonds usually exploit differences in the reduction potential at different locations within and upon cells to release the conjugated drugs. Due to this, different delivery platforms have been designed to achieve different targeted delivery strategies. Redox enzymes reduce disulfide bonds on and inside the cells resulting in drug release [18]. Other bonds like hydrazones, Schiff bases hydrolysis is catalyzed by acid environment. In an acid environment, the bond between the drug delivery system and the drug is broken to release the drug. Apart from direct drug linkage to the delivery systems [70], suitable linkers that are self-immolating such as paminobenzyloxycarbonyl (PABC) can also be involved in the reaction. The purpose of the linkers is to situate the cleavable delivery system away from the drug to allow facile release. Upon cleavage, the linkers rapidly fragment, leading to the release of the drug in a chemically unmodified form [71, 72] (**Table 2**).

## **5. Efficacy and biosafety of drugs due to covalent conjugation**

Particulate drug delivery systems have recorded significant progress in the delivery of small molecular drugs; however, some challenges such as poor drug loading, formulation instability, premature drug leakage, and poor blood circulation are still encountered. This has led to the discovery of newer strategies that can be used to overcome these challenges. Over the years, various research groups have explored the efficacy and biosafety of drugs that were covalently conjugated to nano/micro-delivery systems [73]. These include systems developed from polymers, dendrimers, and peptides among others; usually, the drug is bond to the biomaterial via a linker. The efficacy of drugs covalently conjugated has been significantly improved in terms of drug loading capacity and stability amongst other benefits. Biomaterials such as polymers that are covalently bonded to drugs have shown to be useful drug carriers which help to hold drugs and are tunable to increase the efficacy of the drug [74, 75].

The other advantages drug conjugation provides include increased solubility of the drugs that are insoluble in water, thus enhancing a controlled release of the drug as there will be increased permeability through lipophilic tissues. This will, in turn, lead to an increased effective concentration of the drug at the targeted site [75]. Additionally, drug conjugated covalently to biomaterials are shielded from degradation or deactivation as well as increases the circulation time of the drug [76]. One or more of these advantages offered by drug conjugation has been reported by various groups of researchers. For instance, the efficacy of different poly(ethylene glycol) (PEG)-based anticancer drug conjugates has been extensively explored [73, 77, 78]. The details of the importance of nano/micro-delivery systems such as lysosomes, polymeric micelles, and polymeric nanoparticles in drug delivery applications have also been discussed [79].

In a study [80], hydrazine-based doxorubicin-polymer conjugates were synthesized into doxorubicin-loaded nanoparticles. It was reported that the bioactivity of the drug was largely retained *in vitro*, and there was a tremendous reduction in systemic toxicity of doxorubicin upon nanoparticle conjugation *in vivo* when compared to the physical formulation of the drug. In addition, the nanoparticles prevented the drug from disassembling upon interaction with serum proteins in the blood [81, 82]. The *in vitro* study showed that the doxorubicin-nanoparticle conjugate accelerated the release of the drug in acidic conditions and killed the cancer cell. In the same vein, Modarassi and colleagues investigated the drug release

result to effective therapies as they possess better physicochemical properties that lead to enhanced drug penetration for highly hydrophilic and charged drug molecules [61], enhanced solubility for highly insoluble drugs, and increased residence time for drugs that are easily eliminated via the kidney [62]. For instance, most of anticancer drugs are hydrophobic in nature, and therefore, to produce selfassembled nanostructures with better therapeutic and formulation aspects, hydrophilic molecules or polymers are usually attached to them via a degradable linker to induce amphiphilicity and self-assembly (**Figure 6**) [61]. Self-assembly widely occurs in nature and has been borrowed by science to formulate self-assembled nano/micro-drug delivery systems with better therapeutic outcomes than original

*Schematic representation of self-assembly of drug conjugates and subsequent release mechanism of the drug from*

Covalent linkages alter the absorption, distribution, metabolism, and elimination (ADME) properties of an active drug [64]. Before conjugation, it is paramount to have a complete understanding of the physicochemical, structural relationship activity of the drug candidates. It is also important to understand the ability of the attached groups to cleave, leaving and exposing the functional groups responsible for the activity of the drug [65]. Moreover, once the drug is cleaved from the delivery system, the delivery system should be inactive and nontoxic [66]. Most of the drawbacks from covalent linkages of drugs to the delivery systems are the inability of the drug to cleave from the delivery system. The inability of the drug to detach from the drug delivery system may lower the activity of the drug due to poor bioavailability [67]. Therefore, the chosen covalent linking technique should have

Esterification is a common technique for conjugation because esterases are widely distributed in body tissues that easily cleave the ester bonds leaving the free drug to act. Esterase is a hydrolase enzyme that splits esters into an acid and alcohol in a chemical reaction with water called hydrolysis [68]. The easy cleaving of esters makes the use of ester linkages as an attractive technique. Breakage of amide bonds is via hydrolysis of the carbon-nitrogen bond, and this results in a carboxylic acid and either ammonia or an amine [69]. This cleavage of amides is

**4. Mechanism of drug release from drug conjugates**

the ability to easily cleave to enable the release of the drug.

drug molecules [63].

**144**

*the self-assembly. Adapted with permission from [60].*

*Nano- and Microencapsulation - Techniques and Applications*

**Figure 6.**

*Cleavage mechanism resulting in drug release of common bonds employed in covalent conjugation.*

behavior of doxorubicin that was conjugated onto the structure of nanoparticles. The conjugation was achieved via an acid-labile hydrazone linkage, and the effect of conjugation was compared with the nonconjugated drug [94] (**Figure 7**). The results of the *in vitro* investigation revealed that the release of doxorubicin was dependent on the amount of crosslinker. The higher the amount of crosslinkers, the lower the cumulative drug release in the physically loaded drug. On the other hand, drug conjugation showed that an increase in the amount crosslinker within the structure led to an increased rate and amount of drug released. This implies that the

Another drug PEG conjugate that has shown enhanced drug efficacy is the paclitaxel (PTX) conjugated with polyethylene glycol (PEG-B-PTX), synthesized by Dong et al. [95]. The antitumor efficacy of the stable micelle of about 50 nm, and 13.3 wt% drug load content was investigated against human glioma and breast cancer cells *in vitro*. The conjugate micelle exhibited improved antitumor effects when compared to the clinically used taxol. This result suggests that the drug conjugate can be a superior alternative for current clinically used PTX

nanoformulations which has limitations such as poor *in vivo* stability, premature release, and little improvements in its antitumor efficacy [96, 97]. A detailed *in vivo*

*A) The hydrazone acid-labile DOX release behavior from nano/microparticles at pH 7.4. (B) The hydrazone acid-labile DOX release behavior from nano/microparticles at pH 5.5. Sustained release slow drug release at pH 7.4 when compared to acidic pH. Overall slower release when compared to nonconjugated DOX. Adapted*

efficacy of drug conjugation with respect to drug release is superior to

*Nano/Microparticles Encapsulation Via Covalent Drug Conjugation*

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

nonconjugated drugs.

**Figure 7.**

**147**

*with permission from [98].*

**Table 2.**

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

behavior of doxorubicin that was conjugated onto the structure of nanoparticles. The conjugation was achieved via an acid-labile hydrazone linkage, and the effect of conjugation was compared with the nonconjugated drug [94] (**Figure 7**). The results of the *in vitro* investigation revealed that the release of doxorubicin was dependent on the amount of crosslinker. The higher the amount of crosslinkers, the lower the cumulative drug release in the physically loaded drug. On the other hand, drug conjugation showed that an increase in the amount crosslinker within the structure led to an increased rate and amount of drug released. This implies that the efficacy of drug conjugation with respect to drug release is superior to nonconjugated drugs.

Another drug PEG conjugate that has shown enhanced drug efficacy is the paclitaxel (PTX) conjugated with polyethylene glycol (PEG-B-PTX), synthesized by Dong et al. [95]. The antitumor efficacy of the stable micelle of about 50 nm, and 13.3 wt% drug load content was investigated against human glioma and breast cancer cells *in vitro*. The conjugate micelle exhibited improved antitumor effects when compared to the clinically used taxol. This result suggests that the drug conjugate can be a superior alternative for current clinically used PTX nanoformulations which has limitations such as poor *in vivo* stability, premature release, and little improvements in its antitumor efficacy [96, 97]. A detailed *in vivo*

#### **Figure 7.**

*A) The hydrazone acid-labile DOX release behavior from nano/microparticles at pH 7.4. (B) The hydrazone acid-labile DOX release behavior from nano/microparticles at pH 5.5. Sustained release slow drug release at pH 7.4 when compared to acidic pH. Overall slower release when compared to nonconjugated DOX. Adapted with permission from [98].*

**Table 2.** *Cleavage mechanism*

 *resulting in drug release of common bonds employed in covalent conjugation.*

**Bond**

**146**

**Mechanism**

 **of cleavage**

**Method of hydrolysis** Cleavage in the body is catalyzed by esterases

The bond is cleaved via hydrolysis.

[84–90]

Sometimes catalyzed by enzymes like esterase, hydrolase, serine

and

cysteine proteases, peptidases,

Cleavage is via

Bond cleavage is achieved via acid and base catalysis and redox

potential hydrolysis catalyzed by various classes of reductases

enzyme in the presence of excess reduced glutathione

and thioredoxin

Reductase

Drug release is via

ring-opening

 hydrolysis and thiol exchange

 [92, 93]

 (GSH)

[18]

acid-catalyzed

 hydrolysis

[91]

*Nano- and Microencapsulation - Techniques and Applications*

 antibody Fab-BL 125 and RNA

**References**

[83]

experiment by the same research group further revealed that PEG-B-PTX showed prolonged circulation time as well as enhanced *in vivo* antitumor efficacy (tumor inhibition rate of 89.4%) with low side effects. This observation can be attributed to the favorable pharmacokinetic profile and tumor-specific release of the drug from the drug conjugate. This promising study has prompted other groups of researchers to investigate the efficacy of other stimuli responsive PTX conjugates [99–103]. This same set of researchers [95] went further to demonstrate that hydrophobic drugs can be conjugated with a short water-soluble polymer or peptide chain to make the drug amphiphilic. It was proven that the self-assembled nanovehicles are suitable for the delivery of the drug and even co-delivery of other drugs as reported by other research groups [104, 105]. The effects of this approach are wellcharacterized chemical structures, accurate and reproducible drug loading efficiency (i.e., 100%), fixed, and high drug loading contents. Also, burst release of drugs associated with physically drug-loaded micelles can be prevented [106]. These attributes are very important and favorable for clinical translation; therefore, these conjugates have great potential for clinical application.

Irrespective of the excellent efficacy of any drug conjugates, before their application, it is very important that the biosafety is proven and confirmed to be harmless to the human system. Hence, it has become necessary that developed drug conjugates are evaluated for its biosafety. This has led to the investigation of the toxicity of different drug conjugates by various researchers. For instance, an *in vitro* cytotoxicity study was carried out by Tang and fellow workers, and the cytotoxicity of the drug-conjugate was assessed using the MTT assay method [108]. The study investigated the cytotoxicity effect of sorafenib-polyethylene glycol (PEG) nanoparticles conjugate (SFP) on Hela and HepG2, respectively, after incubation for 48 h at 37°C. The result showed a dose-dependent cytotoxic effect of free sorafenib (SF) and SFP on both cell lines, and no significant difference in cytotoxicity was observed for SF and SFP on both cell lines. Nonetheless, a higher cytotoxicity of SFP was displayed between the concentration ranges of 5–15 μM when compared to free SF. The higher *in vitro* cytotoxicity of SFP observed at those concentrations may be due to the increased intracellular localization of SFP nanoparticles. This suggests that the conjugation of sorafenib with a polyethylene glycol (PEG) nanoparticle does not have a negative influence on the toxicity as seen in the favorably cytotoxic effect on both cell lines. Also, the ability of SFP to serve as

a biosafe anticancer therapeutic agent was further confirmed in the

drug conjugate can be attributed to the outer PEG shell of SFP.

*Nano/Microparticles Encapsulation Via Covalent Drug Conjugation*

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

co-workers, respectively [96, 116].

**149**

hemocompatibility and histological safety results. These nontoxic properties of the

Furthermore, drawbacks such as dose-dependent toxicity have been reported with drug conjugates; therefore, in an attempt to minimize toxicity, Li et al. have evaluated the effect of combining drugs onto a nano-drug delivery system on toxicity [107]. The cell cytotoxicity of the DOX and CPT conjugate versus the free drugs showed enhanced uptake in the cancer cells but reduced in the normal cells exposed to the drug-conjugate. Additionally, it was reported that the side effects of the drugs (doxorubicin and camptothecin) employed in the study decreased by reducing the dosage of the drugs. The toxic side effects of the drugs were alleviated, and it was also observed that the multidrug resistance (MDR) was reversed. This may be attributed to the synergistic effects of multiple therapeutic agents

[113, 114]. Remarkably, to assess the general biosafety of drug conjugates, Ibrahim et al. have gone further into *in vivo* investigation of the drug conjugates developed by their group [115]. The organs of mice exposed to these drug conjugates for 21 days were sliced and analyzed histologically. It was noted that exposure to the tissues (heart, liver, spleen, lung, and kidney) did not result in any tissue damage; hence, the biocompatibility of the formulations was confirmed. Other studies that reported little or no toxic side effects of drug conjugates are Dong and Lu with their

In the cytotoxicity study carried by Lu et al., pullulan which is a natural biocompatible polysaccharide was used to synthesize a novel pH-sensitive nanoparticle drug delivery system for the delivery of doxorubicin (DOX) [116]. The chemical structure of the pullulan/DOX conjugate nanoparticles was assessed using FTIR and 1H NMR and further investigated for *in vitro* drug release and cytotoxicity activities, respectively. The result of the release behavior *in vitro* showed that a faster release of DOX was released from the drug delivery at pH 5.0 than at pH 7.4. It was observed that lower concentrations of the drug (DOX) were more cytotoxic to 4 T1 cells than pullulan/DOX conjugate nanoparticles at a concentration range of 0.01– 5 mg/l. This may be due to the ability of free DOX to readily transport into the cells via passive diffusion [117]. On the other hand, low cytotoxicity was reported for DOX released from the nanoparticle. This may be due to a time-consuming DOX release from nanoparticles and delayed nuclear uptake in 4 T1 cells [118]. Lin et al.

Furthermore, other studies have buttressed the potential excellent effect of drug conjugates in combating diseases as seen in the experiment done by Li et al. [107]. Camptothecin and doxorubicin were loaded onto a polymer, and the efficiency of the drug conjugate was explored. A synergistic drug delivery which improved the anti-cancer efficiency of the drugs was reported. The *in vitro* stability study showed that the drugs were stable with 80% drug loading after 4 weeks at a storage temperature of 4°C. In addition, the *in vitro* studies showed an approximately 30% increase in the cellular uptake of the conjugated drugs into the cancer cells when compared to the free drugs. It is noteworthy that a superior anticancer efficacy was observed in the combined drug conjugate, and the enhanced synergistic effects of about 23.9% was attributed to the good better stability profile, internalization by cells and pH response. Other benefits of the drug conjugate observed from this study include suitable sizes and good water solubility. This led to the greater penetration of the drugs into the solid tumors, thus improving the overall efficacy of the drug conjugate when compared to the free drugs. From these results, it is suggested that DOX-CPT conjugated to nano-delivery systems has the potential to provide synergistic anticancer treatment.

The superior efficacy of drug-conjugated covalently compared to nonconjugated drugs has also been proven by Tang and coworkers via conjugating sorafenib with polyethylene glycol (PEG) nanoparticles. The efficacy of the drug conjugate (SFP) was evaluated on cancer cells in an *in vitro* antitumor experiment [108]. The result showed that SFP had an excellent antitumor activity due to the self-immolative release of the intact drug inside tumor cells caused by the GSH-responsive disulfide linker thus suggests that SFP may be a potential candidate for cancer treatment. Additionally, it was reported that the covalent drug conjugation prevented drug leakage and improved drug stability. SFP is therefore a promising nano/micro-carrier for safe drug delivery which may pave room for new opportunities to explore other drug conjugates. Among other studies, Daniel et al. have reported that drug conjugates especially polymer conjugates are potentially suitable for the delivery of antiviral drugs [109, 110]. A typical example is the synthesis of poly(lactide-co-glycolide) nanoparticles loaded with a combination of reverse transcriptase and protease inhibitors. Such drug conjugates have been reported to be effective in preventing the replication process of HIV replication [111, 112]. This implies that drug conjugates are a promising approach in improving the therapeutic efficacy of therapeutic agents, and this may encourage their clinical translation.

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

experiment by the same research group further revealed that PEG-B-PTX showed prolonged circulation time as well as enhanced *in vivo* antitumor efficacy (tumor inhibition rate of 89.4%) with low side effects. This observation can be attributed to the favorable pharmacokinetic profile and tumor-specific release of the drug from the drug conjugate. This promising study has prompted other groups of researchers to investigate the efficacy of other stimuli responsive PTX conjugates [99–103]. This same set of researchers [95] went further to demonstrate that hydrophobic drugs can be conjugated with a short water-soluble polymer or peptide chain to make the drug amphiphilic. It was proven that the self-assembled nanovehicles are suitable for the delivery of the drug and even co-delivery of other drugs as reported

by other research groups [104, 105]. The effects of this approach are wellcharacterized chemical structures, accurate and reproducible drug loading efficiency (i.e., 100%), fixed, and high drug loading contents. Also, burst release of drugs associated with physically drug-loaded micelles can be prevented [106]. These attributes are very important and favorable for clinical translation; therefore,

The superior efficacy of drug-conjugated covalently compared to

nonconjugated drugs has also been proven by Tang and coworkers via conjugating sorafenib with polyethylene glycol (PEG) nanoparticles. The efficacy of the drug conjugate (SFP) was evaluated on cancer cells in an *in vitro* antitumor experiment [108]. The result showed that SFP had an excellent antitumor activity due to the self-immolative release of the intact drug inside tumor cells caused by the GSH-responsive disulfide linker thus suggests that SFP may be a potential candidate for cancer treatment. Additionally, it was reported that the covalent drug conjugation prevented drug leakage and improved drug stability. SFP is therefore a promising nano/micro-carrier for safe drug delivery which may pave room for new opportunities to explore other drug conjugates. Among other studies, Daniel et al. have reported that drug conjugates especially polymer conjugates are potentially suitable for the delivery of antiviral drugs [109, 110]. A typical example is the synthesis of poly(lactide-co-glycolide) nanoparticles loaded with a combination of reverse transcriptase and protease inhibitors. Such drug conjugates have been reported to be effective in preventing the replication process of HIV replication [111, 112]. This implies that drug conjugates are a promising approach in improving the therapeutic efficacy of therapeutic agents,

Furthermore, other studies have buttressed the potential excellent effect of drug conjugates in combating diseases as seen in the experiment done by Li et al. [107]. Camptothecin and doxorubicin were loaded onto a polymer, and the efficiency of the drug conjugate was explored. A synergistic drug delivery which improved the anti-cancer efficiency of the drugs was reported. The *in vitro* stability study showed that the drugs were stable with 80% drug loading after 4 weeks at a storage temperature of 4°C. In addition, the *in vitro* studies showed an approximately 30% increase in the cellular uptake of the conjugated drugs into the cancer cells when compared to the free drugs. It is noteworthy that a superior anticancer efficacy was observed in the combined drug conjugate, and the enhanced synergistic effects of about 23.9% was attributed to the good better stability profile, internalization by cells and pH response. Other benefits of the drug conjugate observed from this study include suitable sizes and good water solubility. This led to the greater penetration of the drugs into the solid tumors, thus improving the overall efficacy of the drug conjugate when compared to the free drugs. From these results, it is suggested that DOX-CPT conjugated to nano-delivery systems has the potential to provide

these conjugates have great potential for clinical application.

*Nano- and Microencapsulation - Techniques and Applications*

synergistic anticancer treatment.

and this may encourage their clinical translation.

**148**

Irrespective of the excellent efficacy of any drug conjugates, before their application, it is very important that the biosafety is proven and confirmed to be harmless to the human system. Hence, it has become necessary that developed drug conjugates are evaluated for its biosafety. This has led to the investigation of the toxicity of different drug conjugates by various researchers. For instance, an *in vitro* cytotoxicity study was carried out by Tang and fellow workers, and the cytotoxicity of the drug-conjugate was assessed using the MTT assay method [108]. The study investigated the cytotoxicity effect of sorafenib-polyethylene glycol (PEG) nanoparticles conjugate (SFP) on Hela and HepG2, respectively, after incubation for 48 h at 37°C. The result showed a dose-dependent cytotoxic effect of free sorafenib (SF) and SFP on both cell lines, and no significant difference in cytotoxicity was observed for SF and SFP on both cell lines. Nonetheless, a higher cytotoxicity of SFP was displayed between the concentration ranges of 5–15 μM when compared to free SF. The higher *in vitro* cytotoxicity of SFP observed at those concentrations may be due to the increased intracellular localization of SFP nanoparticles. This suggests that the conjugation of sorafenib with a polyethylene glycol (PEG) nanoparticle does not have a negative influence on the toxicity as seen in the favorably cytotoxic effect on both cell lines. Also, the ability of SFP to serve as a biosafe anticancer therapeutic agent was further confirmed in the hemocompatibility and histological safety results. These nontoxic properties of the drug conjugate can be attributed to the outer PEG shell of SFP.

Furthermore, drawbacks such as dose-dependent toxicity have been reported with drug conjugates; therefore, in an attempt to minimize toxicity, Li et al. have evaluated the effect of combining drugs onto a nano-drug delivery system on toxicity [107]. The cell cytotoxicity of the DOX and CPT conjugate versus the free drugs showed enhanced uptake in the cancer cells but reduced in the normal cells exposed to the drug-conjugate. Additionally, it was reported that the side effects of the drugs (doxorubicin and camptothecin) employed in the study decreased by reducing the dosage of the drugs. The toxic side effects of the drugs were alleviated, and it was also observed that the multidrug resistance (MDR) was reversed. This may be attributed to the synergistic effects of multiple therapeutic agents [113, 114]. Remarkably, to assess the general biosafety of drug conjugates, Ibrahim et al. have gone further into *in vivo* investigation of the drug conjugates developed by their group [115]. The organs of mice exposed to these drug conjugates for 21 days were sliced and analyzed histologically. It was noted that exposure to the tissues (heart, liver, spleen, lung, and kidney) did not result in any tissue damage; hence, the biocompatibility of the formulations was confirmed. Other studies that reported little or no toxic side effects of drug conjugates are Dong and Lu with their co-workers, respectively [96, 116].

In the cytotoxicity study carried by Lu et al., pullulan which is a natural biocompatible polysaccharide was used to synthesize a novel pH-sensitive nanoparticle drug delivery system for the delivery of doxorubicin (DOX) [116]. The chemical structure of the pullulan/DOX conjugate nanoparticles was assessed using FTIR and 1H NMR and further investigated for *in vitro* drug release and cytotoxicity activities, respectively. The result of the release behavior *in vitro* showed that a faster release of DOX was released from the drug delivery at pH 5.0 than at pH 7.4. It was observed that lower concentrations of the drug (DOX) were more cytotoxic to 4 T1 cells than pullulan/DOX conjugate nanoparticles at a concentration range of 0.01– 5 mg/l. This may be due to the ability of free DOX to readily transport into the cells via passive diffusion [117]. On the other hand, low cytotoxicity was reported for DOX released from the nanoparticle. This may be due to a time-consuming DOX release from nanoparticles and delayed nuclear uptake in 4 T1 cells [118]. Lin et al.

drug conjugation [120]. Surprisingly, it appears that a substantial number of anticancer drugs and polymers are the most explored in covalent drug conjugation compared to other therapeutic drugs and biomaterials. This may be because polymer-drug conjugates provide more advantages in enhancing stability, increasing water solubility, and prolonging blood circulation [121–123]. Despite these advantages, certain drawbacks such as difficulty to accurately control the reaction site and the degree of conjugation have been associated with polymer-drug conjugates. Thus, the ability to reduce the heterogeneity and batch-to-batch difference of the product remains a challenge [75, 124]. It is therefore suggested that more studies to be done to overcome these challenges; this will provide more information on the efficacy and biosafety of drug conjugates (**Figure 9**). Furthermore, reports from these studies revealed that the efficacy and biosafety of the drugs conjugated onto various nano/micro-delivery systems were significantly enhanced when compared to the free drugs. Other nano/micro-delivery systems and drugs that have been explored are summarized in **Table 3**. The table highlights the key findings of various nano/micro-delivery systems that have been reported by different groups of

*Nano/Microparticles Encapsulation Via Covalent Drug Conjugation*

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

**Drug Target Outcome References**

Paclitaxel poliglumex reduced the systemic exposure to peak concentrations of free paclitaxel. In addition, the drug-conjugate produced similar survival to docetaxel as second-line treatment in NSCLC with less febrile neutropenia and alopecia and greater ease of administration.

*In vivo* studies showed a significant increase in the effectiveness of drugconjugate when compared to

HA-cisplatin conjugate bounded to CD44 expressing cancer cell lines (H1299 and H358). The drug-conjugate was more effective in killing lung tumors in mice when compared to the free drug

the free drug.

[125]

[126]

[127]

cell lung cancer (NSCLC)

Pancreatic cancer

cell lines and mice

scientists.

**Figure 9.**

**system**

*Key benefits derived from drug conjugation.*

Polyglutamic acid Paclitaxel Nonsmall-

Gemcitabine and dodecanol

Hyaluronic acid (HA) Cisplatin H1299, H358

**Nano/micro-delivery**

PEG-b-PCC poly(2 methyl-2-carboxylpropylene carbonate)

polymer

**151**

#### **Figure 8.**

*Biosafety evaluation of free cisplatin and prodrug cisplatin using zebrafish embryos. Survival rates of embryos in the presence of (a) the bare drug and (b) prodrug. Hatching rates of zebrafish embryos after the exposure to (c) the bare drug (d) prodrug. (e) Pictogram representation the embryos with treatment of prodrug at different concentrations over a period of 96 hours.*

improved biosafety of profile of cisplatin by converting it a prodrug. The prodrug has higher survival rate of the zebrafish embryos [119] (**Figure 8).**

From the observations made in these studies, it is clear that drug conjugation impacts positively on the efficacy of the drug as well as its biosafety/toxicity. The biosafety concerns are greatly eliminated or minimized by conjugating drugs to biomaterials. Some unwanted side effects, toxicity, and organ damage associated with the fluctuations that arise from periodic drug administration can be avoided by

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

drug conjugation [120]. Surprisingly, it appears that a substantial number of anticancer drugs and polymers are the most explored in covalent drug conjugation compared to other therapeutic drugs and biomaterials. This may be because polymer-drug conjugates provide more advantages in enhancing stability, increasing water solubility, and prolonging blood circulation [121–123]. Despite these advantages, certain drawbacks such as difficulty to accurately control the reaction site and the degree of conjugation have been associated with polymer-drug conjugates. Thus, the ability to reduce the heterogeneity and batch-to-batch difference of the product remains a challenge [75, 124]. It is therefore suggested that more studies to be done to overcome these challenges; this will provide more information on the efficacy and biosafety of drug conjugates (**Figure 9**). Furthermore, reports from these studies revealed that the efficacy and biosafety of the drugs conjugated onto various nano/micro-delivery systems were significantly enhanced when compared to the free drugs. Other nano/micro-delivery systems and drugs that have been explored are summarized in **Table 3**. The table highlights the key findings of various nano/micro-delivery systems that have been reported by different groups of scientists.

**Figure 9.** *Key benefits derived from drug conjugation.*


improved biosafety of profile of cisplatin by converting it a prodrug. The prodrug

*Biosafety evaluation of free cisplatin and prodrug cisplatin using zebrafish embryos. Survival rates of embryos in the presence of (a) the bare drug and (b) prodrug. Hatching rates of zebrafish embryos after the exposure to (c) the bare drug (d) prodrug. (e) Pictogram representation the embryos with treatment of prodrug at different*

From the observations made in these studies, it is clear that drug conjugation impacts positively on the efficacy of the drug as well as its biosafety/toxicity. The biosafety concerns are greatly eliminated or minimized by conjugating drugs to biomaterials. Some unwanted side effects, toxicity, and organ damage associated with the fluctuations that arise from periodic drug administration can be avoided by

has higher survival rate of the zebrafish embryos [119] (**Figure 8).**

*Nano- and Microencapsulation - Techniques and Applications*

**Figure 8.**

**150**

*concentrations over a period of 96 hours.*


breast cancer

showed a 5.5-fold greater tumor accumulation of drug**6. Effect of covalent drugs conjugation on the pharmacokinetic profile**

Hyaluronic acid, Paclitaxel (PTX) Breast cancer PTX conjugate showed more

*Nano/Microparticles Encapsulation Via Covalent Drug Conjugation*

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

Oxaliplatin Mouse model

of human carcinoma cell line KB.

mucosal

effect of various anticancer drugs such as doxorubicin.

The pharmacokinetic profile of drugs is a very crucial aspect that is considered for clinical application. It is interesting to observe that the pharmacokinetic profile of drugs is becoming better due to covalent drug conjugation as demonstrated by drugs encapsulated to nano/micro-delivery systems. The conjugation of drugs to biomaterials has opened opportunities to alter the pharmacokinetics and biodistribution of the drugs within the human body [120]. The alteration of the pharmacokinetics of the drug offers advantages such as prevention of the rapid clearance or metabolism of the drug. In addition, the drugs are carried to the targeted site of pharmacological action. Drug conjugation to nano/micro-delivery system has shown to be a powerful technique that can alter the pharmacokinetic profile of the drug, thus minimize the side

**Drug Target Outcome References**

conjugate compared to the clinically administered DTX formulation (Taxotere). Drug-conjugate also showed a two-fold improvement in anticancer activity in a murine EMT-6 breast cancer model compared to Taxotere.

enhanced *in vivo* tumor inhibition effects compared

The antitumor efficacy of drug-conjugate was superior to that of oxaliplatin. Also, the animals did not develop acute cold hypersensitivity, which is frequently experienced by patients after oxaliplatin administration.

The drug-conjugate suppressed lung mucosal toxicity after pulmonary delivery, whereas the administration of the free drug-induced significant

toxicity.

[138, 139]

[140]

[141]

to free PTX.

One of the studies that reported the enhanced pharmacokinetic properties of anticancer drug encapsulated to a nanoparticle is that conducted by Vandriess et al. [80]. It was observed that the nanoparticles containing the drug enhanced the drug accumulation and a subsequent reduction of tumor growth in an *in vivo* zebrafish model. Also, another study has revealed that self-assembling drug polymer conjugates which allows a covalent attachment of the drug to the hydrophilic part of the polymer can improve the pharmacokinetic profile of drugs. Thus, covalently

**of the drug**

**Table 3.**

**153**

**Nano/micro-delivery**

PEG-b-poly(glutamic acid) micelle

PEG Alendronate Lung

*Key observations made from other drug conjugates.*

**system**


**Table 3.**

**Nano/micro-delivery**

Poly(styrene-comaleic

methacrylate) (PMMA)

Chimeric peptides

Poly(amidoamine) (PAMAM)

Poly(2-ethyl-2 oxazoline)

**152**

(CPs)

**Drug Target Outcome References**

Arterial infusion therapy with poly(styrene-co-maleic

*In vivo* experiment showed that the Gem-conjugates reduced tumor growth by 68% with little toxicity while free Gem had no effect but significant toxicity.

was significantly greater in the group treated with drugconjugate compared to the standard therapy group at 24 and 48 weeks post-treatment (33.3% vs. 10.5% and 35.7% vs. 10.5%, respectively; P< 0.05 for both).

safe in the lungs and revealed comparable blood coagulation times compared

Increased intratumoral accumulation of the conjugate with a curative effect in 60% of the treated mice was observed

Prolonged lung retention of drug-dendrimer conjugate compared to free drug. Improved chemotherapeutic activity on the lung of mice compared to the free drug

POZ-conjugated rotigotine showed the potential to be viable for subcutaneous treatment for PD patients.

within tumors via the EPR effect, and a significant antitumor activity was observed compared to free

Drug-conjugate showed no signs of toxicity and maximally lowered blood calcium levels when compared to calcitonin alone

Biodistribution studies showed a 5.5-fold greater tumor accumulation of drug-

doxorubicin

to free heparin

[128]

[129]

[130]

[131]

[132]

[133]

[134]

[135]

[136]

[137]

acid)-conjugated neocarzinostatin SMANCS/ Lpd showed to be effective for large renal cell carcinoma

Neocarzinostatin Liver and

*Nano- and Microencapsulation - Techniques and Applications*

Gemcitabine A549 cell-

PEG Interferon α2a/b Hepatitis B The HBsAg clearance rate

Glycol chitosan (GC) Heparin Lungs, mice GC-heparin conjugates were

Lewis lung cancers

melanoma (B16-F10) lung metastases

disease

Glycol chitosan Doxorubicin Drug-conjugate accumulated

rats

breast cancer

Doxorubicin 4 T1 and

Doxorubicin Mice bearing

Rotigotine Parkinson

Carbopol®(CP) Calcitonin A549 cells;

Carboxymethylcellulose Docetaxel EMT-6

renal cancer

derived xenograft murine model

**system**

Poly(methyl

polymer

acid

*Key observations made from other drug conjugates.*
