Antioxidants in Cancer Therapy: Recent Trends in Application of Nanotechnology for Enhanced Delivery

: Recently, the occurrence of cancer has signiﬁcantly increased; it represents the second-most frequent cause of death after cardiovascular diseases. Many dietary antioxidants have shown large impact as e ﬀ ective agents for cancer prevention by reducing oxidative stress, which has been a part in the development of many diseases, including cancer. One of the obstacles in the delivery of antioxidant therapies into the required domain lies in the inadequate delivery of these agents to their intended site of action. Using nanotechnology in delivery of antioxidants leads to increased therapeutic index and higher drug concentration in tumor tissues, thus enhancing anticancer treatment. In this review, we discuss the role of di ﬀ erent antioxidants in cancer therapy and their improved therapeutic e ﬀ ect through their formulation using nanotechnology.


Introduction
Cancer is considered the second most frequent cause of death worldwide after cardiovascular disease. Although great improvements have been made in cancer therapy, it is still a major health concern and, therefore, various investigations have been done to develop new therapeutic approaches [1].
Cancer involves uncontrolled growth of cells and it can develop in any tissue. The basic process of different types of cancer is similar; it begins when a cell has a faster rate of cell division than normal cells. The initiation of cancer is highly linked to oxidative stress that results in DNA damage, instability of genome and cell proliferation [2]. Various researchers have reported the mechanism by which oxidative stress can lead to chronic inflammation, which leads to many diseases including cancer [3].
Compared to normal cells, cancer cells have a concentration of endogenous reactive oxidative stress (ROS) [4,5]. Low concentrations of ROS compounds are required for signal transduction, but a cancer cell has an accelerated metabolism, thus they need high ROS concentrations to maintain their high proliferation rate. They cause damage in mitochondrial DNA, which leads to mutation in the oxidative phosphorylation resulting in increase in production of ROS. It aids in the proliferation of cancer cells, which leads to an increase in tumor growth [6]. That is why cancer cells require high ROS concentrations to maintain their high proliferation rate.
ROS damage in cells depends on their intracellular concentration and on the equilibrium between the ROS and the endogenous antioxidant species. Oxidative stress is generated as a result of the loss of for pH-responsive drug release and targeted cancer therapy [22]. Another study by Liu et al. confirmed the efficacy of antioxidant response via delivery of curcumin using dendrimers as a carrier system in cancer therapy [23].

Micelles
Micelles are self assembled nanosized colloidal dispersions [24,25]. Their surfactant molecules can be aggregated either by cationic, anionic, zwitterionic or non-ionic groups [26] depending on the type of head groups and length of the alkyl chains [27,28]. They consist of both hydrophobic and hydrophilic shells, thus they are used for carrying both hydrophilic and lipophilic molecules [21].
Micelles have attracted attention for their potential use in cancer diagnosis and therapy. A study was done by Masaki et al. showed that antioxidant micelles containing catechol moieties can be used in anti-angiogenic therapy to treat various diseases including cancer [29].
Another study by Esmaili et al. reported that curcumin-loaded micelles showed an increase in curcumin solubility and bioavailability in treatment of cancer [30].

Polymeric Nanoparticles
Polymeric nanoparticles are solid colloidal systems that can carry drugs in different ways, either by dissolving, entrapping, encapsulating or adsorbing the drug onto their matrix. Depending on the formulation method, the obtained polymeric particle may be nanospheres, which are matrix systems in which the drug is dispersed throughout the particles, or nanocapsules, which are vesicular reservoir systems in which the drug is confined to an aqueous or oily cavity surrounded by a single polymeric membrane [31].
In a study by Mady and Shaker on the preparation of ellagic acid-polymeric nanoparticles using the emulsion-diffusion-evaporation method, the results showed that they were able to sustain the diffusion release of ellagic acid and enhance its cytotoxicity (6.9-fold) against the colon adenocarcinoma, thus improved ellagic acid bioavailability and its anticancer efficacy [32]. Rocha et al. also proved the efficacy of using polymeric nanoparticles in delivery of catechin for prostate cancer treatment [33].

Liposomes
Liposomes are spherical vesicles consisting of two layers of lipids with an internal aqueous cavity. They are composed of phospholipids or synthetic amphiphiles incorporated with sterols, such as cholesterol to influence membrane permeability.
Liposomal encapsulation has been reported to alter the biodistribution of compounds, such as doxorubicin [34,35] and vincristine [36] to limit systemic drug-induced toxicities. A study by Koudelka et al. was done to formulate vitamin E liposomes for treatment of cancer, where liposomes could reduce immunotoxicity, thus it was helpful for the combined chemo-and immuno-therapy [37].
Muthu et al. incorporated vitamin E in liposomal formulation for the delivery of vitamin E to target brain cancer cells. Their results showed that liposomes had a great efficacy in targeting cancer cells as a multifunctional carrier [38].

Solid Lipid Nanoparticles
Solid lipid nanoparticles (SLNs) are lipid-based nanocarriers, which are solid in the body temperature and stabilized by emulsifiers [8]. SLNs can protect the incorporated drug from the surrounding harsh environmental situations. Their biodegradability and biocompatibility also make them suitable to be used as a drug carrier systems [39]. They can overcome some of the obstacles of other nano-systems, where their large scale production is lower in cost, and have fewer storage and drug leakage problems in comparison to liposomes [8,40,41].
Various reviews have described the different types of SLNs, characterization methods, preparation techniques, structural properties, factors that may affect their formation and storage stability, drug loading and drug release principles [8,42,43].

Methods of Preparation of SLNs
Several methods have been adapted for the preparation of SLNs, including:

High Pressure Homogenization Technique
This technique includes two strategies, hot homogenization and cold homogenization. Hot homogenization is carried out at temperatures above the melting point of the lipid, while in the cold homogenization process, solubilization of the drug in the lipid is first done, followed by rapid cooling [44]. The cold homogenization can overcome the obstacles of hot pressure homogenization such as degradation [44]. Müller et al. suggested using cold homogenization technique, which is performed at room temperature to increase aqueous solubility of the drug [45,46].

Microemulsion Technique
The concept of microemulsion technique was introduced in the early 1940s by Hoar and Schulman [47]. Microemulsions are thermodynamically stable systems composed of a hydrophilic, a hydrophobic and an amphiphilic component, with the globules are in the nano-size range [48].

Solvent Emulsification Evaporation Technique
In this technique, a water immiscible organic phase containing the lipophilic material (lipid) and the lipophilic drug is emulsified by homogenization in an aqueous phase followed by evaporation of the organic solvent by mechanical stirring at room temperature, resulting in the formation of lipid precipitates as SLNs [49,50].

Solvent Emulsification Diffusion Technique
This technique depends on dissolving the lipid and the drug in a partially water miscible organic solvent (e.g., benzyl alcohol, butyl lactate, ethyl acetate, isopropyl acetate and methyl acetate), then the organic solvent is emulsified using an emulsifying agent dissolved in an aqueous phase under agitation. Once emulsion is formed, SLNs are obtained by evaporation of the solvent under low pressure [51].

Ultrasonication Technique
In this technique, the drug is added to the dissolved lipid, then the aqueous phase added to the dissolved lipid phase and emulsified by sonication at high speed stirring. Ultrasonication technique was initially used for the production of solid lipid nanodispersion [52]. However, one of the drawbacks of this method is the physical instability of the prepared SLNs [53].

Melting Dispersion Technique (Hot Melt Encapsulation Method)
In this technique, the molten solid lipid containing the drug is emulsified into the heated aqueous phase. Following this, the system is allowed to cool down to room temperature resulting in the formation of SLNs. This method is better than the ultrasonication method, but less than the solvent emulsification evaporation method in terms of reproducibility [54,55].

Double Emulsion Technique
Production of SLNs by double emulsion technique involves the dissolution of the drug (mainly hydrophilic drug) in an aqueous solvent (inner aqueous phase) followed by dispersion in lipid containing emulsifier/stabilizer, known as oil phase, to produce primary emulsion (w/o). Following this, a double emulsion (w/o/w) is formed through the introduction of an aqueous solution containing the hydrophilic emulsifier under stirring and finally isolated by filtration [56,57]. Double emulsion technique is suitable for the preparation of protein and peptide-loaded lipid nanoparticles, as it does not involve a heating process. Moreover, the surface of the nanoparticles can be modified to sterically stabilize them through the incorporation of a lipid-PEG derivative, resulting in improved resistance in the gastrointestinal fluids [58].

Solvent Injection Technique
In this technique, the lipid and drug are first dissolved in a water miscible solvent, and then the mixture is injected in an aqueous phase through an injection needle. The resulting dispersion is filtered to obtain the prepared SLNs [53]. Solvent injection technique has several advantages such as the ability of using pharmacologically acceptable organic solvent, easy to handle and fast production process.
Other methods used for preparation of nanoparticles (Table 1).

Desolvation Technique
This technology is applicable for a wide range of polymers by changing the charge and pH by addition of a desolvating agent like ethanol or concentrated organic salt solution [59].

Dialysis Technique
Dialysis technique is a simple and effective technique for preparation of polymeric nanoparticles with narrow distribution. Briefly, drug and polymers are placed inside a dialysis tube/membrane after dissolving with water miscible organic solvents with appropriate molecular weight cut off. The organic phase diffuses out through the dialysis tube/membrane into the aqueous phase which decrease the interfacial tension between them [60].

Nanoprecipitation Technique
Nanoprecipitation technique was first introduced by fessi for preparation of polymeric nanoparticles, where formation of particles is based on the precipitation and subsequent solidification of the polymers due to the interfacial deposition of polymer after displacement of semi polar solvents miscible with water, from a lipophilic solution [61].

Solvent Evaporation Technique
Solvent evaporation technique is commonly used for preparation of polymeric nanoparticles. Briefly, the polymer is dissolved in an organic solvent into which the drug is dissolved/dispersed [62].

Examples on Utilizing Nanotechnology in the Delivery of Antioxidants for Cancer Therapy
The therapeutic benefits of applying nanosystems fabrication techniques in delivering various antioxidants have been listed in Table 2.

Vitamin E
Vitamin E is a lipophilic component in the cell antioxidant defense system and can be obtained from the diet, its important role in the body is related to its antioxidant activity [63]. It has been shown to enhance the cytotoxic effect of anticancer drugs, including 5-fluorouracil [64], vincristine [65] and doxorubicin [66,67]. Studies on animals have shown that parenteral administration of vitamin E resulted in enhancing the anticancer effect of 5-FU [68] and cisplatin [69]. Feng et al. have developed systems of nanoparticles of biodegradable polymers for oral administration of anticancer drugs, with the incorporation of vitamin E using modified solvent extraction/evaporation [70].

Coenzyme Q10 (Ubiquinone)
Co-enzyme Q10 (Co-Q10) is a lipophilic antioxidant that has the ability to prevent lipid peroxidative damage of mitochondrial membranes. A study done by Hurley et al. showed that parenteral administration of Co-Q10 was able to prevent acute doxorubicin-induced cardiotoxicity in animals [71]. In a study done by Nehilla et al. Co-Q10-loaded surfactant-free biodegradable poly (lactic-coglycolic acid) (PLGA) nanoparticles were prepared using nanoprecipitation method, which resulted in sustained drug delivery due to the extended C-oQ10 release [72].

Vitamin C
Vitamin C is a versatile hydrophilic antioxidant that can protect against lipid peroxidation by scavenging ROS in the aqueous phase before initiation of lipid peroxidation. Pohl and Reidy have proven that oral administration of vitamin C to human volunteers lead to protection against chemotherapy induced mutagenesis [73]. Supplementation with vitamin C has significantly reduced the chromosomal damage, indicating that vitamin C could reduce the chemotherapy-induced carcinogenesis. In their study, Duarah et al. [74] studied the loading of Vitamin C into ethyl cellulose nanoparticles by the solvent evaporation method for effective delivery to the skin layers.

β-Carotene
β-carotene is a lipophilic antioxidant produced by microorganisms and plants. In a study done by Teicher et al. β-carotene was found to improve the cytotoxicity effect of melphalan on human carcinoma cells [75] and of cisplatin and dacarbazine on melanoma cells [76]. Another study by Seang Chu et al. reported that β-carotene nanodispersions prepared by emulsification-evaporation method could increase the gastrointestinal absorption of lipophilic drugs [77].

Resveratrol
Resveratrol is a natural polyphenol and phytoalexin, it has numerous therapeutic effects including anticancer, antioxidant and anti-inflammatory effects. However, its poor pharmacokinetic properties, low aqueous solubility, low photostability and extensive first pass metabolism, resulted in its poor bioavailability. In their study, Kampa et al. reported that resveratrol could effectively inhibit the growth of prostate cancer cells [78]. Manna et al. have shown that resveratrol is capable of inhibiting reactive oxidative stress and lipid peroxidation induced by tumor necrosis factor in wide variety of cells [79]. Several studies reported the nanoencapsulation of resveratrol in order to enhance its anti-tumor activity. In their study, Thipe et al. [80] reported the preparation of resveratrol-conjugated gold nanoparticles: in order to increase the anti-tumor efficacy against breast, pancreatic and prostate cancers. Another study by Penalva et al. [81] reported the enhanced oral bioavailability of resveratrol through its encapsulation of casein nanoparticles.

Curcumin
Curcumin is extracted from curcuma rhizomes, through crystallization by extraction with solvent and extract purification. Since inflammation is highly linked to tumor promotion, curcumin is considered as a chemopreventive agent due to its anti-inflammatory and antioxidant effects on carcinogenesis [82]. Although it is a great bioactive agent and natural antioxidant but it has some disadvantages such as water insolubility and low bioavailability [83]. To overcome this problem, curcumin nanoparticles were formulated [84]. Das, Kasoju developed curcumin-loaded tripolymeric composite nanoparticles using ionotropic pre-gelation followed by polycationic cross-linking for the delivery to cancer cells. Results showed that these composite nanoparticles could successfully deliver curcumin into cancer cells [85].

Catechin
Catechin is a herbal flavonoid with great antioxidant activities [86]. However, only few clinical studies are carried out on humans, owing to its very low bioavailability due to rapid elimination from liver by biliary excretion, when administered in vivo [87]. In a study done by Kaur et al. results showed that treatment of mammary adenocarcinoma mice with catechin from green or black tea resulted in an increase in the survival time and decreased the tumor load [88]. Hu, Pan et al. have developed chitosan-tripolyphosphate nanoparticles using cross linking technique as carriers for delivering tea catechins successfully in a controlled manner [89].

Rutin
Rutin (RT) is a natural flavonoid widely distributed in medicinal herbs, food and other products, and possess powerful antioxidant, anti-inflammatory and anticancer properties. Owing to its immense antioxidant potential, it has been reported to exhibit therapeutic potential in diverse variety of cancers including hepatic cancer. Despite its diverse therapeutic merits, RT shows poor aqueous solubility, which results in low oral bioavailability, which further limits its therapeutic potential in cancer therapy. A study which was done to investigate the chemoprotective effects of RT, reported that this antioxidant induced favorable changes in the antioxidant defense system in cancer therapy [90]. In a study by Zhang et al. [91], the antioxidant activity of rutin was improved via incorporation into zein-sodium caseinate nanoparticles.

Baicalin
Baicalin (BA) is a flavonoid extracted from Scutellaria baicalensis Georgi. It has numerous biological activities including anti-inflammatory, antibacterial, antioxidant and anticancer effects [92,93]. Increasing evidence suggests that BA has strong capacity to inhibit tumor growth in various cancers [94,95]. A study by Chen et al. reported that BA effectively inhibited viability of ovarian cancer cells [96]. In addition, BA could retard cancer progression by inhibiting invasion and metastasis of cancer cells [97].
Studies reported that concomitant administration of BA and vitamin C could suppress lung carcinoma and lung metastasis [98,99], and suppress tumor growth in hepatic cancer [100]. Zhao et al. have developed BA loaded cationic solid lipid nanoparticles prepared by emulsion evaporation-solidification at low temperature method for brain targeting as a carrier for drugs through blood brain barrier [23].

Conclusions
Various studies have proved that using antioxidants during cancer therapy is a promising trend to improve response to chemotherapy, avoid/reduce side effects associated with chemotherapy and maintain the cellular integrity, thus maintain the homeostasis of the host immune system. In this review, several antioxidants with different therapeutic benefits in cancer therapy have been discussed. Moreover, several studies focusing on the application of nanotechnology in delivery of these antioxidants have been shown and proven promising results in enhancing their therapeutic benefits in cancer therapy. Therefore, further preclinical and clinical studies are needed to establish the clinical implications of antioxidants doses and timings. -Sustainable -Saving energy, time and additional costs [101] -Hot homogenization has a problem of drug entrapment and crystallization, Cold homogenization minimizes thermal exposure of the drug but does not avoid it completely, due to the melting of the drug-lipid mixture in the initial step [102].
Microemulsion Technique -Spontaneous formation Their formation requires large amounts of surfactant which are irritating at high concentrations [105] -Ease of manufacturing -Scale-up -Thermodynamic stability -Improved drug solubilization and bioavailability [104] [112,113] -Easy handling and a fast production process without technically sophisticated equipment [111]  Conflicts of Interest: The authors declare no conflict of interest.