DISULFIDE BRIDGING: A POTENT STRATEGY FOR ANTI-TUMOR DRUG DELIVERY

DISULFIDE BRIDGING: A POTENT STRATEGY FOR ANTI-TUMOR DRUG DELIVERY

Amardeep Kaur, Shikha Dhiman and Manu Sharma *

M. College of Pharmacy, M. M. Deemed to be University, Mullana, Haryana, India.

ABSTRACT: To increase the efficacy of present anti-tumor agents, tumor-targeting can be proved as a successful approach for effective and safer drug delivery with less toxicity. According to researchers, for an effective tumor targeting module, disulfide linkage plays an important role in providing a medium for conjugating an anti-tumor drug with a carrier like vitamins and fatty acids. Disulfide bonds with unique chemical and biophysical properties can be used as cleavable linkers to deliver chemotherapeutic drugs. From the literature review, it has been observed that a disulfide bond acts as a self-immolating linker, which gets cleaved inside the body in the presence of intracellular glutathione; otherwise, it is stable in blood circulation. In this review, we discussed various strategies available in literature using disulfide as a linker in potent tumor targeting agents. Various successful conjugates are described here, which are currently under clinical trials.

Keywords: Disulfide bridging, Drug release, Glutathione, Tumor targeting

INTRODUCTION: In drug development, one of the most promising strategies which are currently being employed is tumor-targeting drug design. Among the various fatal diseases, the second major cause for the death is cancer. Even after different progressive approaches and discovering new technologies for cancer treatment, researchers are still unable to discover an indefinite treatment to eradicate this malignant condition. Another major problem in chemotherapy is a deficiency of tumor cell specificity. A cytotoxic agent can kill fast-growing tumor cells, and traditional chemotherapy relies on this premise. But these cytotoxic agents lack specificity and results in systemic toxicity, which in return causes severe side effects that are undesirable like hair loss, liver damage, kidneys and bone marrow dysfunction. In order to overcome these issues from the past three decades, drug delivery protocols and systems are being explored. A tumor-targeting drug delivery system is developed in the form of a conjugate, and this conjugate is generated by connecting cytotoxic agents directly or via a suitable linker to tumor recognition moiety. The conjugate must be non-toxic hence;, the linker should be stable in blood circulation and only gets cleaved inside cancer cells. Active cytotoxic agent gets regenerated when conjugate gets cleaved upon cell internalization into cancer cells. Linkers are most frequently classified into three classes, i.e., a) hydrazine linker, b) peptide linker c) disulfide linker.

The requirement of cytotoxic agents and type of cancer helps in the selection of an appropriate linker. Each of the linkers has several advantages and disadvantages and is universal in nature ¹. The disulfide linkers have attracted a lot of attention in the past few years. These disulfide linkers can be cleaved at a higher concentration of glutathione. Because the concentration of glutathione is much higher in tumor cells than normal cells, the use of disulfide linkers is of keen interest for researchers nowadays for synthesizing disulfide-linked anti-tumor conjugates. Efficacy of disulfide linkers as compare to other linkers against several tumor xenografts in pre-clinical models has been proved ². In various chemical and biological agents that show potent reactivity or possess pharmacological activities like anti-tumor activities, disulfide bond (-S-S-) is an extremely valuable functional group ^{3, 9}. It has already been detected that various proteins, oxidized glutathione, and numerous naturally obtained products, including some drugs i.e., Mitomycin, leinamycin consists of disulfide bond ^{10, 11}. In this review, we focused on the benefits of using disulfide linkage in tumor-targeting agents. Disulfide bond can degrade inside the human body by different mechanisms, which are briefly explained. This review can be helpful in a comprehensive understanding of this bond, its various merits and how it can be employed in tumor targeting by serving as a linker.

Principles Underlying Disulfide Linkage: Being a biodegradable linker disulfide has gained the keen interest of researchers in the past years and can be employed in controlled drug release medications. Also, disulfide is easy to prepare in the lab, better tolerated by blood plasma and possesses higher sensitivity inside the tumor environment, and these properties contribute into the importance of this bond as in tumor-targeting agents. Recently, it has been reported that drugs can be released in a spatially controlled manner using stimuli-responsive drug delivery systems. Unique microenvironment properties like acidic pH values, high concentration of certain enzymes of tumor tissues, and a more reductive environment of these tissues can be used to design stimuli-responsive drug delivery systems. Intracellular glutathione concentration is in millimolar range (1-10mM) inside the human body, ¹² but outside cells in common fluids it is at (20-40 µM) levels, ^{13, 15} like plasma, except respiratory tract lower part fluid lining. To act as an interface between extracellular space and cellular membrane, glutathione (GSH) can be translocated to the surface of cells ^{16, 19}. Interestingly, inside cancer cells, GSH concentration is on higher level than that of normal cells. This important fact can be employed to develop anti-tumor drug delivery systems (DDS) ^{20, 24}. Two thiols can result in the formation of a disulfide bond upon oxidation. Under mild oxidative and physiological pH, the disulfide bond is stable but is susceptible for disulfide-thiol exchange reaction ²⁵ and can only be reduced in the presence of GSH ²⁶.

Thus, research is ongoing on reduction responsive drug delivery systems based on these theoretical facts that extracellular GSH concentration is substantially lower than the intracellular glutathione (GSH) concentration ²⁷ and additionally, due to several times higher GSH concentration in tumor tissue than that in normal tissues made this environment more reductive ²⁸. Also, in the extracellular space of proteins, disulfide bonds present are oxidative, but due to the abundance of free thiols inside cells, these disulfide bonds get cleaved, e.g., glutathione (which is most abundant). Glutathione disulfide (GSSG) is another main biological disulfide compound that is formed by oxidation of GSH Fig. 1 ²⁹. The oxidized GSSG can get reduced back due to the presence of NADPH-dependent enzyme glutathione reductase, and it helps in maintaining cellular redox homeostasis ^{30, 32}.

FIG. 1: INTERCONVERSION OF GLUTATHIONE (GSH) AND GLUTATHIONE DISULFIDE (GSSG)

From the literature it has been also seen that this covalent disulfide bond can be cleaved under the presence of reducing agents like dithiothreitol (DTT), mercaptoethanol and tris-(2-carboxyethyl)-phosphine (TCEP) ^{33, 34}. But, even under harsh extracellular conditions, disulfide bond is relatively stable ^{35, 36}. This disulfide bond can get cleaved reversibly inside cells through a thiol-disulfide exchange reaction and it can be described as ³⁷.

2R-SH + R’-SS-R’ ↔ R-SS-R + 2R’SH

This reaction is an SN2 type nucleophilic reaction. Sulfur gets attached by a deprotonated thiolate anion which acts as nucleophile ³⁸. Disulfide bond also plays an important role in protein folding and enhancing their stability as disulfide bond results in a reduction of entropy of denatured state ^{39, 40}. They play a key role in the transformation of molecular oxygen into reactive oxygen species (ROS), which are part of many biological processes ⁴¹.

Some other reviews about disulfide focused on it as therapeutic gene delivery system and thus worked on its evaluation for therapeutic efficacies ^{42, 47}. Recently, disulfide linkers are now being used for intracellular drug release as drug delivery systems ^{48, 52}.

Mechanism of Drug Release via Disulfide Bond: In targeted anti-tumor agents, disulfide bonds act as primary linkers, which help in the conjugation of the drug. It is known that other linkers like amide, ester and hydrazine get cleaved through hydrolytic cleavage, but unlike them, disulfide gets cleaved via electrochemical reduction reaction by yielding respective thiol or through thiol-disulfide exchange reaction ^{53, 55}. Endogenous thiol molecules like cysteine, homocysteine, N-acetylcysteine, glutathione (L-γ-glutamyl-L-cysteinyl-L-glycine; GSH), other cysteine-containing peptides, and thioglycolic acid trigger a chemical mechanism which initiates cleavage reaction ⁵⁶.

FIG. 2: REPRESENTATIVE ENDOGENOUS THIOLS THAT ARE INVOLVED IN DISULFIDE EXCHANGE REACTIONS FOR DRUG RELEASE IN THE CYTOPLASM VIA DISULFIDE CLEAVAGE.

Due to the stability of disulfide linker in intracellular space, targeted drug delivery agent linked with it gets easily taken up because after the endocytosis in the cytoplasm, primarily, a disulfide cleavage occurs. Furthermore, redox machinery present on the cellular surface does not contribute to drug release via disulfide linker cleavage unless otherwise modified, but instead of that, it gets cleaved by thiol triggered intracellular mechanism ⁵³. The mechanism involved in disulfide exchange reactions for drug release in the cytoplasm via disulfide cleavage is explained in Fig. 2.

There was another study about the reduction of disulfide bonds inside the cells, and that serves as a mechanism of disulfide reduction ⁵⁷. One of the reasons for the reduction of many protein disulfide bonds can be thioredoxin (Trx). The reduction occurs via the formation of intermediate mixed disulfide and Trx-sulfate attack on the N-terminal of Trx-Cys. This occurred via the nucleophilic reaction mechanism on the Sulfur group of the terminal, which is related to the mixed disulfide bond. AS there is the supply of NADPH, thioredoxins reductase (TrxR) helps in recycling of Trx Fig. 3A. Glutaredoxin (Grx) catalytically reduces glutathionylated protein thiols via a nucleophilic attack on the G sulfur by Grx N-terminal Cys transfer of protein-bound glutathione to Grx occurs. The catalyzed reduction by NADPH with the help of glutathione oxidoreductase (GOR), the oxidized glutathione (GSSG) is recycled Fig. 3B by following dithiol mechanism protein disulfides are reduced by Grx by attacking on Cys presented on the N-terminal of Grx substituted on disulfide group, which in turn gave rise to a mixed disulfide after nucleophilic attack.

Same as that of the intramolecular procedure followed for Trx, C-terminal Grx Cys also reduced mixed disulfide Fig. 3C ⁵⁸.

FIG. 3: GENERAL MECHANISM FOR DISULFIDE REDUCTION

Disulfide Linkage-Employing Tumor-Targeting Drug Delivery:

Disulfide Linkage in Peptide-Based Strategies for Tumor Treatment: Genetic mutations and epigenetic alterations are being considered as one of the causes of cancer.

On the basis of this theory, Lu et al. have explored on Peptide T7-modified polypeptide with disulfide bonds for targeted delivery of plasmid DNA for gene therapy of prostate cancer by targeting plasmid DNA (pDNA). They employed a cationic gene vector for tumor targeting, called as disulfide crosslinked arginine-aspartic acid peptide modified by HAIYPRH (T7) peptide (CRD-PEG-T7).

The results showed that nanosized positively charged CRD-PEG-T7-plasmid DNA complex Table 1 have efficient cellular efficacy, minimum cellular toxicity and in comparison to CRD-PEG plasmid DNA complex have dual targeting effect. This new modified drug delivery agent had four times higher capacity to target over expressed transferrin receptor (TfR) on tumor cells in comparison with non-modified system. These results ensured that CRD-PEG-T7-plasmid DNA may act as significant gene delivery system by targeting metastatic cancer ⁵⁹. Kotamraju et al. proposed an idea that efficacy of targeted gene cancer therapy can be increased by delivering an anti-tumor agent to the tumor interior using tumor-homing peptides which have properties to penetrate tissues. LyP-1 (CGNKRTRGC) and iRGD (CRGDKGPDC) were used by them for formation of this type of peptides. Conjugation of these peptides with a tumor targeting agent can be done by forming the cyclizing disulfide bond with cysteines presented in lipid structure. LyP-1, iRGD and CRGDC (GACRGDCLGA) peptides with a cysteine or maleimidohexanoic acid added externally at N-terminus of the sequences had been synthesized by them, with retaining tumor-homing properties of the peptide and the biological activity, subsequent conjugation to payloads yielded stable compounds. The expected results were obtained by performing studies on nude mice bearing orthotropic 22Rv1 tumors, Mice bearing MCF10 Ca1A human breast cancer xenograft tumors and intracellular oligonucleotide delivery to HeLa cells which determined that conjugates have the desired activities and efficacies. These conjugates can act as a powerful tool for disulfide-bridged peptide-mediated imaging, targeted drug delivery, and other applications ⁶⁰.

TABLE 1: DISULFIDE AS A LINKER IN PEPTIDE BASED STRATEGIES FOR ANTI-TUMOR PROPERTIES

S. no.	Disulfide Linkage in peptide based strategies	Ref.
1	1	59
2	2 (iRGD peptide with a third cysteine) 3 (LyP-1 peptide)	60

Versatile Disulfide Bond in Nanoparticle-Based Strategies for Tumor Treatment: The development of strategies for the functionalization of nanoparticles is crucial for future clinical use to improve anti-cancer therapies but biological barriers (extracellular and intracellular) can cause hindrance in the delivery of active anti-tumor agents by using this strategy. To explore this angle Wen et al., (2014) researched and stated that to overcome biological barriers (extracellular and intracellular), targeted drug delivery systems are required so that they can deliver the therapeutic drug to a specific biological site. This study showed that extracellular barriers could be overcome by highly stable vehicles, have sustained drug release in the blood circulation, and accumulate at the site of action.

Intracellular barriers determine effective cellular internalization, controllable release, and endosomal escape. Thus, in case to develop a comprehensive targeted drug delivery system, both extracellular and intracellular barriers are critically important.

As the structures of nanoparticles (NPs) can be tailored to release the therapeutic drug at the action site by overcoming the biological barriers, nanoparticles (NPs) based drug and gene delivery systems can be prepared. Wang et al., (2014) prepared newly self-assembled nanomedicines through entirely different mechanisms and explored another prospect of this field.

According to previous studies, stable nanoparticles cannot be prepared by using hydrophobic molecules as they can’t self-assemble themselves; thus, amphiphilic or ionic materials were required for their functionality and stability in-vivo.

To balance the competition between intermolecular forces involved in nanomedicines self-assembly, they developed disulfide-induced nano-medicines (DSINMs) by inserting a single disulfide bond in hydrophobic molecules that improved their stability.

A number of first-line chemotherapy drugs (doxorubicin, paclitaxel, gemcitabine, and fluorouracil), derivatives of small-molecule natural products, and fluorescent probes were developed by using this strategy ⁶². Lattore et al. in 2014, carried out a study which was a general approach for the controlled and selective release of targeted drugs by multi-functionalization of magnetic nanoparticles (MNPs) using anti-cancer drugs (Gemcitabine and Doxorubicin) and targeting moieties. By the formation of disulfide bonds between MNPs and drugs derivatives, functionalization was achieved.

Their strategy was to introduce a pyridyl disulfide group to the anti-cancer drugs, which can react with -SH groups of already activated MNPs.

Under a highly reducing intracellular environment, the drugs get released without any chemical modification by cleavage of the linker. In the intracellular conditions, the release occurred within 5-8 h, while in extracellular conditions, a negligible percentage of release was observed.

This approach can be further used for nanoparticle functionalization in several types of anti-cancer and targeting agents ⁶³.

In bladder cancer, there has been no significant improvement over the last three decades as the drugs associated with it have moderate efficacy but more toxicity. Pan et al. developed nanometer-scale micelles loaded with paclitaxel (PTX) which were found to be bladder cancer-specific and were estimated its anti-cancer activity and toxicity.

Bladder cancer-specific targeting micelles were synthesized that are cross-linked with disulfide bonds to remain stable during blood circulation and coated with PLZ4 bladder cancer targeting ligand for cancer-specific drug delivery.

DC-PNM-PTX showed stability in sodium docecyl sulfate (SDS) solution, but after the addition of glutathione got dissolved within 5 min at a physiological intracellular concentration of 10 mM.

Patient-derived bladder cancer xenografts (PDXs) were used for in-vivo targeting and anti-cancer activity using immunodeficient mice. DC-PNM (disulfide-cross linked PLZ4-nanomicelle) specifically delivered a higher drug load to patient-derived bladder cancer xenografts than to lung cancer xenografts in the same mice after intravenous administration.

At the same dose, DC-PNM-PTX (disulfide-cross linked PLZ4-nanomicelle loaded with paclitaxel) was more effective than free PTX in prolonging the progression-free survival as well as the survival of mice carrying patient-derived xenografts.

The study was also helpful to show that cisplatin resistance was overcome by DC-PNM loaded with PTX and median survival got improved to 69.5days in comparison to 55 days of free PTX ⁶⁴.

The various examples of a versatile disulfide bond in nanoparticle-based strategies are described in Table 2.

TABLE 2: VERSATILE DISULFIDE BOND IN NANOPARTICLE-BASED STRATEGIES

S. no.	Disulfide Linkage in nanoparticle-based strategies	Ref.
1	4 (mPEG-SS-PLL)	61
2	5 (PTX-SS-VE) 6 (DOX-SS-VE) 7 (DOX-SS-SA)	62
3	8, 9 (Drug = DOX or GEM)	63

Disulfide Bond Employed in Anticancer Agents used as the agnostic Agents: The agnostics refer to an appealing new approach to drug development wherein the therapeutic modalities are combined with those associated with diagnostic imaging, and it can be extended to the subcellular level by enhancing efficacy while facilitating imaging. This type of drug delivery system contains both an active drug (for efficacy) and a fluorophore (for ease of monitoring uptake and delivery). In principle, this approach would permit drug delivery and release to be monitored directly. In 2012, Lee et al., with emphasis on this study, synthesized RGD peptide-appended naphthalimide pro- camptothecin (CPT) conjugate using disulfide bond as a cleavable linker, naphthalimide moiety for fluorescence, RGD cyclic peptide for tumor targeting, and CPT as an active tumor agent. Within the endoplasmic reticulum of U87 cells, CPT payload was released via RGD-dependent endocytosis mechanisms as it was revealed by fluorescence-based colocalization under endoplasmic reticulum-selective dye ⁶⁵. In the same way, Maiti et al. in 2013 formed a conjugate in which biotin was used as a cancer-targeting unit, Gemcitabine (GMC), as a model active drug a coumarin moiety as a fluorescent reporter, and a thiol-specific cleavable disulfide bond. Studies were conducted on A549 cells, and the conjugate was released in the lysosome.

TABLE 3: EXAMPLES OF DISULFIDE BOND EMPLOYED IN ANTI-CANCER AGENTS USED AS THERAGNOSTIC AGENTS

S. no.	Disulfide bond employed in anti-cancer agents used as theranostic agents	Ref.
1	10	65
2	11	66
3	12	67

The thiol-induced disulfide cleavage of conjugate occurred based on the mechanism of receptor-mediated endocytosis ⁶⁶. Bhuniya et al. (2014) explored the self-immolative cleavage of disulfide on exposure to biological thiols using this same principle of “all in one” prodrug and pharmacological studies for biotinylated piperazine-rhodol conjugate was performed in which anti-cancer drug SN-38 was used as the active agent.

The viability of the design was tested under controlled chemical conditions, and in-vitro and in-vivo studies were used to monitor the release and delivery of the SN-38 payload and mice xenografts ⁶⁷. The various disulfide bond employed anti-cancer agents used as theragnostic agents are shown in Table 3.

Disulfide Linkage in Copolymer Based Strategy for Tumor Treatment: By using disulfide linkage, a copolymer-paclitaxel conjugate was synthesized and a novel controlled drug delivery system developed by Chen et al. in 2012.

Acrylate derivatives were radically polymerized using copolymer-like polyethylene glycol (PEG), and for developing a structural backbone, carboxyl groups were used, which were further followed by conversion of tert-butyl into carboxyl groups on hydrolyzing Fig. 4.

Carboxyl group presented on paclitaxel was used as an active site for reaction, employing disulfide bond that was covalently linked to backbone of a copolymer and 32 wt% of paclitaxel was loaded.

FIG. 4: DEVELOPMENT OF PACLITAXEL LOADED COPOLYMER VIA DISULFIDE BOND

This drug-loaded copolymer complex in an aqueous solution gets self-assembled into spherical micelle, in which the core represented hydrophobic drug while the shell was hydrophilic PEG. Optical techniques were used to measure the mean diameter of micelles, including transmission electron microscopy and dynamic light scattering, and it was found to be 60 nm. Biocompatibility and suitability to use copolymers as a drug carrier were proved by in vitro cytotoxicity studies.

The synthesized copolymer was proven to be chemotherapeutic towards kidney tumor cells, i.e., OS-CR-2 cells but had not shown any toxicity towards normal human cells. This result showed stability of disulfide inside normal cells but readily cleavable in cancer cells. These properties showed that these copolymers could be proved favorable as they were less toxic and have reduced drug side effects ⁶⁸.

Versatile Disulfide Linkage in Antibody-Drug Conjugates: Disulfide bonds have a bio activating connection in imaging as well as in tumor management therapy.

FIG. 5: ANTIBODY-DRUG CONJUGATES HUC242-DM4 AND HUC242-DM1

The first successful cases of disulfide Linkage in antibody-drug conjugates were reported by Chari et al., including two conjugates Hu C242-DM1 and HuC242-DM4 Fig. 5. Against the antigen-expressing COLO 205 cells during the in-vitro evaluation of both conjugates, it was demonstrated that the conjugates possess tumor-targeting potency, and it was about 1000-fold less cytotoxic toward the antigen-negative human melanoma A-375 cell line while the maytansinoid conjugate HuC242-DM4, bearing a sterically hindered disulfide, was about 2-fold more potent than HuC242-DM1. Based on the promising efficacy, HuC242-DM1 (Cantuzumabmertansine) and HuC242–DM4 was further administered to clinical evaluation in cancer patients ⁶⁹.

FIG. 6: ANTIBODY-DISULFIDE CONJUGATES A) HETEROGENEOUS LYS CONJUGATES, B) DECOUPLING STABILITY AND RELEASE

Disulfides have circulation stability and intracellular release, but in addition, their higher stability caused a corresponding decrease in bond cleavage and release of payload. But, an antibody can make it reversibly stable.

Pillow et al. (2017) examined that by attachment of small molecule in an antibody with unhindered disulfide was stable in circulation yet internalization inside cell and catabolism of antibody generated a rapidly reducible disulfide catabolite Fig. 6. This property can be used for the stable and facile release of payload, and also reversible nature offers an improved way for targeting delivery and therapeutic effects ⁷⁰.

Innovative Disulfide Linkage in Biotin-Anticancer Drug Conjugate: Chen et al. (2010) developed a tumor-targeted advanced drug delivery approach on the basis of mechanism and specificity that will target a particular tumor using receptor-mediated endocytosis according to the vitamin used in conjugate developed. This developed conjugate was made tumor-specific using biotin as a vitamin linked to a taxoid, i.e., SB-T-1214 via cleavable disulfide linker Table 4.

Receptors for biotin were over-expressed on the surface of tumor cells on which site-specific conjugate attacked. The drug was released via glutathione-triggered self-immolated cleavage of disulfide following receptor-mediated endocytosis upon cell internalization. To monitor all the above processes, confocal fluorescence microscopy (CFM) and flow cytometry were done using L1210FR leukemia cells.

Cell specificity was examined using biotin-linked-taxoid-fluorescein on three different cell lines, i.e., a) for which did not have any overexpressed biotin receptors (L1210 cell lines), b) in which negative biotin receptor and healthy mammalian lung fibroblast present (W138 cells), and c) in which overexpressed biotin receptors are present (L1210FR cell lines) and results showed that this molecular probe was highly specific only for L1210FR cells. The anti-tumor activity of the molecular probe was examined against these three cell lines to find the correlation with cell-specific drug delivery. In the same way cell toxicity and specificity for conjugate i.e. biotin-linker-SB-T-1214, was determined against the same three cell lines, and excellent results were obtained ⁷¹. Such strategic design of disulfide linkers can be readily applicable to a range of tumor-targeting drug conjugates. In 2014, another tumor-targeting DDS was developed by Ojima’s group on the basis of 1,3,5-triazine center, and there are three side chains on it, from which one is connected with biotin and the other two connected with two anti-cancer agents bearing a cleavable linker i.e. camptothecin and SBT- 1214 (having different anti-tumor mechanisms).

Usage of two drugs produces synergistic effects and helps in increasing the pharmacological effects of the conjugate. Conjugate gave positive responses towards MX-1, MCF-7, ID8, L1210FR, LCC6-WT, LCC6-MDR, MDA-MB 231, and SkBr3 when examined by CFM and Flow cytometry tests. It also showed impressively low IC₅₀ values (3.22-9.80 nM) for biotin receptor-positive cancer cell lines while high IC₅₀ values (705 nM) in a normal cell line (WI38) during cytotoxicity experiments ⁷². Peptides may have extre

mely diverse sequences, and peptide-based drugs can be designed to have enhanced target selectivity with fewer side effects in various physiological and pathological processes. Considering this into fact, Kim et al. (2014) proposed a work conjugating a peptide with biotin through a disulfide linker. Holliday junction (HJ) inhibitor peptide 2 (KWWCRW) has limited pharmaceutical applications as it has poor membrane permeability, low bioavailability, and metabolic instability.

By cross-linking the peptide to a biotinylated naphthalimide conjugate with a cleavable disulfide linker. Biotin helps increase the bioavailability of the conjugate as it can directly transport biotin-targeting units into tumor cells via a biotin-selective uptake system. HepG2 and WI-38 cell lines were used to check the stability and selectivity of the conjugate.

Also, it was confirmed by dose-dependent cytotoxicity experiments that this conjugate produced a higher anti-tumor effect than the HJ inhibitor peptide-2 itself. 73The structures of disulfide-linked biotin-anticancer drug conjugates are mentioned in Table 4.

TABLE 4: DISULFIDE LINKAGE IN BIOTIN-ANTICANCER DRUG CONJUGATES

S. no.	Disulfide Linkage in Biotin-drug Conjugates	Ref.
1.		71
2.		72
3.		73

Innovative Disulfide Linkage in Folate-Drug Conjugates: Like biotin, folic acid (FA) is another essential vitamin that can be used for synthesizing tumor-targeting agents. Because FA-linked molecules are efficiently bound and internalized by FR-expressing cells, there is the possibility of using FA to target a small molecule drug to FR-over-expressed tumors. On concentrating this concept, Leamon et al. (2005) designed and evaluated a conjugate in which folate was linked with mitomycin C via disulfide linkage, named EC72, and FR-positive cells were used to examine its anti-tumor efficacy. On the coupling of folic acid-γcysteine with 7-N-modified MMC through a cleavable disulfide bond, EC72 was synthesized. This conjugate expressed dose-dependent activity against folate receptor (FR)-positive cell lines in-vitro and also retained higher affinity towards FR-positive cells.

Two reasons that can be considered for EC72’s targeted and specific activity for the FR can be (i) biological activity blockage due to excess folic acid and (ii) unresponsiveness of this drug towards FR-negative cell lines. EC72’s activity was confirmed initially by in-vivo tests in xenograft models. No evidence of myelosuppression or toxicity was observed even on 30 consecutive days of daily dosing of the conjugate to major organs, including the FR-positive kidneys, which made these results significant ⁷⁴.

Reddy et al. in 2006 further carried out investigations on this compound and found that it exhibited no toxicity, but the therapeutic effect was clearly seen against M109 tumors; even Fr-positive kidneys were free from any harmful effects. By comparing this conjugate with EC110 (a folate-MMC conjugate in which amide bond was used for conjugation), they also provided an example of how bearing a cleavable bond like disulfide is important, as EC110 was not able to produce M109 tumor activity.

They also revealed that with an increase in the size of the subcutaneous tumor, the therapeutic potential of EC72 gets decreased. They tried combination therapy using paclitaxel to overcome this limitation, which helped enhance anti-tumor efficacy and decrease toxicity and well-tolerated dose levels. Another more advanced folate-MMC conjugate, named EC118, was also developed bearing two linkers, i.e., disulfide bond, which is reducible, and hydrazone bond, which is acid labile. This resulted in more significant results towards a large number of tumor regressions in the case of M109 tumors, which was apparently less in EC72 ⁷⁵.

In 2007, Leamon et al. worked on another strategy by developing a molecule by using two different active anti-tumor agents with a different mechanism of action, but attached to the same ligand. It was a new type of tumor-targeted agent. This folate receptor (FR)-targeted agent was named as EC0225 and represented the “first in class” multidrug. It was constructed using a single folate molecule, extended by a hydrophilic peptide-based spacer, which was further connected to Mitomycin and Vincaalkaloid units through two separate disulfide linkers.

In-vitro studies of conjugate showed potent, dose-responsive activity and on the administration of well-tolerated dosing regimens produced curative activity against FR-positive xenograft tumors. It was observed that EC0225 can even cure tumors as large as 750 mm³ in volume when used to treat mice ⁷⁶. In the same year, Reddy et al. developed another molecule EC131, a new folate receptor (FR)-targeted drug conjugate, using potent microtubule-inhibiting agent maytansinoid DM1 and combining it with folic acid (FA) via an intramolecular disulfide bond.

EC131 retained its efficacy towards FR-positive cells when tested on cells in culture and gave FR-specific cytotoxicity having IC₅₀ value in the low nanomolar range ⁷⁷. The structures of disulfide-linked folate-anticancer drug conjugates are mentioned in Table 5.

TABLE 5: DISULFIDE LINKAGE IN FOLATE-ANTICANCER DRUG CONJUGATES

S. no.	Disulfide Linkage in Folate-drug Conjugates	Ref.
1.	19 (EC72)	74
2.	20 21	75
	22	76
	23	77

Steric Effects of Disulfide Bond: Reactions that proceed through SN2 type mechanism, steric factors play a major role in their kinetics because of a crowded transition state structure. For the best orientation of their molecular orbitals, middle and leaving moieties should have linear conformation ^{78, 80}. Bulky functional groups can hinder access to attacking thiol, and it increases activation energy. Furthermore, the binding of a substrate can be stabilized by steric interactions ^{81, 82}. Moreover, it has also been seen that steric factors can make disulfide linkage more labile by accelerating their interchange reactions due to increased strain development on disulfide bond ⁸³. When an external -SH group is substituted into a disulfide structure, an SN2 mechanism is preferred theoretically. The rate of disulfide cleavage can be diminished by an increase in steric blockage. Sterics formed by groups adjacent to disulfide bonds affect the drug's biological and non-biological therapeutic effect conjugates ⁸⁴.

Successful Cases of Disulfide Linked Anticancer Agents: As mentioned in research studies, the efficacy of tumor-targeted drug conjugates is due to its therapeutic warhead, hence according to drug payloads, disulfide-linked tumor-targeting agents are classified.

These highly efficient and potent anti-tumor agents that are used for designing tumor-targeting agents are paclitaxel, vinblastine, epothilone, mitomycin C, tubulysin, and camptothecin derivative SN-38 Fig. 7.

FIG. 7: ANTI-CANCER AGENTS USED IN DESIGNING TUMOR-TARGETING AGENTS HAVING DISULFIDE LINKAGE

By using the above-mentioned active cancer treating agents, till date, many drug conjugates bearing disulfide have been reported that have successfully entered into clinical trials.

Vinblastin: In 1958 it was first isolated from Catharanthus roseus and used for the treatment of bladder, non-small cell lung, and brain cancers. 85 By inhibiting the assembly of microtubules and mitotic cell division, vinblastine mechanistically acts as a microtubule-destabilizing agent (MDA). Using the small molecule drug conjugate (SMDC) strategy, it can synthesize highly selective anti-tumor agents that can have lower toxicity for normal human cells. Based on all these theories, some tumor-targeting agents were synthesized like EC0272, EC0265, and EC0276 by the introduction of a methyl group nearby disulfide region.

In comparison to unmodified compounds, these molecular probes have significantly greater stability, and also their rate of disulfide cleavage got decreased. Under reducing conditions, half-lives (t1/2) of EC0265 and EC0272 were 1hour and 0.5 h respectively (8 mM DTT, pH = 7.4, 37 °C). Another derivative, EC0276 (four methyl group-substituted disulfide derivative) have half-life more than 8 h and have stability more than 3 days in the presence of PBS buffer. This stability also affects the cytotoxicity of the compounds.  EC195, a derivative synthesized from vinblastine, also proposed that the active molecule will be released by thioquinone-methide-based 1,6-elimination. But by 1,2-elimination, the parent drug was released faster, as in the case of EC145, than that of 1,6-elimination.  In GSH and DTT, half-lives were 45 min and less than 5 min respectively and had stability in PBS for over 24 h (pH = 7.4, 37 °C). 86-93

Tubulysin: Antibiotic products “Tubulysins” are from mycobacterial family that possesses anti-angiogenic, anti-cancer, and antiproliferative activities, and they target microtubules ^{94, 95}. In the picomolar range, IC₅₀ values of these agents have cytostatic effects on different cancer cell lines ⁹⁶. However, in comparison to their therapeutic potentials, they have a double-edged sword, severe toxicity. To overcome the drawbacks, tubulysin was conjugated with folatevia disulfide linker (EC0305) ⁹⁷. Another tubulysin folate conjugate EC0510 has a similar IC₅₀ value to EC0305 was synthesized. In KB cellular tumor mouse models, both of these conjugates showed different efficacies. With 80% complete reversion by animals, EC0510 exhibited excellent anti-cancer activity at 0.5 μmol/kg dose. On the other side, at the same dosage, moderate tumor regression was showed by EC0305. But as the dosage increased from 0.5 to 1.0 μmol/kg it showed a promising anti-tumor effect, and in the span of 80 days of study, all mice tumors were disappeared.

Analog of tubulysin B, EC0317, which was also a folate conjugate and methyl- ether analog, was also synthesized using disulfide bond. This conjugate was less active in comparison to EC0305 even at a dose of 2.0 μmol/kg.  Even against FR-positive M109 and 4T1-cl2 models, EC0305 possessed better anti-tumor activities than Vinblastine conjugate EC145 ⁹⁸. Different spacers were used for modification in tubulysin-folate conjugates. A second-generation spacer was used for conjugate EC1456, which further moved to phase I clinical trials for advanced solid cancer ⁹⁹. Without off-target binding 2-[3-(1,3-Dicarboxypropyl) ureido] pentanedioic acid (DUPA), a ligand of prostate-specific membrane antigen (PSMA) can be selectively attached for imaging and therapeutic efficacy against prostate cancer cells. With a limited expression on normal tissues, most prostate cancers (PSMA) are highly expressed. DUPA-TubBH was one such molecule that is used as a PSMA-targeted anti-tumor agent; PSMA-positive LNCaP cells got killed by this conjugate (IC₅₀ = 3 nM) and with no loss in body weight and it eliminated almost established tumor xenografts in nude mice ¹⁰⁰.

With an undisclosed structure, EC1169 a derivative of DUPA-TubBH, was developed using a releasable disulfide linker in TubBH structure and act as a high-affinity PSMA-targeting ligand which further progressed to phase I clinical trials against prostate cancer ¹⁰¹. Conjugate CRL-L1-TubBH was also generated by conjugation of Tubulysin B with cholecystokinin 2 receptor (CCK2R) ligand, which specifically inhibits CCK2R-positive tumors and with negligible cytotoxicity against normal cells but have excellent receptor-specific anti-tumor effect ¹⁰².

Paclitaxel: Another clinically effective first-line chemotherapeutic agent is Paclitaxel (PTX), which can help treat different types of tumor and also cause promotion of tubulin assembly into microtubules ¹⁰³. But several shortcomings like poor solubility in water, less selectivity, and other toxic effects included neurotoxicity, hyper-sensitivity, nephrotoxicity, and cardiotoxicity, limiting its pharmacological applications.

Octreotide as a ligand was used to overcome the above-mentioned drawbacks, which bound explicitly to somatostatin receptors 2 and 5 (SSTR2 and SSTR5) and resulted in higher efficacy towards cancer cells. Thus, a tumor-targeting agent was designed as Octreotide (Phe)-polyethylene glycol–paclitaxel [OCT (Phe)-PEG-PTX] bearing a disulfide bond.

This conjugate had higher cytotoxicity towards NCI-H446 cells (overexpression of SSTR) and less towards WI-38 cells (normal cells without SSTR expression) and also had a pH-dependent profile. Also, in-vivo studies showed that OCT (Phe)–PEG–PTX had better anti-cancer potential (66.3% inhibition in tumor weight) with low systemic toxicity than mPEG-PTX conjugate (43%) and commercially available PTX (Taxol, 54.3%) in an xenograft mouse model of NCI-H446 cancer cells ¹⁰⁴.

Mitomycin C: An antibiotic Mitomycin is usually known as mitomycin C (MMC), is obtained from the broth of Streptomyces caespitosus ^{105, 106}. This compound has a very wide anti-tumor spectrum and showed its effectiveness towards pancreatic cancer, breast cancer, gastric cancer, cervical cancer, neck, head, and bladder cancer ¹⁰⁷. But fast clearance and cellular toxicity limited its clinical use ¹⁰⁸. EC72 is one of the conjugates which was synthesized by using folic acid as targeting ligand, and it showed an effective dose-response towards FR-positive cell lines in in-vitro studies. At the same time, it does not produce any toxicity or pathological degeneration in animals.

In another research, the disulfide linker of EC72 was changed with carbon chain and a non-releasable conjugate (EC110) was synthesized but it was not able to create any potent anti-tumor effect at same dose like EC72, which proved that disulfide is important for effective drug delivery. But increased in subcutaneous tumor size the therapeutic effect of EC72 also got decreased. 74 One more folate-MMC conjugate (EC118) was also constructed in order to elevate tumor efficacy by the use of two linkers i.e.disulfide bond and an acid-labile hydrazone bond. Against subcutaneous M109 tumors, this conjugate showed more potency in comparison to EC72. Also, anti-tumor efficacy improved by using a combination of MMC and paclitaxel ⁷⁵. This multidrug approach can result in great deposition of the drug inside tumor mass and for that, EC0225 was synthesized comprising both PTX and MMC and made it FR-expressing tumor-specific ⁷⁶. This folate-targeted multidrug conjugate on passing pre-clinical studies initiated towards Phase I clinical trials (CTI: NCT00441870) for patients who have refractory or metastatic tumors and do not get treated by standard therapy.

Others: Another class of anti-cancer drugs that target microtubules isepothilones ¹⁰⁹. Ixabepilone (Ixempra), one of these analogs, got approved in 2007 by the FDA and was used to treat metastatic and breast cancers that weree early not responding towards standard chemotherapies ¹¹⁰. Endocyte Inc. and Bristol Myers Squibb together developed BMS-753493 conjugate, i.e., Epofolate.  By using epothilone, a semi-synthetic analog, Epofolate was constructed as folate conjugate. But due to unknown reasons phase, II clinical trials of the conjugate were terminated.¹¹¹. In another conjugate, conjugation of folic acid was done with Camptothecin using releasable disulfide carbonate linker and hydrophilic peptide spacer.

This conjugate proved higher efficacy towards folate receptor-expressing cells and also helped to prevent cell proliferation of KB cells having IC₅₀ values of 10 nM ¹¹². To validate the use of SMDC in targeting tumor-associated phosphatidylserine (PS) in the tumor microenvironment (ZnDPA)-SN38 conjugate was developed ¹¹³. By attaching folic acid to maytansinoid, an anti-microtubule agent, a cleavable disulfide bond EC131 conjugate was designed.  With an IC₅₀ in the low nanomolar range, this compound has shown a high affinity for FR-positive cells in the pre-clinical investigation. The pre-clinical studies were conducted on BALB/c mice and human KB models in FR-positive M109 cells ⁷⁷. Another conjugate was synthesized when HDAC (Histone Deacetylase) inhibitor (NCH-31) via a disulfide bond attached to folic acid. Against FR-positive MCF-7 breast cancer cells, the compound showed good growth-inhibitory activity ¹¹⁴.

TABLE 6: SUCCESSFUL CASES OF DISULFIDE-LINKED ANTI-CANCER AGENTS

S. no.	Structure with Compound Name	Ref.
1.	Vinblastine as active drug
		86, 93
	NCT01170650 (In phase III)
2.	Tubulysin as active agent
		97
		98
		98
	NCT01999738 (In Phase I)	99
	NCT02202447 (In phase I)	100
		101
3.	Paclitaxel as active agent
		105
4.	Mitomycin C as active agent
	EC72	74
		75
	NCT00441870 (In phase I)	76
5.	Others
	NCT0050017 (In Phase I)	111
		112
		77
		114

CONCLUSION: Due to the rapid development and expansion of targeted drug design, interest in disulfide-based biodegradable linkers is growing at a faster rate. With improved anti-tumor efficacy, numerous potent conjugates have been presented, which have increased tumor inhibition and reduced side effects and better tumor selectivity. Several more of these targeted conjugates are under clinical or pre-clinical study.

Disulfide linkage-based tumor-targeting conjugates development has definitely opened the gate for a drug delivery system that is more effective and cell-specific. Several strategies can be employed in synthesizing a tumor-targeting conjugate having disulfide linkage with monoclonal antibodies (mAb), glucose, biotin and folic acid. By using disulfide bond linkage, the carrier and chemotherapeutic agent together act as bi-functional conjugate. Even though there are several successful cases of conjugates bearing disulfide bonds and have superiority in targeted drug delivery, much has to be done for further improvements.  In conclusion, disulfide bonds have provided significant tumor-targeting conjugates that improve the drug’s anti-cancer efficacy with a reduction in side effects. And further investigation on the design of disulfide linkage will bring more progress in the development of targeted tumor drug delivery systems.

ACKNOWLEDGEMENT: This work is funded by SERB-DST (Department of Science and Technology), Government of India. We are thankful to them for providing grants and fellowships to the research scholar.

CONFLICTS OF INTEREST: The author does not have any conflict of interest with anyone.

REFERENCES:

1. Zhang S, Hou Y, Chen H, Liao Z, Chen J, Xu BB and Kong J: Reduction-responsive amphiphilic star copolymers with long-chain hyperbranched poly(ε-caprolactone) core and disulfide bonds for trigger release of anti-cancer drugs. European Polymer Journal 2018.
2. Wang Q, Guan J, Wana J and Li Z: Disulfide based prodrugs for cancer therapy. RSC Advances 2020; 10: 24397-09.
3. Ohri R, Bhakta S, Fourie-O'Donohue A, Dela Cruz-Chuh J, Tsai SP, Cook R, Wei B, Ng C, Wong AW and Bos AB: High-throughput cysteine scanning to identify  stable antibody conjugation sites for maleimide and disulfide-based linkers. Bioconjugate Chemistry 2018; 29: 473-85.
4. Pei Z, Chen C, Chen J, dela Cruz-Chuh J, Delarosa R, Deng Y, Fourie-O’Donohue A, Figueroa I, Guo J and Jin W: Exploration of pyrrolobenzodiazepine (pbd)- dimers containing disulfide-based prodrugs as payloads for antibody-drug conjugates. Molecular Pharmaceutics 2018; 15: 3979-96.
5. Zhang D, Yu SF, Khojasteh SC, Ma Y, Pillow TH, Sadowsky JD, Su D, Kozak KR, Xu K, Polson AG, Dragovich PS and Hop C: Intratumoral payload concentration correlates with the activity of antibody-drug conjugates. Molecular Cancer Therapy 2018; 17: 677-85.
6. Li K, Dong W, Qiu L, Liu Q, Lv G, Peng Y, Xie M and Lin J: A new GSH-responsive prodrug of 5-aminolevulinic acid for photodiagnosis and photodynamic therapy of tumors. European Journal of Medicinal Chemistry 2019; 181: 111582.
7. Tsuchikama K and An Z: Antibody-drug conjugates: recent advances in conjugation and linker chemistries. Protein Cell 2018; 9(1): 33-46.
8. Li N, Guo W, Li Y, Zuo H, Zhang H, Wang Z, Zhao Y, Yang F, Ren G and Zhang S: Construction and anti-tumor activities of disulfide-linked docetaxel dihydroartemisinin Nanoconjugates. Colloids and Surfaces B: Biointerfaces 2020; 191: 111018.
9. Li X, Wu Z, Xu L, Chi CL and Chen BQ: Design, synthesis and anti-tumor evaluation of novel naphthalimide Derivatives. Medicinal Chemistry Research 2020; 29: 180-88.
10. Yang Q, Deng Z, Wang D, He J, Zhang D, Tan Y, Peng T, Wang XQ and Tan W: Conjugating aptamer and mitomycin c with reductant-responsive linker leading to synergistically enhanced anti-cancer effect. Journal of the American Chemical Society 2020; 142: 2532-40.
11. Xu GJ, Xu L, Yang P, He F, Yang D, An G and Ansari MBB: Redox responsive UCNPs-DPA conjugated NGO-PEG-BPEI for cancer theragnostic. Dalton Transa 2018.
12. Du Y, Wang Z, Wang T, He W, Zhou W, Li M, Yao C and Li X: Improved anti-tumor activity of novel redox-responsive paclitaxel- encapsulated liposomes based on disulfide phosphatidylcholine. Molecular Pharmaceutics 2020; 17: 262-73.
13. Chang C, Worley BL, Phaeton R and Hempel N: Extracellular glutathione peroxidase gpx3 and its role in cancer. Cancers 2020; 12: 2197.
14. Singh N, Nayak J, Sahoo SK and Kumar R: Glutathione conjugated super paramagnetic Fe3O4-Au core shell nanoparticles for pH controlled release of DOX. Materials Science & Engineering C 2019; 100: 453-65.
15. LvH, Zhen C, Liu J, Yang P, Hu L and Shang P: Unraveling the potential role of glutathione in multiple forms of cell death in cancer therapy. Oxidative Medicine and Cellular Longevity 2019.
16. Ibrahim A, Twizeyimana E, Lu N, Ke W,Mukerabigwi JF, Mohammed J, Japir AWM and Ge Z: Reduction-responsive polymer prodrug micelles with enhanced endosomal escape capability for efficient intracellular translocation and drug release. ACS Applied Biomaterials 2019; 2: 5099-09.
17. Wei X, Mo X, An F, Ji X and Lu Y: 2′,4′-Dihydroxy-6′-methoxy-3′,5′- dimethylchalcone, a potent Nrf2/ARE pathway inhibitor, reverses drug resistance by decreasing glutathione synthesis and drug efflux in BEL-7402/5-FU cells. Food and Chemical Toxicology 2018.
18. Lee JH, Kim C, Lee SG, Sethi G and Ahn KS: Ophiopogonin d, a steroidal glycoside abrogates stat3 signaling cascade and exhibits anti-cancer activity by causing gsh/gssg imbalance in lung carcinoma. Cancers 2018; 10: 427.
19. Jabir M, Sahib UI, Taqi Z, Taha A, Sulaiman G, Albukhaty S, -Shammari AA, Alwahibi M, Soliman D, Dewir YH and Rizwana H: Linalool-loaded glutathione-modified gold nanoparticles conjugated with calnn peptide as apoptosis inducer and nf-κb translocation inhibitor in skov-3 cell line. Intern J of Nanom 2020; 15: 9025-47.
20. Ju P, Hu J, Li F, Cao Y, Li L, Shi D, Hao Y, Zhang M, He J and Ni P: A biodegradable polyphosphoester‐functionalized poly (disulfide)s nanocarrier for reduction‐triggered intracellular drug delivery. Journal of Material Chemistry B 2018.
21. Lu J, Chen Q, Ding X, Wen J, Zhang Y, Li H, Xu Y, Li F, Chen SS and Sun S: BSA modified, disulfide-bridged mesoporous silica with low biotoxicity for dual responsive drug delivery. Micro porous and Mesoporous Materials 2019.
22. Shuang B, Yong-E G, Xiaoqian M, Xiaoxiao S, Meili H, Peng X, Yuejun K and Zhigang X: Reduction stimuli-responsive unimolecular polymeric prodrug based on amphiphilic dextran-framework for anti-tumor drug delivery. Carbohydrate Polymers 2018.
23. Wang Y, Wang X, Deng F, Zheng N, Liang Y, Zhang H, He B, Dai W, Wang X and Zhang Q: The effect of linkers on the self-assembling and anti-tumor efficacy of disulfide-linked doxorubicin drug-drug conjugates nano-particles. Corel 2018.
24. Sun B, Luo C, Yu H, Zhang X, Chen Q, Yang W, Wang M, Kan Q, Zhang H, Wang H, He Z and Sun J: Disulfide bond-driven oxidation/reduction-responsive prodrug nanoassemblies for cancer therapy. Nano Letters 2018.
25. Gauthier F, Bertrand JR, Vasseur JJ, Dupouy C and Debart F: Conjugation of doxorubicin to sirna through disulfide-based self-immolative linkers. Molecules 2020; 25: 2714.
26. Mo S, Zhang X, Hameed S, Zhou Y and Dai Z: Glutathione-responsive disassembly of disulfide dicyanine for tumor imaging with reduction in background signal intensity. Theranostics 2020; 10(5): 2130-40.
27. Guo X, Cheng Y, Zhao X, Luo Y, Chen J and Yuan W: Advances in redox‑responsive drug delivery systems of tumor microenvironment. J of Nanobiotechn 2018; 16: 74.
28. Raza A, Hayat U, Rasheed T, Bilal M and Iqbal HMN: Redox-responsive nano-carriers as tumor-targeted drug delivery systems. European Journal of Medicinal Chemistry 2018.
29. Claeson AS, Gouveia-Figueira S, Stenlund H and Johansson AI: A standardized protocol for comparable analysis of GSH/GSSGby UHPLC-ESI-MSMS for human plasma. Journal of Chromatography B 2018; 67-72.
30. Mejia SA, Gutman LAB, Camarillo CO, Navarro RM, Becerra MCS, Santana LD, Cruz M, Perez EH and Flores MD: Nicotinamide prevents sweet beverage-induced hepatic steatosis in rats by regulating the G6PD, NADPH/NADP+ and GSH/GSSG ratios and reducing oxidative and inflammatory stress. European Journal of Pharmacology 2018; 5(818): 499-07.
31. Zhang L, Li J, Dong X, Meng D, Zhi X, Yuan L and Yao L: PSAT1 regulated oxidation-reduction balance affects the growth and prognosis of epithelial ovarian cancer. Onco Targets and Therapy 2020; 13: 5443-53.
32. Yuana B, Wanga H, Caib J, Penga Y, Niua Y, Chena H, Baia L, Zhanga S, Jina J, Liub L and Xu C: A novel oxidation-reduction method for highly selective detection of cysteine over reduced glutathione based on synergistic effect of fully fluorinated cobalt phthalocyanine and ordered mesoporous carbon. Sensors & Actuators: B. Chemical 2019; 288: 180-87.
33. Zhaoa E, St-Jeanb F, Robinsona SJ, Siroisb LE, Pelletta J and Al-Sayaha MA: Identification of an acetonitrile addition impurity formed duringpeptide disulfide bond reduction using dithiothreitol and Tris (2-carboxyethyl) phosphine. Journal of Pharmaceutical and Biomedical Analysis 2019; 174: 518-24.
34. Lee MV, Kaur S and Saad OM: Conjugation site influences antibody-conjugated drug pk assays: case studies for disulfide-linked, self-immolating next-generation antibody drug conjugates. Analytical Chemistry 2020; 92: 12168-75.
35. Jiang Z and Thayumanavan S: Disulfide-containing macromolecules for therapeutic delivery. Israel Journal of Chemistry 2020; 60: 1-9.
36. Zhang P, Hu J, Bu L, Zhang H, Du B, Zhu C and Li Y: Facile preparation of reduction-responsive micelles based on biodegradable amphiphilic polyurethane with disulfide bonds in the backbone. Polymers 2019; 11: 262.
37. Gevrek TN, Cosar M, Aydin D, Kaga E, Arslan M, Sanyal R and Sanyal A: Facile fabrication of a modular ‘catch and release’ hydrogel interface: harnessing thiol-disulfide exchange for reversible protein capture and cell attachment. Applied Material and Interfaces 2018.
38. Arslan M, Sanyal R and Sanyal A: Thiol-reactive thiosulfonate group containing copolymers: facile entry to disulfide-mediated polymer conjugation and redox-responsive functionalizable networks. Polymer Chemistry 2020.
39. Arai K, Ueno H, Asano Y, Chakrabarty G, Shimodaira S, Mugesh G and Iwaoka M: Protein folding by water-soluble cyclic diselenides with novel oxidoreductase and isomerase activities. Chem Bio Chem 2018.
40. Lv JM, Lu SQ, Liu ZP, Zhang J, Gao BX, Yao ZY, Wu YX, Potempa LA, Ji SR, Long M and Wu Y: Conformational folding and disulfide bonding drive distinct stages of protein structure formation. Scientific Reports 2018; 8: 1494.
41. Aggarwal V, Tuli HS, Varol A, Thakral F, Yerer MB, Sak K, Varol M, Jain A, Khan MA and Sethi G: Role of reactive oxygen species in cancer progression: molecular mechanisms and recent advancements. Biomolecules 2019; 9: 735.
42. Laskar P, Somani S, Campbell SJ, Mullin M, Keating P, Tate RJ, Irving C, Leung HY and Dufes C: Camptothecin-based dendrimersomes for gene delivery and redox-responsive drug delivery to cancer cells. Nanoscale 2019; 11: 20058.
43. Kim Y, Uthaman S, Nurunnabi M, Mallick S, Oh KS, Kang SW, Cho S, Kang HC, Lee Y and Huh KM: Synthesis and characterization of bioreducible cationic biarm polymer for efficient gene delivery. International Journal of Biological Macromolecules 2018; 110: 366-74.
44. Lu M, Xing H, Cheng L, Liu H, Lang L, Yang T, Zhao X, Xu H and Ding P: A dual-functional buformin-mimicking poly(amido amine) for efficient and safe gene delivery. Journal of Drug Targeting 2020.
45. Nam JP, Kim S and Kim SW: Design of PEI-conjugated bio-reducible polymer for efficient gene delivery. International Journal of Pharmaceutics 2018.
46. Ullah I, Zhao J, Rukh S, Muhammad K, Guo J, Ren X, Xia S, Zhang W and Feng Y: PEG-b-poly (disulfide-L-lysine) based redox-responsive cationic polymer for efficient gene transfection. Journal of Material Chemistry B 2019; 7: 1893-05.
47. Li I, Ma YJ, Wang Y, Chen BZ, Guo XD and Zhang CY: Dual redox/pH-responsive hybrid polymer-lipid composites: synthesis, preparation, characterization and application in drug delivery with enhanced therapeutic efficacy. Chemical Engineering Journal 2018; 341: 450-61.
48. Bej R, Achazi K, Haag R and Ghosh S: Polymersome formation by amphiphilic poly glycerol-b-polydisulfide-polyglycerol and glutathione triggered intracellular drug delivery. Bio Macromolecules 2020.
49. Jazani AM, Arezi N, Shetty C, Hong SH, Li H, Wang X and Oh JK: Tumor-targeting intracellular drug delivery based on dual acid/reduction-degradable nanoassemblies with ketal interface and disulfide core locations. Polymer Chemistry 2019; 10: 2840.
50. Oh JK: Disassembly and tumor-targeting drug delivery of reduction-responsive degradable block copolymer nanoassemblies. Polymer Chemistry 2019.
51. Zhao J, Yan C, Chen Z, Liu J, Song H, Wang W, Liu J, Yang N, Zhao Y and Chen L: Dual-targeting nano-particles with core-cross linked and pH/redox bio responsive properties for enhanced intracellular drug delivery. Journal of Colloid and Interface Science 2019; 540: 66-77.
52. Xue Q, Ye C, Zhang M, Hua X and Cai T: Glutathione responsive cubic gel particles cyclodextrin metal-organic frameworks for intracellular drug delivery. Journal of Colloid and Interface Science 2019; 551: 39-46.
53. Pham SH, Choi Y and Choi J: Stimuli-responsive nanomaterials for application in anti-tumor therapy and drug delivery. Pharmaceutics 2020; 12: 630.
54. Liang Y, Li S, Wang X, Zhang Y, Sun Y, Wang Y, Wang X, He B, Dai W, Zhang H, Wang X and Zhang Q: A comparative study of the anti-tumor efficacy of peptide-doxorubicin conjugates with different linkers. Journal of Controlled Release 2018.
55. Chang S, Wang Y, Zhang T, Pu X, Zong L, Zhu H, Zhao L and Feng B: Redox-responsive disulfide bond-bridged mpeg-pbla prodrug micelles for enhanced paclitaxel biosafety and anti-tumor efficacy. Frontiers in Oncology 2019; 9: 823.
56. Deng Z, Hu J and Liu S: Disulfide-based self-immolative linkers and functional bioconjugates for biological applications. Macromolecular Rapid Communication 2019; 1900531.
57. Deng Z, Yuan S, Xu RX, Liang H and Liu S: Reduction-triggered transformation of crosslinking modules of disulfide-containing micelles with chemically tunable rates. Angewandte Chemie International Edition 2018.
58. Felber JG, Zeisel L, Poczka L, Scholzen K, Busker S, Maier MS, Theisen U, Brandstädter C, Becker K, Arnér ESJ, Thorn-Seshold J and Thorn-Seshold O: Selective, modular probes for thioredoxins enabled by rational tuning of a unique disulfide structure motif. Journal of the American Chemical Society 2021; 143: 8791-03.
59. Lu Y, Jiang W, Wu X, Huang S, Huang Z, Shi Y, Dai Q, Chen J, Ren F and Gao S: Peptide T7-modified polypeptide with disulfide bonds for targeted delivery of plasmid DNA for gene therapy of prostate cancer. International Journal of Nanomedicine 2018; 13: 6913-27.
60. Kotamraju VR, Sharma S, Kolhar P, Agemy L, Pavlovich J and Ruoslahti E: Increasing tumor accessibility with conjugatable disulfide-bridged tumor-penetrating peptides for cancer diagnosis and treatment. Breast Cancer Basic and Clinical Research 2015; 9: 79-87.
61. Wen H and Li Y: Redox sensitive nanoparticles with disulfide bond linked sheddable shell for intracellular drug delivery. Medicinal Chemistry 2014; 748-55.
62. Wang Y, Liu D, Zheng Q, Zhao Q, Zhang H, Ma Y, Fallon JK, Fu Q, Haynes MT, Lin G, Zhang R, Wang D, Yang X, Zhao L, He Z and Liu F: Disulfide bond bridge insertion turns hydrophobic anti-cancer prodrugs into self-assembled nanomedicines. Nano Letters 2014.
63. Latorre A, Couleaud P, Aires A, Cortajarena AL and Somoza A: Multifunctionalization of magnetic nanoparticles for controlled drug release: A general approach. European Journal of Medicinal Chemistry 2014; 82: 355-62.
64. Pan A, Zhang H, Li Y, Lin T, Wang F, Lee J, Cheng M, Dall-Era M, Li T, White R, Pan CX and Lam KS: Disulfide-cross linked nanomicelles confer cancer-specific drug delivery and improve efficacy of paclitaxel in bladder cancer. Nanotechnology 2016; 27: 9.
65. Lee MH, Kim JY, Han JY, Bhuniya S, Sessler JL, Kang C and Kim JS: Direct fluorescence monitoring of the delivery and cellular uptake of a cancer-targeted rgd peptide-appended naphthalimide theragnostic prodrug. Journal of the American Chem Soc 2012; 134: 12668-74.
66. Maiti S, Park N, Han JH, Jeon HM, Lee JH, Bhuniya S, Kang C and Kim JS: Gemcitabine-coumarin-biotin conjugates: a target specific theranostic anti-cancer prodrug. Journal of the American Chemical Society 2013; 135: 4567-72.
67. Bhuniya S, Maiti S, Kim EJ, Lee H, Sessler JL, Hong KS and Kim JS: An activatable theranostic for targeted cancer therapy and imaging. Angewandte Chemie International Edition 2014: 53: 1-7.
68. Chen W, Shi Y, Feng H, Du M, Zhang JZ, Hu J and Yang D: Preparation of copolymer paclitaxel covalently linked via a disulfide bond and its application on controlled drug delivery. The J of Physical Chemis B 2012; 116: 9231-37.
69. Chari RVJ: Targeted cancer therapy: conferring specificity to cytotoxic drugs. Accoun of Chem Res 2008; 41: 98-07.
70. Pillow TH, Sadowsky JD, Zhang D, Yu SF, Rosario GD, Xu K, He J, Bhakta S, Ohri R, Kozak KR, Ha E, Junutula JR and Flygare JA: Decoupling stability and release in disulfide bonds with antibody-small molecule conjugates. Chemical Science 2017; 8: 366-70.
71. Chen S, Zhao X, Chen J, Kuznetsova L, Wong SS and Ojima I: Mechanism-based tumor-targeting drug delivery system. Validation of efficient vitamin receptor-mediated endocytosis and drug release. Bioconjugate Chemistry 2010; 21: 979-87.
72. Vineberg JG, Zuniga ES, Kamath A, Chen YJ, Seitz JD and Ojima I: Design, synthesis, and biological evaluations of tumor-targeting dual-warhead conjugates for a taxoid-camptothecin combination chemotherapy. Journal of Medicinal Chemistry 2014; 57: 5777-91.
73. Kim T, Jeon HM, Le HT, Kim WT, Kang C and Kim JS: A biotin-guided fluorescent-peptide drug delivery system for cancer treatment. Chemical Comm 2014; 50: 7690-93.
74. Leamon CP, Reddy JA, Vlahov IR, Vetzel M, Parker N, Nicoson JS, Xu LC and Westrick E: Synthesis and biological evaluation of ec72: a new folate-targeted chemotherapeutic. Bioconjugate Chemis 2005; 16: 803-11.
75. Reddy JA, Westrick E, Vlahov I, Howard SJ, Santhapuram HK and Leamon CP: Folate receptor specific anti-tumor activity of folate-mitomycin conjugates. Cancer Chemotherapy and Pharmacology 2006; 58: 229-36.
76. Leamon CP, Reddy JA, Vlahov IR, Westrick E, Dawson A, Dorton R, Vetzel M, Santhapuram HS and Wang Y: Pre-clinical anti-tumor activity of a novel folate-targeted dual drug conjugate. Molecular Pharmaceutics 2007; 4: 659-67.
77. Reddy JA, Westrick E, Santhapuram H, Howard SJ, Miller ML, Vetzel M, Vlahov I, Chari RVJ, Goldmacher VS and Leamon CP: Folate receptor–specific anti-tumor activity of ec131, a folate-maytansinoid conjugate. Cancer Research 2007; 67: 6376-82.
78. Nikitin K, Jennings EV, Sulaimi SA, Ortin Y and Gilheany DG: Dynamic cross-exchange in halophosphonium species: first direct observation of stereochemical inversion in the course of an sn2 process. Angewandte Chemie International Edition 2017.
79. Petrov AI and Dergachev VD: Palladium(II) ion mediated disulfide/thiolate interconversion: predicting the disulfide group state from first principles. The Journal of Physical Chemistry A 2019; 123: 4873-82.
80. Putzu M, Gräter F, Elstner M and Kuba T: On the mechanism of spontaneous thiol–disulfide exchange in proteins. Physical Chemistry Chemical Physics 2018.
81. Depuydt M, Messens J and Collet JF: How proteins form disulfide bonds. Antioxidants & Redox Signaling 2011; 15: 49-66.
82. Ito K and Inaba K: The disulfide bond formation (Dsb) system. Current Opinion in Struc Bio 2008; 18: 450-58.
83. Wunderlich M, Jaenicke R and Glockshuber R: The redox properties of protein disulfide-isomerase (DsbA) of Escherichia-coli result from a tense conformation of its oxidized form. J of Molecular Biology 1993; 233: 559-66.
84. Zhuang C, Guan X, Ma H, Cong H, Zhang W and Miao Z: Small molecule-drug conjugates: A novel strategy for cancer-targeted treatment. European Journal of Medicinal Chemistry 2019; 163: 883-95.
85. Qu Y, Safonova O and Luca VD: Completion of the canonical pathway for assembly of anti-cancer drugs vincristine/vinblastine in Catharanthusroseus. The Plant Journal 2019; 97: 257-66
86. Yang J, Chen H, Vlahov IR, Cheng JX and Low PS: Characterization of the pH of folate receptor-containing endosomes and the rate of hydrolysis of internalized acid-labile folate-drug conjugates. Journal of Pharmacology and Experimental Therapeutics 2007; 321: 462-68.
87. Ajaj KA, El-Abadla N, Welker P, Azab S, Zeisig R, Fichtner I and Kratz F: Comparative evaluation of the biological properties of reducible and acid-sensitive folate prodrugs of a highly potent doxorubicin derivative. European Journal of Cancer 2012; 48: 2054-65.
88. Parker N, Turk MJ, Westrick E, Lewis JD, Low PS and Leamon CP: Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Analytical Biochemistry 2005; 338: 284-93.
89. Moudi M, Go R, Yien CYS and Nazre M: Vinca alkaloid. International J of Preventive Medicine 2013; 4: 1231-35.
90. Li J, Sausville EA, Klein PJ, Morgenstern D, Leamon CP, Messmann RA and LoRusso P: Clinical pharmacokinetics and exposure-toxicity relationship of a folate-Vinca alkaloid conjugate EC145 in cancer patients. The Journal of Clinical Pharmacology 2009; 49: 1467-76.
91. Yang J, Chen H, Vlahov IR, Cheng JX and Low PS: Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging. Proceedings of National Academy of Sciences United States of States of America 2006; 103: 13872-77.
92. Leamon CP, Vlahov IR, Reddy JA, Vetzel M, Santhapuram HK, You F, Bloomfield, Dorton R, Nelson M, Kleindl P, Vaughn JF and Westrick E: Folate-vinca alkaloid conjugates for cancer therapy: a structure-activity relationship. Bioconjugate Chemistry 2014; 25: 560-68.
93. Low PS, Henne WA and Doorneweerd DD: Discovery and development of folic-acid-based receptor targeting for imaging and therapy of cancer and inflammatory diseases. Accounts of Chemical Research 2008; 41: 120-29.
94. Rios-Doria J, Harper J, Rothstein R, Wetzel L, Chesebrough J, Marrero A, Chen C, Strout P, Mulgrew K, McGlinchey K, Fleming R, Bezabeh B, Meekin J, Stewart D, Kennedy M, Martin P, Buchanan A, Dimasi N, Michelotti E and Hollingsworth R: Antibody-drug conjugates bearing pyrrolobenzodiazepine or tubulysin payloads are immunomodulatory and synergize with multiple immunotherapies. Cancer Research 2017.
95. Sani M, Lazzari P, Folini M, Spiga M, Zuco V, Cesare MD, Manca I, Dall’Angelo S, Frigerio M, Usai I, Testa A, Zaffaroni N and Zanda M: Synthesis and superpotent anti-cancer activity of tubulysins carrying non-hydrolysable n-substituents on tubuvaline. Chemistry of European Journal 2017; 23: 5842-50.
96. Dračaa D, Mijatovića S, Krajnovića T, Pristovb JB, Đukićc T, Kaluđerovićd GN, Wessjohanne LA and Maksimović-Ivanića D: The synthetic tubulysin derivative, tubugi-1, improves the innate immune response by macrophage polarization in addition to its direct cytotoxic effects in a murine melanoma model. Experimental Cell Research 2019; 380: 159-70.
97. Leamon CP, Reddy JA, Vlahov IR, Dorton R, Bloomfield A, Vetzel M, Klein PJ, Westrick E, Xu L and Wang Y: Enhancing the therapeutic range of a targeted small‑molecule tubulysin conjugate for folate receptor‑based cancer therapy. Cancer Chemotherapy Pharmacol 2017; 79: 1151-
98. Rana A and Bhatnagar S: Advancements in folate receptor targeting for anti-cancer therapy: A small molecule-drug conjugate approach. Bioorganic Chemi 2021; 112: 104946.
99. Reddy JA, Dorton R, Bloomfield A, Nelson M, Dircksen C, Vetzel M, Kleindl P, Santhapuram H, Vlahov IR and Leamon CP: Pre-clinical evaluation of EC1456, a folate-tubulysin anti-cancer therapeutic. Scie Rep 2018; 8: 8943.
100. Patel TK, Adhikari N, Amin SA, Biswas S, Jha T and Ghosh B: Small molecule drug conjugates (SMDCs): an emerging strategy for anti-cancer drug design and discovery. New Journal of Chemistry 2021; 45: 5291.
101. Morris M, Vogelzang NJ, Sartor O, Armour A, Groaning M, Messmann R, Robarts A, Petrylak DP, Tolcher A, Gordon MS and Babiker H: Phase 1 study of the PSMA-targeted small-molecule drug conjugate EC1169 in patients with metastatic castrate-resistant prostate cancer (mCRPC). Annals of Oncology 2017; 28(5): 273.
102. Wayua C, Roy J, Putt KS and Low PS: Selective tumor targeting of desacetyl vinblastine hydrazide and tubulysin b via conjugation to a cholecystokinin-2 receptor (cck2r) ligand. Molecular Pharmacology 2015; 12: 2477-83.
103. Zhu L and Chen L: Progress in research on paclitaxel and tumor immunotherapy. Cellular & Molecular Biology Letters 2019; 24: 40.
104. Li J, Wang F, Sun D and Wang R: A review of the ligands and related targeting strategies for active targeting of paclitaxel to tumours. Journal of Drug Targeting 2016.
105. Guo X, Ni J, Xue J and Wang X: Phyllanthusemblica fruit extract potentiates the anti-cancer efficacy of mitomycin C and cisplatin and reduces their genotoxicity to normal cells in-vitro. Biomed & Biotechnol 2017; 18(12): 1031-45
106. Baird L and Yamamoto M: NRF2-dependent bio activation of mitomycin C as a novel strategy to target KEAP1-NRF2 pathway activation in human cancer. Molecular Cellular Biology 2020.
107. Bruzaca EES, Lopes IC, Silva EH, Carvalho PAV and Tanaka AA: Electrochemical oxidation of the anti-tumor antibiotic mitomycin C and in situ evaluation of its interaction with DNA using a DNA-electrochemical biosensor. Microchemical Journal 2017; 133: 81-89.
108. Patil Y, Amitay Y, Ohana P, Shmeeda H and Gabizon A: Targeting of pegylated liposomal mitomycin-C prodrug to the folate receptor of cancer cells: Intracellular activation and enhanced cytotoxicity. J of Controlled Release 2016.
109. Cheng H, Huang H and Huang G: Synthesis and anti-tumor activity of epothilone B. European Journal of Medicinal Chemistry 2018.
110. Cao DS, Jiang SL, Guan YD, Chen XS, Zhang LX, Zhang Y, Chen AF, Yang JM and Cheng Y: A multi-scale systems pharmacology approach uncovers the anti-cancer molecular mechanism of Ixabepilone. European Journal of Medicinal Chemistry 2020; 199: 112421.
111. Shena H, Wanga L, Chena W, Menard K, Hong Y, Tian Y, Bonacorsic SJ, Humphreysa WG, Leeb FY and Gan J: Tissue distribution and tumor uptake of folate receptor-targeted epothilone folate conjugate, BMS-753493, inCD2F1miceafter systemic administration. Acta Pharmaceutica Sinica B 2016.
112. Henne WA, Doorneweerd DD, Hilgenbrink AR, Kularatne SA and Low PS: Synthesis and activity of a folate peptide camptothecin prodrug. Bioorganic & Medicinal Chemistry Letters 2006; 16: 5350-55.
113. Liu YW, Shia KS, Wu CH, Liu KL, Yeh YC, Lo CF, Chen CT, Chen YY, Yeh TK, Chen WH, Jan JJ, Huang YC, Huang CL, Fang MY, Gray, Pak KY, Hsu TA, Huang KH and Tsou LK: Targeting tumor-associated phosphatidylserine with new zinc dipicolylamine-based drug conjugates. Bioconju Chemistry 2017; 28: 1878-92.
114. Suzuki T, Hisakawa S, Itoh Y, Suzuki N, Takahashi K, Kawahata M, Yamaguchi K, Nakagawa H and Miyata N: Design, synthesis and biological activity of folate receptor-targeted prodrugs of thiolate histone deacetylase inhibitors. Bioorganic & Medicinal Che Letters 2007; 17: 4208-12.
     

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     How to cite this article:

     Kaur A, Dhiman S and Sharma M: Disulfide bridging: a potent strategy for antitumor drug delivery. Int J Pharm Sci & Res 2022; 13(5): 1935-58. doi: 10.13040/IJPSR.0975-8232.13(5).1935-58.

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