Resveratrol‐loaded nanomedicines for cancer applications

Abstract Background Resveratrol (3, 5, 4′‐trihydroxystilbene), a natural polyphenol and phytoalexin, has drawn considerable attention in the past decade due to its wide variety of therapeutic activities such as anticancer, anti‐inflammatory, and antioxidant properties. However, its poor water solubility, low chemical stability, and short biological half‐life limit its clinical utility. Recent findings Nanoparticles overcome the limitations associated with conventional chemotherapeutic drugs, such as limited availability of drugs to the tumor tissues, high systemic exposures, and consequent toxicity to healthy tissues. This review focuses on the physicochemical properties of resveratrol, the therapeutic potential of resveratrol nano‐formulations, and the anticancer activity of resveratrol encapsulated nanoparticles on various malignancies such as skin, breast, prostate, colon, liver, ovarian, and lung cancers (focusing on both in vitro and in vivo studies). Conclusions Nanotechnology approaches have been extensively utilized to achieve higher solubility, improved oral bioavailability, enhanced stability, and controlled release of resveratrol. The resveratrol nanoparticles have markedly enhanced its anticancer activity both in vitro and in vivo, thus considering it as a potential strategy to fight various cancers.

The incidence of "French Paradox" demonstrated that consumption of red wine decreases the incidence of cardiovascular diseases despite the intake of a high-fat diet. 3 After this highly publicized "French paradox," resveratrol has gained increased popularity in the scientific community, leading to numerous publications on the investigation of its biological activities. In a study by Soleas et al, the anticarcinogenic properties of four polyphenols were compared. Polyphenols such as catechin, quercetin, gallic acid, and trans-resveratrol were administered to the mouse twice a week for 18 weeks. 4 The percentage of tumor inhibition and the number of mice developing one or more tumors were compared among different polyphenols. It was observed that the administration of trans-resveratrol showed much higher absorption compared to catechin and quercetin.
Absorption of trans-resveratrol is approximately 20-fold more effective than catechin. 5 By considering the concentration of polyphenols in their respective dietary sources, it was concluded that trans-resveratrol is the most effective anticancer polyphenol available in red wine. 4 Resveratrol (3, 5, 4 0 -trihydroxystilbene, a phytoalexin), a natural polyphenol, is found in a wide variety of plants, such as peanuts, blueberries, cranberries, legumes, rhubarb, grapes, eucalyptus, and various grasses. Although resveratrol is naturally occurring, it can only be isolated in a few milligram quantities per kilogram of the plant material, for example, grape skin. Therefore, resveratrol has been chemically synthesized in its purest form for biological use. It has a wide variety of pharmacological activities such as cardioprotection, platelet de-aggregation, antioxidant, anti-inflammatory, and vasorelaxant properties. 6 It also shows antiviral activity against human immunodeficiency virus and the herpes simplex virus, 7,8 and enhances the antiviral activity of zidovudine, zalcitabine, and didanosine. 9 One of the main biological activities of resveratrol is that it exhibits anticancer activities against various cancers, which was first reported by Jang et al in the year 1997. 10 According to a report from the World Health Organization (WHO), cancer is the second leading cause of death worldwide, accounting for nearly 9.6 million deaths in the year 2018. 11 Naturally occurring polyphenols have been used both as an adjunct therapy and chemopreventive dietary supplement for decades. In addition, the anticancer activity of resveratrol is reported to be enhanced when used as combination therapy with other chemotherapeutic drugs. [12][13][14] Several studies have reported the antiproliferative effects of resveratrol in vitro, but the literature is lacking in correlating these results in animal models to enable human application. The low aqueous solubility, chemical instability, and poor absorption across biological membranes limit resveratrol's usage as a chemopreventive or therapeutic agent. Although resveratrol is currently marketed in various traditional dosage forms (tablets, capsules, and powders), there is a lack of sufficient data on its efficacy against cancer prevention and treatment. 15 To overcome these limitations, nanoparticle-based formulations have been developed for enhanced absorption and to deliver the optimal concentrations of resveratrol to the tumor target tissue. The novel nano-formulations for resveratrol delivery include polymeric nanoparticles, liposomes, micelles, metallic nanoparticles, and solid lipid nanoparticles. These systems increase water solubility, stability, and permeation across biological membranes and provide enhanced permeation and retention effect (EPR) in the tumor sites. 16 The present review attempts to gather comprehensive information on the nanomedicine approach in treating a larger body of cancers. Various challenges in the formulation and delivery of resveratrol and the in vitro and in vivo effects of nanoformulation on each cancer type are presented. Figure 1 shows a rapidly growing trend of resveratrol nanoformulations in cancer for the past decade, showing hundreds of publications in the scientific literature.

| PHYSICOCHEMICAL PROPERTIES AND PHARMACOKINETICS OF RESVERATROL
Resveratrol has a molecular weight of 228.25 g/mol and a melting point of 254 C. It is a creamy white powder, a hydrophobic compound with a log P O/W = 3.1, 17 and low aqueous solubility of 30 μg/mL. 18 Resveratrol demonstrates a solubility-limited absorption across biological membranes, and hence it is categorized as a "Class II" compound according to the Biopharmaceutical Classification System. A double bond linking two phenolic rings in the resveratrol structure facilitates the formation of trans-and cis-isomers, among which the trans-isomer is the most stable form. 19 The trans-and cis-isomers exhibit differences in the spectrophotometric UV absorption levels, enabling their identification, and these can be distinguished clearly in nuclear magnetic resonance spectroscopy due to their chemical shifts. Trans-resveratrol is more biologically active and is converted to the cis-isomeric form upon exposure to UV light. 20,21 The trans-resveratrol is stable for months when protected from light in a wide pH range. 21 The pKa of transresveratrol corresponding to 1, 2, and 3 phenolic groups are 8. 99, 9.63, and 10.64, respectively. 22 Based on the stable nature and biological activity, when the structure of resveratrol is not specified, the compound is generally referred to as trans-resveratrol.
Despite its chemopreventive properties, resveratrol poses various pharmacokinetic challenges due to its low bioavailability and chemical instability. Following oral administration, resveratrol is well absorbed (75%) by the intestinal epithelium through passive diffusion. However, it is extensively metabolized in the intestine and liver (glucuronidation and sulfate conjugation) to form metabolites such as trans-resveratrol-3-O-glucuronide and trans-resveratrol-3-sulfate, respectively. 23 It can also be found as free resveratrol forming F I G U R E 1 Number of publications in the past decade retrieved using the search terms "Resveratrol", "nano", and "cancer" used together from Web of Science (accessed on 10th December 2020) complexes with the low-density lipoproteins, plasma proteins such as albumin, thus leaving only trace amounts of free resveratrol in the systemic circulation. 24 It was found that the order of abundance of resveratrol metabolites in the systemic circulation is glucuronides, followed by sulfates, followed by the free resveratrol. 25 Thus, resveratrol has a very short plasma half-life of only 8 to 14 minutes, 26 and reaches peak plasma concentrations at 1 hour (following ingestion) and 6 hours (following enteric recirculation of resveratrol metabolites). 27 The most significant route of excretion is via urine or feces.
However, the excretion of sulfates through urine is higher (84%) than glucuronides and free resveratrol (trace amounts to 17%). 28 In addition, resveratrol, and its metabolites are also found in feces, where only small quantities of sulfates (<1%) are excreted via feces. 28 Following intravenous administration of resveratrol, the terminal elimination half-life ranges between 7.8 and 35 minutes. 29 In a study by Walle et al (2004), following the intravenous administration of the low dose of 14 C-labelled resveratrol, high absorption rate followed by a rapid decline in the peak plasma concentration after 1 hour indicates that the distribution is rapid and only trace amounts of resveratrol enter the enterohepatic circulation. The calculated t 1/2 after oral and i. v administration is 9.2 and 11.4 hours, respectively, 28 indicating the superiority of i.v compared to oral administration in overcoming some of the limitations. However, elimination rate and clearance are still faster due to the metabolic instability favoring the conjugation of resveratrol with either glucuronic and/or sulfonic acid. 30 This rapid metabolization impairs the anticancer efficacy of resveratrol, which could be addressed by developing various nanoformulations with sustained and site-specific resveratrol delivery.

| NANOTECHNOLOGY FOR DELIVERY OF RESVERATROL
One of the main drawbacks of current cancer therapy is the lack of targeted delivery to the cancer tissue. Due to the high toxicity of conventional chemotherapeutic agents and poor drug delivery, nanomedicines have emerged as a novel tool to improve cancer treatment. Based on the drug's physicochemical properties, nanoformulations with improved stability, greater circulation half-lives, enhanced intratumor deposition, and controlled release can be achieved. [31][32][33] These properties can be optimized to improve antitumor activity and reduce toxicity to nontarget, healthy tissues. 34,35 Furthermore, imaging probes can be included in the nanoparticles so that the side effects of drugs can be predicted in certain patients by providing data on potential nontarget accumulation sites in healthy tissue. 36 Nanomedicines are generally defined as particles that are complex systems consisting of at least two components, one of which is the active ingredient. These nanomedicines typically have particles around 100 to 200 nm nanometers in size and are associated with other pharmaceutical ingredients for stabilizing the formulation or altering the pharmacokinetics (ADME-absorption, distribution, metabolism, and elimination) and improving the drug delivery to the tumor sites. 37,38 Targeted drug delivery or active targeting describes the specific interaction between the drug carrier and target cells, usually through specific ligand-receptor interactions, 39 that generally facilitate the intracellular uptake of nanoparticles. The efficiency of ligand-receptor binding depends on various factors such as its availability, selective expression of receptor on the target cells, and shedding of the receptor following ligand binding. 36,40,41 Despite very low bioavailability, considering its therapeutic benefits, resveratrol is available as a dietary supplement in the form of oral dose products such as tablets, capsules, and powders. Nanomedicines have been used to improve bioavailability, reduce metabolism, and improve the delivery of resveratrol. 9,[42][43][44] In addition, they offer advantages such as enhanced tumor targeting, improved solubility, and chemical stability for resveratrol. 45 A wide range of nanomaterials has been employed in the development of resveratrol cancer therapeutics such as lipids, synthetic polymers, glycan, and proteins. Figure 2 shows various resveratrol nanocarrier systems for cancer treatment. Different biocompatible and biodegradable polymers can be utilized in the preparation of nanoparticles. In general, these nanoparticles are coated with polyethylene glycol (PEG) on their surface, a process called as PEGylation.
The PEGylated particles remain in the circulation for extended periods, resist biotransformation reactions, and selectively accumulate in the tumors through the EPR effect. 46 This higher accumulation in tumor tissues and lower retention in healthy tissues lead to better efficacy with minimal side effects. 47 More recently, with the help of surface engineering, nanoparticles are conjugated with various targeting ligands such as peptides, antibodies, and aptamers, which allow to reach the targeted tumor directly and release the payload for enhanced efficacy and reduced toxicity. 46 Liposomes are spherical vesicles containing an aqueous core and phospholipid bilayer and can be differentiated into small unilamellar, giant unilamellar, large unilamellar, and multilamellar based on their size and the number of bilayers. 48 They can incorporate hydrophilic drugs in the aqueous core and lipophilic drugs in the phospholipid bilayer. Moreover, liposomes can provide protection against photodegradation for various drugs and biochemicals. For example, trans-resveratrol encapsulated in liposomes remained intact (70% retained) for 16 minutes when exposed to UV light compared to the free drug (10% retained). 49 Polymeric nanoparticles are another type of drug delivery system where the drug is either conjugated or dispersed within the polymer matrix, protects the drug from degradation, provides a sustained release, and improves bioavailability. 50 However, another carrier system referred to as solid lipid nanoparticles (SLNs) provides combined benefits of both polymeric nanoparticles and lipid emulsions. They are the spherical vesicles containing lipid core surrounded by hydrophilic surfaces. Generally, hydrophobic drugs such as resveratrol can be easily incorporated into the lipid core. 51 SLNs have been reported to be superior to liposomes in increasing the chemical stability of resveratrol. It is found to protect against oxidation, hydrolysis, and photodegradation while enhancing the bioavailability of resveratrol. 52 In addition, surface properties can be altered to modify the uptake. In a study by Teskac et al (2010), cellular uptake, transport, and internalization of resveratrol-loaded SLN were investigated, which showed that the particles crossed the cell membrane in less than 15 minutes. 52 Another type of nanocarrier system is cyclodextrins (CD), which are cyclic oligosaccharides containing lipophilic core and hydrophilic surface. They are typically between 1 and 2 nm and form inclusion complexes with the drugs and can enhance the solubility, bioavailability, and stability of the drug. 53 In a study by Venuti et al (2014), resveratrol sulfobutylether β-CD complex (1:1) showed increased solubility and cytotoxicity compared to the cyclodextrin without resveratrol, which showed no effect on cell viability. 54 Based on the targeting principle, nanotherapeutics are classified into passive targeting, active targeting, stimuli-responsive systems, and theranostics. Both active and passive targeting strategies are used to improve the targeting of anticancer drugs. However, distribution is dependent on the physicochemical properties of a drug and is limited by its penetration into the tumor tissue. 55,56 Particles that have longcirculation half-lives and remain in the systemic circulation for extended periods of time have been able to exploit the EPR effect and accumulate passively. 57,58 However, optimal activity is dependent on the stability of drugs within the nanoparticle, drug-carrier release kinetics, tumor vascular extravasation, and uniform intra-tumor distribution. Active targeting strategies have been employed to improve intracellular uptake and better control targeting to specific cell populations. In contrast, stimuli sensitive nanomedicines can be designed to release the drug upon a trigger, for example, drugs such as doxorubicin, for which delivery is not pH-sensitive, can be conjugated with a pH-sensitive nanomedicine in order to increase the cellular uptake and intracellular drug release. 59 These nanomedicines also decrease tumor resistance to anticancer drugs, thus mediating the stimuli-responsive drug release and endocytic drug uptake. 60,61 In the below sections, we will discuss resveratrol delivery systems for various cancer types.

| ANTICANCER ACTIVITY OF RESVERATROL NANOFORMULATIONS
Chemoprevention is defined as a reduction or prevention of cancer risk by ingestion of either synthetic or natural compounds with low toxicity that is able to suppress, delay, or reverse carcinogenesis. 62 Resveratrol has been found to possess many chemoprevention and chemotherapeutic properties. 63,64 Resveratrol acts by various cellsignaling pathways such as cell cycle arrest, suppression of cell proliferation, induction of apoptosis, reduction of inflammation, and inhibition of adhesion, invasion, and metastasis. [65][66][67] Resveratrol's mechanism of action has been widely studied through in vitro, 68 and in vivo experiments. 63,69 It has been well reported that resveratrol's antitumor activity is due to multiple mechanisms, including proapoptotic, antiproliferative, anti-inflammatory, and antiangiogenesis. For example, in stem-like cells derived from breast cancer cells, resveratrol induces apoptosis by downregulating fatty acid synthase and enhancing proapoptotic genes such as DAPK2 and BNIP3. 70 Mechanism of action in in vivo studies is much more complicated, where resveratrol has been shown to affect a number of molecular targets based on formulation, cancer type, stage of the disease, dose, and duration of resveratrol present at the target site of action. 71 Both reactive oxygen species (ROS) and reactive oxygen metabolites (ROM) are involved in the generation of oxidative stress in vivo. 72,73 Physiological levels of ROS are essential for transcriptional and posttranscriptional cell signaling. 74 However, excessive production and accumulation of ROS may induce the modification of cellular proteins and nucleic acids with deleterious effects such as DNA damage, inflammation, and promotion of tumor growth. 75 Evidence suggests that resveratrol serves as a free radical scavenger because of its ability to promote the activity of various antioxidative F I G U R E 2 Different types of resveratrol-loaded nanoparticles for cancer prevention and therapy T A B L E 1 Co-delivery of resveratrol nano-formulations with various chemotherapy drugs enzymes. 76 It has the potential to inhibit lipid peroxidation (induced by Fenton reaction), decrease the oxidative chain complex, scavenger of ROS, and other free radicals. 75

| Skin cancer
Among all human malignancies, skin cancer is the most common form, 112 accounting for more than three million cases every year in the United States alone. 113 The development of skin cancer is related to two main factors, ultraviolet B (UVB) radiation exposure and nuclear factor kappa B (NFkB). Resveratrol, due to its antioxidant properties, can block the damage caused by UVB exposure, thus inhibiting UVB-induced lipid peroxidase or blocking UV-mediated activation of NFkB. 10 Tyrosinase is an essential enzyme for melanin production. Inhibiting this enzyme activity was found to be very effective in controlling melanoma cell growth. 112 Application of resveratrol, both before and after the UVB exposure drastically reduced the skin damage and decreased skin cancer incidence. 114 It is able to reduce tyrosinase activity by 30 to 45%. 115 Resveratrol also inhibits tumor progression by suppressing the growth of various cancer cell types by inhibiting DNA polymerase, deoxy-ribonucleotide synthesis, and inducing cell cycle arrest. 116

| In vitro studies
In a study by Rigon et al (2016), in vitro biological activity of transresveratrol loaded SLNs was evaluated in various skin disorders.
Resveratrol-loaded SLNs showed a mean particle size less than 200 nm, zeta potential of 3 mV, and permeated 45% of resveratrol after 24 hours. The nanoparticles also achieved tyrosinase inhibitory activity, greater than or equal to that of the positive control, kojic acid, and proved to be nontoxic in HaCat keratinocytes. 112  Abbreviations: COX-2, Cyclooxygenase-2; ERK1/2, extracellular signal regulated kinases; HER-2, human epidermal growth factor receptor 2; HERG-β1, heregulin-beta 1; HO-1, hemeoxygenase-1; MAPK, mitogen-activated protein kinases; MMP-9, matrix metalloproteinase-9; mTOR, mammalian target of rapamycin; NF-kB, nuclear factor kappa; NO, nitric oxide; NOS, nitric oxide synthase; PARP, Poly(ADP-ribose) polymerase; PI3K/Akt, Phosphatidlyinositol-3kinase; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor. studies. Jang et al (1997) first reported the chemopreventive role of resveratrol in skin cancer in mice treated with a carcinogen. 10 In SKH-1 hairless mice, topically applied resveratrol showed greater inhibition of UVB mediated skin inflammation, induction of cyclooxygenase and ornithine decarboxylase, and generation of hydrogen peroxide in the skin. 120 Similarly, resveratrol's anticancer activity against multiple UVB exposures and the involvement of survivin was studied in SKH-1 hairless mouse skin. It was found that topical pretreatment of resveratrol resulted in inhibition of UVB exposure mediated cell proliferation and phosphorylation of survivin. 121   Molecular targets that are activated due to loss of PTEN, including p-Akt, cyclin D1, and androgen receptor, were downregulated by coencapsulation, suggesting that these liposomes can target multiple mechanisms. One such mechanism is due to enhanced binding of coadministered liposomes to the albumin, enhancing its transportation into the bloodstream and thus improving its therapeutic efficacy. 102

| Colon cancer
According to WHO, colon cancer is the fifth most common cause of cancer deaths worldwide. 139 About 95% of the colorectal cancer cases are caused by common dietary and environmental factors. 140 Some of the significant factors that influence colorectal cancer are old age, smoking, high alcohol consumption, diabetes mellitus, obesity, and low fiber intake. 141 Due to poor bioavailability and substantial accumulation of resveratrol in the colon, it is considered as the most convenient target for application. 142 Arunachalam and coworkers reported that the NFkB pathway is the main contributor to colon cancer. Resveratrol reverses the activation of NFkB, which is responsible for inducing inflammatory cytokines. 143

| In vivo studies
Resveratrol-loaded PEG-polylactic acid-based polymeric nanoparticles were designed to suppress the glucose metabolism and tumor growth both in vitro and in vivo. 105 These nanoparticles showed an increased apoptotic cell death and 18 F-fluorodeoxyglucose ( 18 F-FDG) uptake and reduced ROS compared to control in CT26 colon cancer cells.
Whereas in CT26 tumor-bearing mice, 18 F FDG uptake was reduced with retardation of tumor growth and improved survival rate compared to empty nanoparticle-injected control. 105 In another study, a novel self-microemulsifying formulation (SMEDDS) (based on Capryol 90, Cremophor EL) containing curcumin together with resveratrol was developed to address its poor aqueous solubility, improve their absorption, and delivery across colon cancer cells. This co-delivery system showed higher antioxidant and cytotoxic activity than the nanoemulsion with either curcumin or resveratrol alone, demonstrating synergistic cytotoxic action due to the co-delivery formulation.
Following oral administration of nanoemulsion to rabbits, the total plasma concentrations of curcumin and resveratrol increased by 10and 6-fold, respectively, compared to the unformulated drug combination. The nanoformulation achieved increased solubility, protection from degradation, and improved the absorption of resveratrol and curcumin. The possible mechanism of action is due to the presence of the drug in the dissolved form in the SMEDDS, and due to its smaller particle size, the surface interfacial tension is increased, thereby enhancing the rate and extent of oral absorption. 92

| Liver cancer
Liver cancer is the fourth leading cause of all cancer-related deaths worldwide, which is reported to have relatively high mortality and morbidity in men. 148 Treatment is often challenging due to the high systemic toxicity of chemotherapeutic drugs, leading to discontinuation of the treatment. Therefore, nano-formulations and targeted delivery of anticancer agents are highly beneficial to enhance the efficacy, increase the uptake and internalization of drugs into tumors, and reduce the toxicity in healthy tissues.

| In vitro studies
Resveratrol-loaded chitosan nanoparticles were surface-modified with either biotin or both biotin and avidin. 149 The nanoparticles containing both avidin and biotin demonstrated a size range <200 nm and superior cytotoxicity in HepG2 cells compared to biotin alone. 149 These nanoparticles enhanced the target specificity of resveratrol-loaded chitosan nanoparticles in hepatocarcinoma. In addition, it was observed that the drug-loaded nanoparticles surface modified with both avidin and biotin had a higher liver targeting index (2.70) and more potent cytotoxicity against HepG2 cells than nanoparticles surface modified with biotin alone. 149 Similarly, resveratrol-loaded ionically crosslinked chitosan nanoparticles were prepared to improve the stability, solubility, and hepatic tumor targeting of resveratrol. The nanoparticles were able to improve the long-term stability and drug release in simulated tumor pH 6.5 than at physiological pH 7.4. Moreover, the antioxidant activity of resveratrol was maintained even after UV light irradiation. The nanoparticles were efficiently taken up by hepatocellular carcinoma (SMMC 7721) cells, showed similar antiproliferative activity in SMMC 7721 cells, and lower cytotoxicity in normal hepatocyte cells (L02) compared to free resveratrol. All the abovementioned advantages might be due to the smaller size of chitosan nanoparticles (200 nm), accumulating the drug at the cancer site by EPR effect. 106 In another study, resveratrol nanosuspension composed of poloxamer 188 was prepared using a high-pressure homogenizer, and in vitro anti-hepatocarcinoma effects relative to free resveratrol was evaluated. The particle size of the nanosuspension was 159 nm, and the zeta potential was −2.1 mV.
The nanosuspension inhibited the proliferation of HepG2 cells (2.5-fold lower IC50 values) than the bulk resveratrol. 150 Therefore, resveratrol-loaded nanoformulations were found to be very promising for the treatment of hepatocarcinoma. In a mouse xenograft model, the gold-resveratrol nanoparticles reduced the tumor growth by decreasing the expression of vascular endothelial growth factor (VEGF) and promoting apoptosis in tumor tissue. In addition, there was no observable organ toxicity in the heart, liver, kidney, and spleen as assessed by histological studies. Moreover, the gold nanoparticles effectively increased the uptake of resveratrol into cells and localized near mitochondria. Therefore, these nanoparticles possess significantly better antitumor efficacy than resveratrol alone, both in vitro and in vivo. 151 Similarly, glycyrrhizic acidconjugated human serum albumin nanoparticles loaded with resveratrol for liver tumor targeting was prepared by high-pressure homogenization. The particle size of 108 nm, PDI of 0.001, encapsulation efficiency of 83.6% were observed, and resveratrol in the nanoparticles was found to be in an amorphous state. The nanoparticles showed a sustained release pattern and 2-fold higher cytotoxicity and greater cellular uptake as compared to free resveratrol. Moreover, the nanoparticles were labeled with near-IR fluorophore Cy5 to monitor the in vivo body distribution of nanoparticles in H22 tumor-bearing mice. The near-IR fluorescence images showed an enhanced distribution of nanoparticles to the liver tumors and a sustained-release pattern. This study demonstrates the influence of formulation parameters such as human serum albumin concentration, homogenization speed, pressure, duration, and water to the organic phase volume ratio on the particle size and drug loading efficiency.

| In vivo studies
Moreover, this study also showed the beneficial effect of combining the drugs with albumin to prevent deposition of the drug at the injection site and obtain a slow release of the drug.

| Ovarian cancer
Ovarian cancer is the primary cause of cancer-related deaths among all gynecological cancers, and chemotherapy is the most common treatment in many cases. However, if the tumor is well-differentiated and confined to the ovary, surgery is the first choice of treatment.
The anticancer activity of resveratrol was due to inhibition of STAT3 signaling. 158 The role of autophagy was also found in resveratrolinduced apoptotic cell death in OVCAR-3 and CAOV-3 human ovarian cancer cells. Resveratrol causes the generation of ROS, causing autophagy and subsequent apoptosis. 159 Resveratrol is shown to lower the glucose uptake significantly and levels of phosphorylated Akt and mTOR in epithelial ovarian cancer cells. 160

| In vitro studies
The chemotherapeutic activity of resveratrol-loaded bovine serum albumin nanoparticles in human SKOV3 ovarian cancer cell lines was investigated. The nanoparticles induced apoptosis in a dosedependent manner, and the translocation of apoptosis-inducing factor (AIF) from mitochondria to cytoplasm occurred even before the Cyto C. Moreover, binding of Bax to the mitochondria was essential for the release of AIF and Cyto C. Thus, it was concluded that resveratrol-loaded bovine serum albumin nanoparticles induced apoptosis through AIF apoptosis pathway, which is considered as an alternative to the caspase-dependent apoptosis pathway. This is the first study to investigate the mechanism for caspase-independent apoptotic pathway. While AIF protein is an important factor in caspase-independent pathway, further research is required to understand the mechanism by which AIF causes DNA ladder formation and also the association between early response signal and apoptotic signal. 110 In another study, resveratrol-loaded herbal extract (Angelica Gigas Nakai) (AGN) based nanoparticles were prepared using the nanocrystal concept. 161 Nanoparticles are converted from crystalline to amorphous states by emulsification and solvent evaporation methods. The nanoparticles showed a particle size of 224 nm and negative zeta potential values. properties. 162 Due to the complexity in cancer cell signaling, the therapeutic benefit of specific inhibitors that can target only one network is limited. However, resveratrol is considered as a potent anticancer agent because it has both chemopreventive and chemotherapeutic effects by targeting multiple molecular pathways. 163 Moreover, resveratrol affects all three stages (initiation, promotion, and progression) of carcinogenesis, and it is shown to induce the apoptotic pathway through several mechanisms. 164 In addition to its anticarcinogenic activity as a single agent, its coadministration with other chemothera- showed low encapsulation efficiency ranging from 0.6% to 3.1%. The formulation with tween 80 alone had the lowest encapsulation efficiency and increased when two or more surfactants were utilized. This is due to enhanced solubilization of resveratrol in the combination of surfactants. Therefore, it is important to consider the formulation parameters such as surfactant composition and the drug-lipid ratio in order to achieve higher encapsulation of the drug in the lipid core. 165 In contrast, conventional polymeric micelles have reasonable encapsulation efficiency; however, they have poor stability in vivo.
This is due to the physical assembly of conventional polymeric micelles leading to micelle disintegration. Therefore, it is important to consider alternatives such as disulfide-mediated crosslinked micelles, which can be reversely disintegrated by reducing agents 166 or to generate unimolecular micelles, constructed by coupling of L-lactide with mPEG, thus forming a highly stable unimolecular micelle. 167 Likewise, polymeric nanoparticles had good drug loading efficiency and stability; however, scale-up of the current synthetic technique is a major challenge hindering its commercial availability. 168  Other clinical studies focused on supplementation of resveratrol (2.5 g/day for 29 days) for reducing the levels of IGF-1 and IGFBP3. 172 Overall, all these clinical trials had a very small patient sample size, highlighting the scarcity of human data for resveratrol, thus focusing on the need for more research into the safety and efficacy of resveratrol. In addition to the completed trials, some of the ongoing trials focused on determining the optimal dose of resveratrol formulation that will result in effective plasma levels (NCT00433576) in colon cancer (ClinicalTrials.gov). This is important because the optimal dose of resveratrol is yet to be established.
All these trials provide us with key suggestions for future clinical studies to make the most effective treatment regimens. Based on the analysis of these clinical trials, it is essential to focus on (a) the exact mechanism of action of resveratrol, (b) establishing the optimal dose, and (c) developing novel formulations of resveratrol with other agents. Considering all these parameters in the future will likely result in bringing the resveratrol nanoformulations from bench to bedside.

| CONCLUSION
To overcome the extreme heterogeneity of cancer cells, an ideal therapeutic agent should target multiple biochemical pathways while limiting toxicity to healthy tissues. Among the naturally occurring anticancer agents, resveratrol has the potential to be an effective chemopreventive agent due to its ability to interact with several molecular targets involved in carcinogen metabolism, cell proliferation, apoptosis, etc. Resveratrol was also shown to modulate various signal transduction pathways. Despite being a potential chemopreventive agent, resveratrol presents various limitations such as low chemical stability and poor absorption and tumor delivery. In order to effectively overcome these shortcomings and to improve the bioavailability and cellular uptake of resveratrol, novel nanostructured delivery systems have been recently evolved. Various nanotechnology approaches were attempted for resveratrol for its beneficial effects in cancer chemoprevention as well as therapy. Despite these challenges in nanoformulations, the authors foresee a growth in the resveratrol nanomedicines in the coming years, due to the advancements in nanoengineering and bioengineering methods.

DATA AVAILABILITY STATEMENT
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.