Recent Findings on Thymoquinone and Its Applications as a Nanocarrier for the Treatment of Cancer and Rheumatoid Arthritis

Cancer causes a considerable amount of mortality in the world, while arthritis is an immunological dysregulation with multifactorial pathogenesis including genetic and environmental defects. Both conditions have inflammation as a part of their pathogenesis. Resistance to anticancer and disease-modifying antirheumatic drugs (DMARDs) happens frequently through the generation of energy-dependent transporters, which lead to the expulsion of cellular drug contents. Thymoquinone (TQ) is a bioactive molecule with anticancer as well as anti-inflammatory activities via the downregulation of several chemokines and cytokines. Nevertheless, the pharmacological importance and therapeutic feasibility of thymoquinone are underutilized due to intrinsic pharmacokinetics, including short half-life, inadequate biological stability, poor aqueous solubility, and low bioavailability. Owing to these pharmacokinetic limitations of TQ, nanoformulations have gained remarkable attention in recent years. Therefore, this compilation intends to critically analyze recent advancements in rheumatoid arthritis and cancer delivery of TQ. This literature search revealed that nanocarriers exhibit potential results in achieving targetability, maximizing drug internalization, as well as enhancing the anti-inflammatory and anticancer efficacy of TQ. Additionally, TQ-NPs (thymoquinone nanoparticles) as a therapeutic payload modulated autophagy as well as enhanced the potential of other drugs when given in combination. Moreover, nanoformulations improved pharmacokinetics, drug deposition, using EPR (enhanced permeability and retention) and receptor-mediated delivery, and enhanced anti-inflammatory and anticancer properties. TQ’s potential to reduce metal toxicity, its clinical trials and patents have also been discussed.


Introduction
As per the WHO, approximately 80% of the global population utilizes indigenous systems of medicine for their primary health care [1]. Recently, various potential phytocandidates such as β-elemene, brazilin, bufalin, cardamonin, cryptotanshinone, isogarcinol, curcumin, celastrol, lapachol, nobiletin, oroxylin A, thymoquinone, resveratrol, torilin, and swertiamarin have been identified to have pharmacological properties [2]. Thymoquinone (TQ) is a crucial active ingredient obtained from the black seed of the plant Nigella sativa (NS) and Caramcarvil, with potential antioxidant and anti-inflammatory activities [3]. It holds a wide range of other therapeutic properties, including hepatoprotective, cardioprotective, anticancer, antidiabetic, and antimicrobial properties [4]. Moreover, TQ also nullifies oxidative stress and prevents any damage to the tissue or cellular environment [5].
The seeds of N. sativa contain a combination of volatile oils (0.40-0.45%), fixed oils (>30%, wt/wt) with two terpene alkaloids and eight fatty acids. Dithymoquinone, TQ, trans-anethol, (2-isopropyl-5-methylbenzo-1, 4-quinone), limonine, carvone, nigellidine, hedrin and p-cymene are some of the majorly identified terpenes. Moreover, the seeds also contain isoquinoline (nigellicimine-N-oxide and nigellicimine) and indazole alkaloids (nigellicimine and nigellidine) [6]. TQ exists in tautomeric forms in which the keto fraction (~90%) majorly exerts pharmacological actions [7]. The 2D and 3D structures of TQ are depicted in Figure 1. TQ is a pharmacologically active agent used as a therapeutic agent as well as for preventive measures [8]. Oral dosing of Nigella sativa (NS) seeds at a quantity of 2 gm daily can effectively treat diabetes, as per reports [9]. However, it is associated with various pharmacokinetic issues that halt its pharmacodynamic activities. TQ is a hydrophobic molecule with low aqueous solubility and is associated with thermal instability and photosensitivity [10], which makes it systematically less bioavailable. Moreover, the bioavailability of TQ is mainly dependent upon its administration route. The absolute bioavailability (BA) of TQ in rabbits after oral (20 mg/kg PO) and IV (5 mg/kg) administration revealed a *58% lag time of 23 min with slower absorption and rapid elimination rates [11]. It is an acidic molecule with a pKa value of 5.1 [12] that is extensively degraded in the aqueous medium, especially at higher pH concentrations [1]. Low aqueous solubility, bioavailability, thermal, and photodegradability are some major drawbacks in utilizing its maximum potential as therapeutic.
Orally administered TQ is biotransformed into hydroquinone by DT-diaphorase (a quinine reductase enzyme) [13]. Enzyme glutathione and NADPH (nicotinamide adenine dinucleotide phosphate oxidase) quinine oxidoreductase converted it into glutathionyldihydrothymoquinone and thymohydroquinone, respectively, via the redox mechanism [14]. TQ catalyzes in a two-step one-electron reduction or a two-electron one-step reduction. In one-electron two-step reduction of TQ, microsomal NADH cytochrome-b5 reductase, mitochondrial NADH ubiquinone oxidoreductase, and microsomal NADPH cytochrome P450 reductase convert TQ into semiquinone, which is further biotransformed into thymohydroquinone [15,16]. Conversely, a one-step two-electron reduction directs the conversion of TQ into thymohydroquinone [17]. Semiquinone of TQ is also known to possess oxidative stress-producing capabilities in cancerous tissues. Superoxide anion produced via oxidation can be nullified by TQ administration [18]. Due to the lack of detoxifying enzymes, which is quite common in cancer cells, the accumulated superoxide may exert the pro-oxidant effect of TQ [19]. The physiological catalysis of TQ is summarized in Figure 2.

Method for Literature Search and Studies Selection
The authors searched a number of electronic databases, namely Science Direct, Scopus, PubMed, US National Library of Medicine Clinical Trials (https://clinicaltrials.gov; accessed on 12 January 2021), and the Clinical Trial Registry of India (http://ctri.nic.in/; accessed on 12 January 2021). The following keywords were selected based on MeSH terms: thymoquinone, nanoparticle, nanocarrier, targeted nanoparticle, rheumatoid arthritis, nano, inflammation, cancer, neoplasm, toxicity, and antioxidant. These keywords were searched individually and in combination. At the first stage of screening, only English language articles were selected if the title, abstract, or full text contained the word "thymoquinone". The initial database search found 5522 articles: 1389 from PubMed, 2191 from Scopus, 1933 from Science Direct, 7 from ClinicalTrials.gov, and 3 from the Clinical Trial Registry of India. In this process of analysis, 4240 articles were excluded due to them being indexed in two or more databases and were considered as duplicates. The remaining 1282 articles were screened out by analyzing the article's title and abstract according to the inclusion criteria. After the second stage of screening, only 184 studies (158 experimental articles, 16 patents, 10 clinical txrials) were found to be appropriate according to the inclusion criteria. Studies of clinical trials in humans of any age, gender, or nationality, case-control studies, cohort studies, and randomized, double-blind, placebo-controlled, and parallel-group trials were considered for the review. Studies demonstrating the safety and efficacy of thymoquinone in in silico models were excluded for this review. Conference abstracts without full data or experimental information, letters to editors, and opinion papers, with potential influences of funding sources on the study results, were also excluded. The progression of thymoquinone articles from the year 2011 to 2021 is reported in Table 1. Furthermore, our review analysis indicated that TQ alone or in combination has beneficial roles in arthritic inflammation and various type of cancers.

Rheumatoid Arthritis
Rheumatoid arthritis (RA) is recognized by the way the body's immune system attacks the lining of the joints and results in significant mortality and morbidity rates. The lifetime risk of developing RA is increasing globally such as in the United States-1.7% (1 in 59) for men and 3.6% (1 in 28) for women; it varies within gender and individuals over time [20]. It is a long-lasting degenerative joint disease with an unknown etiology; however, it is thought to have multifactorial pathogenesis, including genetic and environmental defects as well as impaired immune regulation [21]. RA is characterized by joint inflammation, synovial membrane hyperplasia, excessive chemokines infiltration, leukocyte migration, and autoantibody production [22]. In the altered immune system, T cells fail to control inflammation and may initiate RA or other immune-related disease [23]. Additionally, the cell metabolism, whose primary work is to combat against the autoantigen attack, does not respond properly and effectively in RA conditions, which leads to chronic inflammation. Long-term RA inflammation alters cytokine release; overexpression of pro-and antiinflammatory cytokines results in bone and cartilage damage. Patients with rheumatoid arthritis have unusual autoantibodies such as anticitrullinated antibodies and Rheumatoid Factor (RF), etc., that continue to circulate in the blood and thus, target their own body tissues, leading to polyarticular inflammation of the synovial membrane, wrists and feet along with nodule formation [24]. The synovium of RA is persistently upregulated by the induction of several chemokines and cytokines, including TNF-α, IL-1, IL-6, IL18, IL-15, and IL-12 [25]. Moreover, Toll-like receptors (TLR7, TLR4, TLR3, and TLR2) are also found to be upregulated in arthritic synovium [26] along with other inflammatory molecules, followed by the destruction of cartilage and bone [27]. Osteoclasts, the major bone resorbing cells, are mainly responsive to autoantibodies and inflammatory cytokines, in particular IL-1, IL-6 and TNF, which all induce osteoclast differentiation either directly or by inducing receptor activator of nuclear factor kappa B ligand (RANKL) activity [28]. Subsequently, stimulation of lymphocytes triggers cellular proliferation, differentiation, and also increased inflammatory cytokine synthesis (TNF-α, IL-7, and IL-1) [23,29]. In addition, RA patients also show pulmonary, cardiovascular, and other systemic complications [30].
The diversified pathogenesis of RA demands pharmacological and non-pharmacological approaches and sometimes, rotating interventions to achieve satisfactory therapeutic outcomes as well as patient compliance. Appropriate knowledge about the disease, preventive measures, optimized therapeutic regimens, and treatment goals for patients and health care providers might produce an appropriate impact upon RA amelioration.
Nevertheless, various non-pharmacological and pharmacological approaches, including recognition and avoidance of causative factors, non-steroidal anti-inflammatory drugs (NSAIDs), immunosuppressant therapies, herbal therapies (plant extract, oils), and physical measures (physiotherapy), have been used, either alone or in combination for the management of acute to chronic RA. The initial phase of RA can potentially be treated with NSAIDs; however, the chronic phase requires intensive therapies of disease modifying antirheumatic drugs (DMARDs), including modern biologics [31]. DMARDs such as methotrexate, leflunomide, sulfasalazine, and mycophenolate, etc., and modern biologics that specifically target cytokines and inflammation-inducing cells are used to ameliorate pain and prevent bone damage [31]. Besides their potential application in RA, DMARDs and biologics are also associated with numerous side effects such as TNF-α inhibition, which is associated with the risk of tuberculosis [32]; and tocilizumab, which is associated with a risk of lower intestinal perforation [33]. A large number of patients are resistant to current drugs with only 20-30% reaching low disease activity status and none of them can completely cure RA [31].
Currently, there is no absolute regimen for RA and cancer management owing to a multifaceted pathogenic interaction between a patient's immunity, gene abnormality, and environmental susceptibility. Recently, bioactive compounds of natural origin such as thymoquinone and their nanoformulations were utilized for the treatment of cancer and rheumatoid arthritis. The potential of TQ and its nanoformulations-based targeted delivery for the management of cancer and arthritis is critically analyzed and reported in the following sections.

Thymoquinone Works as an Anti-Arthritic
Thymoquinone is a naturally occurring bioactive molecule reported to ameliorate rheumatic conditions in multiple pathways. TQ (10 mg/kg body weight) significantly downregulated the elevated level of Toll-like receptor (TLR) and other inflammatory cytokines (TNF-α, IL-1, and IL-6) in a Freund's complete adjuvant (FCA)-induced arthritis rat in vivo model [34]. TQ (5 mg/kg body weight) significantly downregulated the level of pro-inflammatory mediators (IL-1b, IL-6, TNF-α, and prostaglandin E 2 ) to reduce arthritis scoring and bone leaching in collagen-induced arthritis in a Wistar rats in vivo model [35]. In an in vivo study, Boudiaf et al. demonstrated that TQ (10-50 mg/kg, intraperitoneal) potentially inhibits N-formyl-methionyl-leucyl phenylalanine-induced neutrophil functions, and superoxide production [36]. In another in vitro study, the inhibition of phospho-p38 and phospho-JNK expression by TQ (0.1-5 µM) through apoptosis-regulated signaling kinase 1(ASK1) was reported to ameliorate rheumatic tissue damage [37]. Similarly, phosphorylation of p38 mitogen-activated protein kinase was blocked by TQ as investigated in both in vitro (isolated human RA fibroblast-like synoviocytes, dose 0-10 mM) and in vivo (rat adjuvant-induced arthritis, dose 5 mg/kg/day of TQ) studies; besides this, LPSinduced overexpression of inflammatory markers such as interleukin-1beta (IL-1b), TNF-α, cyclooxygenase-2, nuclear factor-kappa B-p65 metalloproteinase-13, and prostaglandin E 2 (PGE2) were also regulated [8,38]. TQ decreases receptor-activated nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis (in vitro in RAW 264.7 cells, TQ dose: 2.5, 5, and 7.5 µM) by inhibiting mitogen-activated protein kinase signaling and NF-κB (nuclear factor kappa light chain enhancer of activated B cells) as well as prevention of LPS (lipopolysaccharides)-induced bone erosion at a dose of 5 mg/kg as investigated in an in vivo C57/BL6 male mice model [39]. A similar potential of TQ (dose: 10 µM) in LPS-activated BV-2 murine microglial cells were also reported in an in vitro model [40]. TQ (intra-articularly injection of 0.3 mL; 10 mmol/L) also upregulated the expression of MMP-1 (matrix metalloproteinase-1) (tissue inhibitors) and downregulated MMP-13 in both rabbit chondrocytes and animal models of osteoarthritis induced by anterior cruciate ligament transaction [41]. The anti-inflammatory potential of TQ (dose of 2.5 mg/kg and 5 mg/kg) was found to be comparable with methotrexate (MTX) in an in vivo model of Freund's incomplete adjuvant-induced arthritis [42]. Similar results were also observed to decrease carrageenan-induced inflammation in an in vivo rat model with an intraperitoneal dose of TQ (10 and 50 mg/kg) [36]. Moreover, the immunomodulatory effects of TQ (10 mg/kg of body weight, intraperitoneally) are almost similar to the therapeutic effects of MTX (0.5 mg/kg of body weight, intraperitoneally) as investigated in an FCA-induced arthritic in vivo model in rat [34]. An in vivo study by Pop et al. [43] has reported the anti-inflammatory and analgesic potential of NS oil in oral doses of 1, 2, and 4 mL/kg in comparison with diclofenac (5 mg/kg), as investigated in the carrageenan and Freund's adjuvant-induced inflammatory in vivo model. In the same study, the antioxidant effects were studied and a decrease in malondialdehyde levels as well as oxidized glutathione was recorded. When caspase-1 cleaves, it leads to an increase in pro-inflammatory markers: for example, IL-1β, IL-18, post-NLRP3 (NOD-like receptor family pyrin domain containing 3) inflammasome activation [44]. TQ has been reported to block this cascade of events. [45]. The anti-arthritic mechanism of TQ is diagrammatically represented in Figure 3 and the applications of thymoquinone in the treatment of inflammation and arthritis are recorded in Table 2. ↓IL-6, ↓IL-1β ↑antioxidant and ↑anti-inflammatory potential [46] Abbreviations

Encapsulated TQ Nanocarriers in the Treatment of Arthritic Inflammations
The tumorigenic tissues and RA synovium exhibit likeness; for instance, EPR and hypoxia happen in both. The aim and strategies of nanoparticulate delivery for tumors can be similar to that of RA. The altered fenestrated synovial membrane in RA and tumor EPR could be a potential object for nanoparticulate-based drug delivery. The fenestrated and leaky vasculature of the synovial membrane favors penetration and retention of NP [47]. The nanocarriers have specific targeting ability to the inflammatory cells and thereafter, efficiently downregulate the pro-inflammatory sequence of events and can ameliorate RA indications and consequent bone damage; for example, macrophages increase at the arthritic inflammatory site and can engulf nanoparticles, resulting in passive targeting [48]. Moreover, NPs could also decrease the dose and off-target toxicities, thereby enhancing treatment potential for arthritic drug delivery [49,50]. To improve the stability and oral bioavailability of TQ, various nanoformulations, including an oral phospholipidic nanomatrix (particle size > 100 nm) [1], topical ethosomes (particle size 105.2 ± 8.0) [51], and liposomal chitosan gel [52], were developed which enhanced the therapeutic efficacy of TQ as investigated in a carrageenan-induced paw inflammation model. The phospholipidic nanomatrix made up of lipidic core and surfactant mixture enhances TQ aqueous solubility and intestinal absorption relative to TQ suspension [1]. Besides this, lipidic NPs are directly taken up by intestinal lymph and deliver the drugs directly into the bloodstream, which leads to avoidance of the first-pass metabolism process. As a result, lipidic NPs enhance the anti-inflammatory potential of TQ, vis TQ suspension as observed in the carrageenan-induced paw edema rat model.

Neoplasm and Its Pathogenesis
A large group of individuals are diagnosed with cancer annually, being the second leading cause of mortality worldwide [53]. Its pathogenesis is very complex and is often difficult to identify, and most of the time, it is multifactorial. The tendency to multiply some groups of cells beyond their limit leads to abnormal development in a specific body part, which is called neoplasm or cancer [54]. Generally, metastasis-suppressor genes are involved in the inhibition of motility, invasiveness, colony formation, growth arrest, differentiation, proliferation, adhesion to extracellular matrix components, cell-cell adhesion and aggregation, and the immune sensitivity of cells [55,56]. All of these tasks require precise timing, which is controlled by a variety of cellular functions. Signaling, transcriptional activation, integrin expression and signaling, cell adhesion, and motility, cell communication, cytokine stress-induced signaling, serine protease expression, and nucleotide diphosphate kinase activity are among these functions [57]. Failing any of the above-said factor or group of factors may initiate cancer genesis [58]. Epigenetic changes also play a crucial role in disease initiation. Lower levels of H3K4me2, H3K18ac and H3K9me are linked to a poor prognosis in prostate, lung, and kidney cancers, respectively; similarly, higher levels of H3K9ac expression in lung cancer patients are linked to a shorter survival period [59,60]. Thymoquinone has recently been shown to modulate epigenetic machinery, such as histone acetylation and deacetylation, DNA methylation, and demethylation, all of which are significant epigenetic changes that may lead to carcinogenesis [61]. TQ has antineoplastic activity against human tumors, antioxidant effects and anti-inflammation in animal models and cell culture systems, chemopreventive effects, and most notably, anti-multidrug-resistant variants of human malignant cell [62].

TQ Nanocarrier for the Treatment of Cancer
Many drugs do not reach the antineoplastic drug pipeline because of low aqueous solubility, high toxicity, large doses, and shorter half-life. Nanoformualtions provide opportunities to improve the pharmacokinetics of these drugs for precise treatment at the molecular level with reduced off-target effect [164,165]. The tumor tissues that exhibit enhanced permeability and retention (EPR) and hypoxia-like properties could be utilized for targeted drug delivery. The NPs take advantage of the EPR effect and accumulate in the cancer cells, providing maximum therapeutic efficacy with minimum off-target effect [166]. The nanoformulations, including polymeric (natural/synthetic), lipidic (liposomes, niosomes, ethosomes, cubosomes, solid lipid nanoparticles (SLN), nanoemulsion, and microemulsion), pretentious (bovine serum albumin, human serum albumin) and metallic (silver, gold, iron, etc.), in combination with surface modification, are utilized for targeted delivery of therapeutic drugs in tumor sites [167,168]. NPs deliver drugs at the selective tumor site utilizing multiple approaches, including passive targeting and active targeting. Some of them are explained in the following sections to deliver TQ at the target site. Applications of TQ nanocarriers and surface-modified TQ nanocarriers for the management of cancer and inflammation are reported in Tables 5 and 6, respectively. Moreover, therapeutic importance of TQ-loaded nanoparticulate-based therapies for RA management is also reported in Table 5 with comparison to the conventional formulations and pure TQ.

Passive Targeting Approach in Cancer Drug Delivery Passive Targeting Utilizes the Tumor Microenvironment for Drug Delivery
Tumor vasculature is different from normal cell vasculature. Blood vessels of cancer tissue have comparatively larger fenestration with poor lymphatic drainage system, which results in enhanced retention and permeation of the nano-sized particulate matter [169]. Based on the delivery site, the size and surface of the NPs can be modulated. NPs' size and surface architecture modulation also avoid reticuloendothelial system (RES) uptake and make it circulate for a long period of time. This could be explored in passive drug delivery. Various strategies depicting passive targeting of TQ via nanoparticles are reported in Figure 5.

Passive Targeting through Long-Circulating Nanocarriers
Chitosan-grafted lipid nanocapsules [170] and PEGylated liposomes [171] were reported for the co-delivery of TQ and docetaxel (DTX) against drug-resistant breast cancer. Chitosan grafting improved cellular uptake and escaped endosomal effect; PEGylation increased circulation time of the dual payload [172], resulting in increased cytotoxicity against triple-negative breast cancer (TNBC) cells (MDA-MB-231 and MCF-7). A longcirculating PEGylated vitamin E lipidic nanocapsule loaded with TQ and DTX was also investigated against resistant breast cancer cells (MCF-7 and MDA-MB-231) [173]. PEGylation in vitamin E lipidic nanocapsules inhibits p-glycoprotein efflux, re-sensitizes the resistant TNBC cells and provides enhanced antimetastatic effects with reduced multiple side effects. Co-encapsulation of TQ with DTX improved loading efficiency into PEGylated liposomes and vitamin E lipidic nanocapsules as well as the chemosensitivity of DTX against breast cancer cells (MCF7 and MDA-MB-231).
PLGA-PEG-Pluronic TQ NPs were designed for sustained delivery of TQ into tamoxifenresistant breast cancer cells (UACC 732, MCF-7) [174]. TQ-NPs reduce the dose and synergize tamoxifen chemoprevention potential with selective tumor cell toxicity. PEGylated LMW chitosan nanocapsules selectively deliver TQ into cancer cells (MCF 7 cells) [175] as chitosan (with pKa 6-6.5) solubilizes in the inter, as well as intracellular acidic microenvironment of cancer cells, thereby delivering TQ in a targeted manner. Passive Targeting through Surface Charge and Size of NPs Nanocarriers overcome TQ pharmacokinetics issues and deliver it at the specific site with enhanced efficacy. A co-liposphere of Cabazitaxel (CBZ) and TQ was made of vitamin E-TPGS tricaprin, and egg phosphatidylcholine improved cellular internalization, which potentiates dose-dependent apoptosis as well as anticancer efficacy against MDA-MB-231 and MCF-7 cell lines [176]. The poly-L-lysine (PLL) and polyethylene glycol surfacedecorated nanocontainers (NC-PLL) complex of diethylaminoethyl dextran/xanthan gum enhanced intracellular accumulation of TQ [177]. The positive surface charge of the NC-PLL significantly favored nanocontainer binding on the negatively charged cell membrane as compared to nonmodified nanocontainers, resulting in negatively charged NC-PEG. NC-PLL dominated in terms of cytotoxic efficacy, as investigated in MCF-7, likely due to enhanced accumulation in cancer cells.
Mesoporous silica NPs (TQ-MSNPs) improved TQ aqueous solubility and photostability as well as reduced the therapeutic dose (8-fold), which delayed cell migration and enhanced cytotoxic and apoptotic potential, as evaluated in the MCF-7 and HeLa cell lines [178]. The core-shell NPs of mesoporous silica delivered TQ to glioma cells selectively, which triggered cytochrome c, increased caspase-3 activation, and cell cycle arrest at the G2/M phase [179]. Chitosan-coated PLGA NPs containing TQ enhanced cytotoxic potential when compared with surface-decorated TQ-poly(lactic co-glycolic acid) NPs and TQ alone; this was investigated through the MDA-MB-231 and MCF-7 cell lines [180]. The antimetastatic potential of TQ was enhanced by chitosan nanoparticles against HepG2 cell lines through longer duration inhibitory actions when compared with free TQ [181]. TQ-NLC-NPs accumulated in cancer cells and inhibited their proliferation through time and dose-dependent modulation in the cellular morphology, as investigated in HepG2 cancer cells [182]. The polymeric NPs of methoxy poly(ethylene glycol)-b-poly(-caprolactone) improved the systemic bioavailability of TQ (1.3-fold) with slower elimination rates, which provides greater antiproliferative efficacy against varieties of pure cell cultures of human carcinoma (PANC-1, MCF-7, and Caco-2) [78,183]. The nanoarchitecture of polymeric shells increased TQ solubility, intestinal absorption, and bioavailability rates, resulting in higher cancer cell selectivity compared to free TQ. A soy phytosomal formulation of TQ with a dual release pattern (initial burst followed by prolonged release) revealed excellent anticancer activity against a lung cancer cell line (A539) [184]. The sustained release of TQ from phytosome accumulates TQ in the G2-M and pre-G1 phases of cancer cells, which initiate dose-dependent apoptosis and cell necrosis activities via caspase-3 activation. A Soluplus ® -Solutol ® HS15 micelles formulation enhanced the antimigratory efficacy of TQ (1.5-10 µM) through improving aqueous solubility (10 times) and encapsulation efficacy, as investigated in SH-SY5Y human neuroblastoma cells [185]. The synergistic potential of TQ loaded in cockle-shell-derived aragonite CaCl 3 -NPs was reported with doxorubicin to reduce cellular migration in mammary gland carcinoma stem cells (MDA MB 231) [186]. A cubosomal formulation of TQ improved cellular accumulation, which leads to increased apoptotic activity migration in mammary gland carcinoma cell lines (MDA-MB-231 and MCF-7) [187]. Chitosan-coated TQ-PLGA-NPs accumulated in melanoma cancer cells (A375) by taking advantage of the EPR effect and positive surface charge of chitosan, which facilitate binding with the negatively charged cell membrane and induce cellular retention as well as time-dependent cytotoxicity [188]. TQ loading into niosomes improved cellular internalizations with controlled release of TQ, which markedly inhibits the migration of pro-inflammatory markers in mammary gland carcinoma with respect to pure TQ [10].

Active Targeting Receptors Based Active Targeting
A variety of surface receptors have been found to be upregulated in certain physiological conditions, including cancer, and are widely utilized for delivery via surface-decorated nanoparticles (NPs). The surface-coated NPs can target those cells which overexpress specific receptors on their surface and because of this, the nanoparticles attach to these [10]. The same is shown in Figure 6. The ligands which are used for surface modification include hyaluronic acid, anisamide, transferrin, folic acid, and many more utilized for active targeting of TQ into cancer. These have been reported in the following sections. This receptor is overexpressed in various types of cancers, including colon, brain, breast, lung, prostate, and kidney [189,190]. Anisamide is a benzamide analog, which exhibits a higher affinity towards sigma receptor-expressing cells [191] Anisamide-conjugated polymeric nanocapsules of eudragit-S100 delivered TQ into the colon-specific region through binding with overexpressed colonic sigma receptor [192]. The RNA aptamer, A10-coated planetary ball-milled starch NPs of TQ exclusively delivered drug into docetaxel-resistant prostate cancer cell lines (C4-2B-R and LNCaP-R) through overexpressed prostate-specific membrane antigen and inhibited drug efflux, which improves cancer potential [193]. The PEG and PCL, in the ball-milled NPs, decrease non-specific binding to the cell membrane and allow prolonged circulations. Hyaluronic acid (HA)-decorated Pluronic ® NPs of TQ accumulated in TNBC cells through selective binding with overexpressed CD44 receptor of cancer cells [194]. Pluronic-enhanced TQ encapsulation and HA facilitate CD44 targeting and make it have prolonged circulation, which reduced the dose for cell migration by modulating both miR-361/Rac1 and RhoA/actin stress fibers and the miR-361/VEGF-A mechanism that attenuate angiogenesis and metastasis of TNBC cells. Radio-iodinated NPs of folic acid-chitosan specifically bind to overexpressed folate receptors of human ovarian cancer cells (SKOV3) and improve anticancer efficacy through improved cellular internalization and retention [195]. A PEGylated-PLGA-TQ-NP surface decorated with transferrin potentiated anticancer efficacy of TQ through specific binding with the overexpressed transferring receptor on tumor cells, which decreases dose and improved cellular accumulations of NPs through EPR, as investigated in lung carcinoma A549 cells [196]. The as1411-conjugated nanodroplets delivered TQ into cancer cells through specific binding with overexpressed nucleolin on the cancer cells surface as investigated in MDA-MB-231 cells [197]. The as1411-conjugation facilitates rapid cellular uptake and dose-dependent cytotoxicity via nucleolin-stimulated Rac1 activation [198].
PI3K/Akt activation in cancer cells leads to resistance to traditional chemotherapeutics [199]. pH-sensitive gold niosomes of TQ along with Akt-siRNA were utilized to deliver TQ into tamoxifen-resistant breast cancer cells as well as knockdown of Aktoverexpression [96,200]. These niosomes resensitized cancer cells to TQ through Akt silencing and enhanced apoptosis by inhibiting MDM2 expression as well as inducing p53 [200].

Stimulus-Responsive NPs for Active Targeting
Designing stimuli-responsive NPs for active targeted drug delivery is dependent upon tumor microenvironments such as pH, hyperthermia, catalytic enzymes, or external stimuli such as pressure, ultrasonication, or magnetic field. The stimuli-responsive NPs retain their physicochemical properties, including structure, during their circulation. They are stimulated upon exposure to small changes in the tumor microenvironment or external stimuli and undergo rapid changes (aggregation, permeability, and disruption) to release the encapsulated drug. Various TQ-loaded stimuli-responsive NPs with enhanced anticancer potentials have been discussed in the following sections. A TQ-loaded Fe3SO4 NPs surface decorated with ethylene glycol and polyvinylpyrrolidone (PVP) pH-dependently delivered TQ in TNBC cells (MDA-MB-231) [201]. PVP surface decoration improved water solubility and delivered drugs in the acidic environment, which maximized tumoricidal efficiency.
Eudragit L-100-coated nanoconjugates of chitosan, HPMC, and PVA pH dependently delivered TQ into the colon for cancer management [202]. This study finds that at pH 7 concentration, eudragit L-100 dissolves and chitosan becomes degraded by anaerobic bac-

Stimulus-Responsive NPs for Active Targeting
Designing stimuli-responsive NPs for active targeted drug delivery is dependent upon tumor microenvironments such as pH, hyperthermia, catalytic enzymes, or external stimuli such as pressure, ultrasonication, or magnetic field. The stimuli-responsive NPs retain their physicochemical properties, including structure, during their circulation. They are stimulated upon exposure to small changes in the tumor microenvironment or external stimuli and undergo rapid changes (aggregation, permeability, and disruption) to release the encapsulated drug. Various TQ-loaded stimuli-responsive NPs with enhanced anticancer potentials have been discussed in the following sections. A TQ-loaded Fe 3 SO 4 NPs surface decorated with ethylene glycol and polyvinylpyrrolidone (PVP) pH-dependently delivered TQ in TNBC cells (MDA-MB-231) [201]. PVP surface decoration improved water solubility and delivered drugs in the acidic environment, which maximized tumoricidal efficiency.
Eudragit L-100-coated nanoconjugates of chitosan, HPMC, and PVA pH dependently delivered TQ into the colon for cancer management [202]. This study finds that at pH 7 concentration, eudragit L-100 dissolves and chitosan becomes degraded by anaerobic bacteria. The bacterial fermentation end-product butyrate forms polysaccharides with anticancer potential; TQ is released with butyrate and reaches into cancer cells, showing higher cytotoxicity. A technetium-99m ( 99m Tc)-labeled TQ formulation was designed for theranostic application against skeletal muscle malignancy (rhabdomyosarcoma) [203]. The 99m TC with TQ synergizes anticancer potential through rapid internalization and slower externalization, which enhanced theranostic applications. A fluorescent liposome co-delivered TQ and curcumin into lung cancer cells (A549) and potentially inhibited cellular proliferation compared with TQ or curcumin alone or the lipidic formulation of either of them, probably due to improved internalization [204]. A TQ-capped magnetic nanoparticle of iron oxide improved endocytotic internalization in breast cancer cells (MDA-MB-231 cells) and displayed a potent synergistic chemo-photothermal effect compared with free TQ [205]. Guar gum microvehicles rapidly release TQ in the intracellular acidic environment of cancer cells (pH~5.5) compared to physiological pH (~7.4), due to breakdown of the interlinking bonds in an acidic environment, leading to prolonged TQ release, with synergistic anticancer activity, as investigated in HepG2 cell line [206].   Abbreviations: NS-Nigella sativa; EE-entrapment efficiency; DTX-docetaxel; DOX-doxorubicin; TQNPs-thymoquinone nanoparticle; PLGA-poly(lactic-co-glycolic acid); Cur-curcumin; SNEDDS-self-nanoemulsifying drug delivery systems. Table 6. Surface-modified TQ nanocarrier in the management of cancer (↓: decrease, ↑: increase).

Role of TQ in Toxicity Reduction
TQ is systemically well-tolerated with a large safety profile dose (LD 50 , 2.5 g/kg) [3] and has the potential to reduce oxidative stress and systemic toxicity as the dose increases. The intravenous dose of 25 mg/kg thymoquinone nanostructured lipid carrier (TQ-NLC) was found safe in female Sprague Dawley rats [221]. It shows antiproliferative effect at 20 µM, genotoxicity at concentration ≥1.25 µM, and cellular narcosis at between 2.5 and 20 µM concentrations in the rat hepatocyte [222]. TQ (10 mg/kg) ameliorated sodium arsenate (20 mg/kg)-induced neurotoxicity by increasing the levels of norepinephrine, dopamine, superoxide dismutase, and catalase, and decreases serotonin, nitrate, and tumor necrosis factor alpha (TNF-α) levels in the cerebellum, cortex, and brain stem regions [223]. In another study, the neuroprotective effect of TQ (10 mg/kg/day) was observed on electromagnetic radiation-induced oxidative stress [224]. Similarly, glutamate and iron oxide nanoparticle-induced toxicity were also attenuated by TQ [5]. A combined formulation of Costus speciosus, Fumaria indica, Cichorium intybus, and TQ (CFCT) (25 mg/kg per oral) decreases cisplatin-induced hepatorenal toxicity in rats through membrane stabilization and decreasing aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase serum levels [225].

Recent Update on Patents of Thymoquinone
The latest patent literature search on thymoquinone and its loaded nanocarriers reported potential applications in the prevention, balancing, and treatment of multiple physiological conditions such as cancer, inflammations, dermal disorders, anxiety, and stress-related disorders; treatment of female urinary tract infections; and management of immunological diseases, etc. TQ was patented alone and in combinations for the treatment of inflammatory symptoms, including the eicosapentaenoic acid pathway [226]. Additionally, TQ and H5WYG peptide-loaded nanomicelles were also patented for targeted cancer drug delivery [227] and TQ-loaded nanodroplet emulsions for cancer targeting [228]. TQloaded nanocarriers are not limited to cancer targeting. Aminoglycoside-thymoquinoneloaded nano-liposomal formulations have been patented for aminoglycoside antibiotic delivery [229]. Authors rightfully assume an increase in patent outcomes when pure thymoquinone is converted to nanocarrier-loaded thymoquinone for various pharmacological applications. The patents illustrating the pharmacological significance of thymoquinone and related nanocarriers are recorded in Table 7.

Clinical Trials OF Thymoquinone
TQ has the potential to correct various physiological conditions of the body. It is widely investigated from dietary supplementation to chemoprevention. To date, a total of 10 clinical trials (Table 8) of thymoquinone claiming its effect on malignant lesions, aphtha, chronic periodontitis, type 2 diabetes mellitus, oral submucous fibrosis, pediatric major thalassemia, and supportive care in patients with COVID-19 are ongoing worldwide, the details of which are mentioned in Table 8. Moreover, recently, a clinical trial of TQ was registered to analyze efficacy and safety for best supportive measures (Guidelines on Clinical Management of COVID-19 issued by MOHFW, India) against COVID-19 patients. The confirmed COVID-19 patients were assigned as Cohort A and Cohort B. Cohort A patients received 50 mg TQ once a day for 14 days along with the best supportive measure, while Cohort B patients received the best supportive measure only. The trial was primarily evaluated for virologic (change in positive COVID-19 status on days 8 and 15) and clinical outcomes (proportion of patients on WHO progression scale 0 to 10 on days 8 and 15). A human trial (CTRI/2020/12/029514) of TQ tablets (dose of 50 mg; 25 mg; 12.5 mg) was registered to measure safety and tolerability and to analyze pharmacokinetic behavior in normal healthy adults under fasting conditions. A trial (NCT04686461) of thymoquinone extract is underway to investigate the effects against arsenical keratosis. In this trial, TQloaded topical ointment was used to treat 34 patients with arsenical keratosis at two-week intervals. The TQ ointment formulation was found to reduce the keratotic nodular size as well as improvement of the lesion calculated using the Likert Scale.

Conclusions and Prospects
TQ is a molecule that has multifaceted modes of action, including anti-arthritic and antineoplastic activities through modulating inflammatory and apoptotic pathways. However, its biological instability, rapid metabolism, poor water solubility, narrow bioavailability, inadequate cellular availability, and lack of targeting halt its transition from research to clinical application. Extensive literature analysis revealed that nanotechnology upgraded drug delivery patterns in cancer and arthritic disease through significant improvement in pharmacokinetics and target-oriented active molecules delivery, while decreasing their off-target side effects. To maintain the biological stability of TQ during formulation design or delivering alone, site-specific availability is among the major challenges to utilizing its maximum therapeutic potential in arthritis and cancer management.
The role of TQ individually and its diverse types of nanoformulations for targeted delivery to tumorigenic cells and synovial tissues, with longer circulating time and higher synovial accumulation, improved anti-inflammatory and anticancer potential. The nanoformulation delivery of TQ results in significantly enhanced targeting payload and promising upgrades to its anti-inflammatory and anticancer efficacy.
Nanoparticles are emerging carrier systems for the delivery of a wide range of therapeutic molecules. NPs are extremely attractive due to their important properties (size surface area and charge). Their use, as a drug carrier system or in theranostic applications including personalized medicine, might pave the way for a future strategy of prevention and counteraction of multiple diseases.
In this review, we vitally analyzed and reported the possible mechanistic approach of thymoquinone, such as the downregulation of various cytokines, inflammatory factors, and apoptotic pathways for the management of rheumatoid arthritis and cancer. Moreover, their toxicity reduction potential was also reported. An extensive review of their patent and clinical trials worldwide was also reported.
With the deep dive that we undertook in this review, it was revealed that formulations can transform the applicability of the nanocarrier-based formulation of thymoquinone; however, these studies can be dynamic. Significant dots in research have been recognized that need to be connected: various pre-clinical and human trials are taking place worldwide to ascertain the applicability of thymoquinone in humans; there are a lack of comparative findings on various nanoformulations to optimize the best regimen for TQ delivery against rheumatoid arthritis and cancer; the nonavailability of toxicity/safety data for thymoquinone-loaded NPs and human studies specifically exploring the pharmaceutical importance of nanoparticulate systems on arthritic and cancer milieu. Nuclear factor of activated T-cells, cytoplasmic 1 ROS Reactive oxygen species fMLF N-Formylmethionine-leucyl-phenylalanine eEF-2K; Eukaryotic elongation factor-2 kinase NLRP3 NACHT, LRR, and pyrin domain-containing protein 3 DOX Doxorubicin SOD Superoxide dismutase LC-3 Light chain 3-II PPAR-γ Peroxisome proliferator-activated receptor gamma UHRF1 Ubiquitin-like, containing PHD and RING finger domains-1 p-mTOR Phosphorylated mechanistic target of rapamycin NFκB Nuclear factor kappa-light-chain-enhancer of activated B cells 4E-BP1 Eukaryotic translation initiation factor 4E-binding protein 1 eIF4E Eukaryotic translation initiation factor 4E p70S6K Ribosomal protein S6 kinase beta-1 also known as p70S6K kinase PI3K Phosphoinositides, including 3-kinase IRAK1 Interleukin-1 receptor-associated kinase 1 FAK Focal adhesion kinase Hes1 Hairy and enhancer of split-1 VEGF Vascular endothelial growth factor IRAK1 Interleukin-1 receptor-associated kinase 1,