Heterocyclic Anticancer Compounds: Recent Advances and the Paradigm Shift towards the Use of Nanomedicine’s Tool Box

A simple glance at FDA databases reveals the structural significance of nitrogen-based heterocycles in the drug design and engineering of pharmaceuticals, with nearly 60% of unique small-molecule drugs containing a nitrogen heterocycle [24]. Recently, Edon Vitaku and colleagues comprehensively compiled the structural diversity, substitution patterns and frequency of nitrogen heterocycles among U.S. FDA approved pharmaceuticals. Noteworthy, the average number of nitrogen atoms per drug, being around 2.3, while in those containing a nitrogen heterocycle an increase to 3.1 nitrogen atoms per drug is evidenced [24]. Ultimately, the structure dynamics involved in nitrogen-based heterocycles (and other classes of heterocycles), alongside with fundamental aspects such as ring size and aromaticity, translates into a vast array of chemical structures by which their molecular mechanisms of action can vary significantly [2,5,14].

Indoles and indole derivatives for instance comprehend one of the most versatile and common nitrogen-based heterocyclic like fragments that are frequently used in the synthesis of fundamental FDA approved drugs for common pathological conditions, ranking in the ninth position of the top 25 most frequent nitrogen heterocycles among the U.S. FDA approved drugs [24]. There has been particular emphasis on the synthesis of indole derivatives in recent decades due to the virtually endless possibilities for architectural design of polycyclic structures by the incorporation of multiple fused heterocyclic scaffolds in an attempt to achieve promising new heterocycles with chemical and biomedical relevance [25,26]. The indole structure plasticity observable in drug rational design is translated to the wide range of biological targets that these have been found to affect often ranging from topoisomerase inhibitors to G2/M abrogators and others [27]. However, application of the indole basic core structure for the synthesis of several potent tubulin polymerization inhibitors has lately been receiving increasing attention, in particular in oncology [26,27,28,29], often being present among renowned FDA approved drugs and others reported in clinical trials (Figure 2).

In a recent study, 25 new combretastatin 2-(1-acetyl-1H-indole-3-yl)-3-(phenyl) propenoic analogues were evaluated for their antiproliferative properties in a small panel of tumor cell lines, including THP-1 (leukemia), A-549 (lung), IGROV-1 (ovary), HEP-2 (liver), MCF-7 (breast), and DU-145 (prostate). Among the synthetized compounds containing the indole moiety, two have been reported to elicit a significant antiproliferative activity, compound (Z)-2-(1-acetyl-1H-indol-3-yl)-3-(4-(dimethylamino) phenyl) propenoic acid (6) and 3-(N-acetyl-1H-indol-3-yl)-8-acetoxy-2H-chromen-2-one (7) (Figure 3) [29]. With an half maximal inhibitory concentration (IC₅₀) of 0.80 and 0.37 μM in THP1 and MCF7 tumor cell lines respectively, evidenced by compound 6, and an IC₅₀ of 3.60 μM against MCF7, evidenced by compound 7, the anti-cancer properties of these compounds were comparable to that of the conventional and clinically employed chemotherapeutic drug Paclitaxel (Taxol^®) [29]. Moreover, the authors address the inhibition of tubulin polymerization by both compounds and their physical binding to the colchicine binding site between α and β interface of tubulin [29].

Another indole compound, 1,4-bis(di(5-hydroxy-1H-indol-3-yl)methyl)-benzene (SK228) (8) (Figure 4), was evaluated for its potential as a pharmaceutical compound for cancer treatment through a well-designed in vitro and in vivo experimentation [30]. SK228 was capable of inhibiting cell growth of human lung and esophageal cancer cell lines with proven IC₅₀ values ranging from 0.28–1.86 µM. Moreover, a series of DNA interaction studies including Comet assay, DNA-intercalating assay and ROS probe showed that SK228 caused severe DNA damage in binding to the minor groove. Plus, conformational changes to plasmid DNA after exposure to increasing doses of compound, adducts and intercalation of the SK228 metabolites with nucleobases are thought to be equally responsible for its general toxicity and mutagenic potential [30]. Data relative to the induction of programed cell death mechanisms demonstrated that SK228 was able to promote phosphatidilserine flipping from the inner to the outer membrane, cytochrome c release from mitochondria, and ultimately caspase activation (-9; -3), which together suggest the activation of apoptosis mechanisms through the intrinsic pathway. Moreover, complementary studies (e.g., Western Blot, immunocytochemistry and trans-well invasion assays) showed that SK228 severely reduced A549 and CL15 tumor cell line’s invasion characteristics through FAK/Paxillin pathway disruption, thus providing support for SK228 clinical translation as an inhibitor of highly metastatic tumor cells [30]. Conformational changes to the linear form of the plasmid allowed to infer about the compound’s ability to intercalate within DNA through adduct formation. Adducts of such tamoxifen metabolites with glutathione and nucleobases are thought to be responsible for its general toxicity and mutagenic potential [30].

Imidazole fragments have been recently attracting much attention due to their roles as attractive scaffolds for biologically active heterocyclic drugs [24,31]. In general terms, physicochemical properties like hydrogen bond donor-acceptor capability, π-π stacking interactions, co-ordination bonds with metals as a ligands, and van der Waals, polarization and hydrophobic forces have caused the increasing interest in these fragments. These properties accountable for their reactivity enable derivatives to readily bind with a series of biomolecules, including several enzymes and nucleic acids [31,32]. A vast screening study of a series of new 2,4,5-trisubstituted and 1,2,4,5-tetrasubstituted imidazoles led by Sanjay Malhotra group against the National Cancer Institute’s 60 human cancer cell line panel enabled the isolation of 2,20-(2-(3-(cyclopentyloxy)-4- methoxyphenyl)-1-isobutyl-1H-imidazole-4,5-diyl)dipyridine (9) (Figure 5) for its particular reactivity against A549 human lung cancer cell line, [32]. Further studies carried out with A549 epithelial cancer cells revealed the compound’s ability to profoundly inhibit cell proliferation, upon exposure to increasing dosages (with a proven IC₅₀ of 9 μM). Most significantly, cell proliferation inhibition was found to be linked with an overall loss of cell morphology, inhibition of cell migration capabilities and the ability to induce a cell cycle arrest through senescence. Moreover, this work highlights the compound’s ability to inhibit anchorage independent growth [32].

Piperidine and pyridine complexes comprehend two of the most common heterocyclic fragments present in FDA approved drugs [24]. Several N-(piperidine-4-yl)benzamide derivatives revealed a potent antitumor activity. For example, the structure N-(1-(2, 6-difluorob- benzyl)-piperidine-4-yl)-4-phenoxybenzamide (10) (Figure 6) was found to be the most effective against hepatocarcinoma cell line—HepG2 cells—exhibiting an IC₅₀ of 0.25 μM, and the ability to regulate AMPK phosphorylation, activate downstream signaling proteins, and arrest cell cycle in a p53/p21-dependent way [33].

Also, among nitrogen-based heterocycles more complex scaffolds have been gaining terrain in medicinal chemistry studies over the last decade. For example, triazolothiadiazoles and triazolothiadiazines, which are cleverly designed polycyclic scaffolds arranged by combining triazoles to thiadiazoles or thiadiazines, have become important biologically relevant scaffolds in cancer [34]. Linking benzimidazole basic scaffold to other heterocyclic moieties including fused rings has led Asif Husain and colleagues to the synthesis of benzimidazole hybrid heterocycles clubbed with triazolo-thiadiazoles and triazolo-thiadiazines, in an attempt to produce improved pharmacological compounds [35]. Compound screening against several cancer cell lines has demonstrated a broad spectrum of antiproliferative activity, although among the synthetized derivatives 1-(1H-benzo[d]imidazol-2-yl)-3-(6-(2,4-dichlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl) propan-1-one), (11) (Figure 7), elicited the most significant effects with reported 50% growth inhibition (GI₅₀) values ranging from 0.20–2.58 μM [35]. Previous studies from the same group have already demonstrated the same rational process by synthetizing benzimidazole heterocycles bearing oxadiazole and triazolo-thiadiazoles cores, also with proven anticancer activity. In this study, 3-(5-(4-amino-2,6-dibromophenyl)-1,3,4-oxadiazol-2-yl)-1-(1H-benzo[d]imidazol-2-yl)propan-1-one, (12) (Figure 7), is highlighted among the several compounds by proving to be the most active synthetized derivative, evidencing GI₅₀ values ranging from 0.49–48.0 μM, against the National Cancer Institute 60 human cancer cell lines panel [36].

More recently, novel 1,2,4-triazoles, triazolothiadiazines and triazolothiadiazoles have been synthetized and their anticancer potential evaluated [37]. Kamel and coworkers reported to have obtained seven compounds with considerable cytotoxic activity against a broad spectrum of cancer cell lines (NUGC; DLD1, HA22T, HEPG2, HONE1, MCF7) amongst which the compound 6-(4-chlorophenyl)-3-(pyridin-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole, (13) (Figure 8), presented an IC₅₀ of 25 nM against gastric cancer cell line (NUGC). Noteworthy, toxicological testing employing normal fibroblast cells (WI38), in order to assess potential side effects, demonstrated significant differences, being approximately 400-fold less toxic to normal cells compared to the NUGC cell line [37].

Despite their potential, no particular correlation between the nitrogen-based heterocycle fragments and potential families of target molecules seems to exist. Nonetheless, the bulk of these fragments or pharmaceutical drugs in which they are incorporated appear to be responsible, or to take part in coordination to major biomolecules in key regulatory pathways. Tubulin coordination/inhibition, DNA cleavage, ROS induction, and cell cycle arrest through inhibition of cyclins are several possible targets that support the previous statement but are most likely not restricted to these. The perspective covered here has only taken into consideration the most recent developments regarding the application of nitrogen-based heterocycles as potential chemotherapeutic cancer drugs, given that the vast structural diversity does not allow to present all currently investigated compounds nor the majority of the nitrogen-based heterocycle scaffolds currently incorporated into renowned pharmaceuticals. For a more complete review on other major nitrogen-based scaffolds (e.g., piperidines, pyridines, piperazines, cephems, pyrolidines, pyrazoles, purines, pyrimidines, and others), their structural diversity, substitution patterns and role as fundamental components of FDA approved pharmaceuticals, the reader is referred to a more comprehensive review on this matter [24].

Paclitaxel (PTX, Taxol^®), an oxygen-based heterocycle drug with an incorporated oxetane ring, has emerged as a key drug in cancer therapy [38]. The therapeutic effect of Paclitaxel is carried out by preventing the de-polymerization of microtubule polymers in a process similar to that of microtubule associated proteins (MAPs), except for the irreversible aspect of it, which leads to the progression inhibition of mitosis in cancer cells. Notwithstanding the fundamental progress that has been made in cancer therapy since the discovery of PTX, several issues still remain to be addressed. Namely, cellular alterations of tubulin structure and the amplification of efflux pumps have been strongly correlated with PTX and the acquisition of multidrug resistance profiles, burdening oncology related research and clinical treatments. Hypersensitivity, haematological and neurotoxicity are included as other equally related systemic side effects reported for PTX in literature [38]. Similarly to PTX, many other conventional chemotherapeutic drugs fit into this profile, where the therapeutic advantages are often overcome by the dreadful side effects, and hence alternatives to surpass these obstacles are required.

Since 2010, about 8% of all heterocycles with anticancer properties approved by FDA are oxygen-based heterocycles [22]. Cabazitaxel (14) and Eribulin (15) (Figure 9) were the latest drugs to be approved.

Cabazitaxel, or Jevtana^® its commercial name, is indicated for patient treatment with castrate resistant metastatic prostate cancer. As a taxane derivative, cabazitaxel works as microtubule inhibitor and prevents cell division [39]. Conversely, Eribulin (Halaven^®) is a non taxane drug that acts as a microtubule inhibitor and it is used to treat patients with metastatic breast cancer [40]. Yadaguiri and coworkers synthesized different cyclic compounds bearing coumarins, oxygen-based heterocycles that showed high antiproliferative activity against several human cancer cell lines (including A549 (human alveolar adenocarcinoma cell line), HeLa (Human cervical cancer cell line), MDA-MB-231 (human breast adenocarcinoma cell line), MCF7 (human breast adenocarcinoma cell line)) compared to healthy the human cell line HEK293 (Human embryonic kidney cell line). It is particularly noteworthy that the compounds, 8,9-dihydro-7H-5-oxa-benzo[6,7] cyclohepta [1,2-a]naphthalen-6-one, (16) and 11,12-dimethoxy-8,9-dihydro-7H-5-oxa-benzo [6,7] cyclohepta [1,2-a]naphthalen-6-one (17) (Figure 10), were found to elicit promising cytotoxicity against A549, HeLa, MCF7 and MDA-MB-231. Compound 16 reported IC₅₀ values ranging from 3.35–16.79 μM in the mentioned cell lines, while its counterpart, compound 17, was found to be particularly active against HeLa and MDA-MB-231 with IC₅₀ values of 6.72 and 4.87 μM, respectively [41]. As polyphenolic compounds with natural occurrence mainly in plants, these present a wide variety of modulatory and cytoprotective functions, thus offering a potential avenue for the synthesis of innovative compounds with a desirable combination of properties for anticancer drugs and superior pharmaceutical characteristics for clinical application [42,43].

Auranofin, a gold containing heterocyclic compound recognized by World Health Organization as a rheumatic arthritis therapeutic agent, was recently approved by FDA for Phase II clinical trials to treat ovarian cancer [44,45]. Auranofin which contains a tetrahydropyran ring was recently identified as a proteasomal deubiquitinase inhibitor and as a tumor growth inhibitor [45]. See-Hyoung Park and co-workers additionally suggested that auranofin activates pro-apoptotic caspase-3 in a FOXO3 dependent regulation through the induction of apoptotic proteins such as Bax and Bim and the decrease of anti-apoptotic mediator Bcl-2 in SKOV3 cell line (ovarian carcinoma cell line with a null p53 mutation) [46].

Y. Murti et al. showed that flavanones (18) (Figure 11), a natural product with extensive biological activities such as antibacterial, antifungal, analgesic and antioxidant properties with low toxicity, also possess striking anticancer activities against HT29 (human colon adenocarcinoma), MCF7 (human breast adenocarcinoma) and A498 (human kidney adenocarcinoma). Interestingly, among all the derivatives screened and analyzed, those containing furan rings (oxygen-based rings) demonstrated outstanding anticancer activity against all cell lines [47]. More notably, heterocycle flavanone derivative containing a furan ring, furfuraldehyde 19 (Figure 11), exhibited IC₅₀ values of 75.9, 51.0 and 59.3μM for HT29, MCF7 and A498, respectively, thus providing pre-clinical evidence of a reasonably good anticancer compound despite the fact that the mechanism of action of this particular derivative is still uncertain. In similarity with coumarins, flavones might equally provide a sustainable building scaffold for potential new and improved pharmaceutical anticancer compounds.

Benzofurans represent another class of oxygen-based heterocycles, and as coumarins, benzofurans are equally found in nature and are known for their extensive array of biological activities [48]. Recent studies have highlighted the cytotoxic effect of benzofuran-based compounds in cancer cell lines. Minho Choi and colleagues have synthetized several benzofuran derivatives and evaluated their cell viability in several cell lines such as HCT15 (colon), ACHN (renal), NUGC-3 (gastric), MM231 (breast), PC-3 (prostate) and NCI-H23 (lung) with the purpose of developing novel scaffolds as both specific anticancer agents and inhibitors of NF-κB transcriptional activity. Results revealed high toxicities with low compound concentrations and the ability to inhibit the NF-κB transcriptional activity [49]. Perhaps most noteworthy, insights into the structure activity relationship of benzofuran derivatives, revealed that the compound N-(40-hydroxy)phenylamide 21 (Figure 12), was both the most cytotoxic compound among the synthetized derivatives and the most efficient at inhibiting NF-κB activity.

Another promising heterocycles are the ones containing not only oxygen but also nitrogen in their ring structure. Felipe Rodrigues and co-workers study the antiproliferative effect of 23 different derivatives of mefloquine-oxazolidine, cyclic compounds containing oxygen and nitrogen. Following cell viability assays in HCT8 (colorectal), OVCAR8 (ovarian), HL60 (peripheral blood) and SF295 (nervous system), IC₅₀ values obtained ranged from 0.59–4.79 µg/mL This was the first study to evaluate the anticancer properties of mefloquine-oxazolidine derivatives [50]. Another research performed by Saulo Andrade et al. analyzed oxazolidine complexes as potential anticancer agents. As a part of a previously reported work based on the core structure of a series of 2,3,4-trisubstituted oxazolidines (22) (Figure 13), the authors synthetized several oxazolidine derivatives with the aim of understanding the potential therapeutic effect of oxazolidine rings as building blocks for the development of chemotherapeutic cancer drugs. The rationale behind compound synthesis was based on previous observations regarding substituent groups. Interestingly, substituent groups at 3 or 4-position were found to be paramount for compound activity, and while ortho-substituted compounds were found to be inactive in targeting cancer cells S isomer were the most potent, often being 10 times more active than their enantiomers, By exploiting these characteristics and by exploiting the importance of the oxymethylene spacer between benzene and oxazolidine rings the synthetized compounds were found to exhibited in general high cytotoxic activities with low IC₅₀ concentrations against a cancer cell line panel composed of, HL60, JURKAT (peripheral blood), MDA-MB-231 (mammary gland) and LNCaP (prostate) [51]. More significantly, the derivative (S)-tert-butyl 2,2-dimethyl-4-(1-(4-nitrophenyl)vinyl)oxazolidine-3-carboxylate (23) (Figure 13) exhibited a cytotoxic activity similar to the remaining derivatives, however, with exception for LNCaP cell line for which it presented high affinity with a IC₅₀ value in the micromolar range (11 μM). Moreover, under the assayed conditions and given the cancer cell line panel, the activity of the compound 23 was proven to be specific for cancer cells, as it did not affect VERO or PBMC cell survival and proliferation, which in a possible translation into the clinic might suggest low side effects.

Additionally, other studies to evaluate anticancer properties of oxazolidine compounds were carried out always showing high toxicities with low IC₅₀ values, thus suggesting that these families of oxygen, nitrogen-based heterocycles are a new emerging area of investigation [52,53].

The findings presented here for many of the oxygen-based heterocycles currently under pre-clinical studies indicate the occurrence of significantly different toxicities depending on the type of oxygen-based heterocycle, its overall structure, ligands, ring size and aromaticity. The evidence that oxygen-based heterocycles scaffolds have been demonstrating promising anticancer activities is most likely to be continuously exploited by pharmaceutical industries, in light of Cabazitaxel and Eribulin examples, in order to develop superior chemotherapeutic cancer drugs. Nonetheless, current research is yet to determine the exact mechanism by which many of these new heterocycle drugs/scaffolds exert their therapeutic effects.

Resulting from the derivation of homocyclic hydrocarbons from the substitution of the ring carbon atom by sulfur heteroatoms, their relevance comes from the significant changes in cyclic molecular structure engraved by differences in electron configurations, unshared pairs of electrons and ultimately the electronegativity between heteroatoms and carbon [54,55]. Physicochemical properties and reactivity of sulfur containing heterocycles are hence deeply conditioned by the overall electron configuration as well as by the versatile chemistry of the sulfur atom itself. The covalently bonded sulfur is determinant in many biological systems and is often known to form metal complexes with metal ions [56]; e.g., the proteins’ fundamental building blocks cysteine and methionine hold sulfur as key for the overall tertiary structure [57]. Other regulatory roles in biological systems range from the incorporation as a key component in several vitamin cofactors, sugars and nucleic acids to play important roles in regulating translation by sulfuration of transfer RNA [57].

Given the importance of sulfur in biological systems and its increasing interest as a regulatory agent, the rationality behind the employment of sulfur-based heterocycle drugs comes to light. Said and Elshihawy synthesized N-(3-cyano-5,6-dihydro-4H-cyclopenta (b) thiophene derivatives and evaluated their antiproliferative activity against human breast adenocarcinoma cell line (MCF7) [58]. In this work, the newly synthetized thiophene derivative complexes, N-(3-Cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2- yl)-2-(4-(N-(pyrimidin-2yl) sulfamoyl,sodiumsalt) phenylamino) acetamide (24) and 4-(5,6-dihydro-7H-cyclopenta (4:5) thieno (2,3-d)-1,2,3- triazin-4-ylamino)phenol (25) (Figure 14), exhibited the strongest inhibitory effect with IC₅₀ values of 30.8 and 38.7 nM, respectively. Furthermore, the inhibition of ATP recognition binding sites of tyrosine kinase receptors was suggested as the main possible mechanism of action by these complexes, mimicking the similar processes carried out by gefitinib and dasatinib drug molecules in the biological context [58].

In a similar study, Alsaid Mansour group reported the synthesis of novel series of thiophenes having different biologically active moieties with a noteworthy antiproliferative activity comparable to that of doxorubicin [59]. Biologically active sulfonamide, isoxazole, benzothiazole, quinoline and anthracene moieties linked to the respective thiophene derivatives exhibited IC_50s lower than 50 µM [59]. Complexes (Z)-4-(3-oxo-3-(thiophen-2-yl)prop-1-enylamino)-N-(thiazol-2-yl)benzenesulfonamide (26), (Z)-4-(3-oxo-3-(thiophen-2-yl)prop-1-enylamino)-N-(1-phenyl-1H-pyrazol-5-yl)benzenesulfonamide (27), (Z)-4-(3-oxo-3-(thiophen-2-yl)prop-1-enylamino)-N-(pyrimidin-2-yl)benzenesulfonamide (28) and (Z)-3-(4-methoxybenzo[d]thiazol-2-ylamino)-1-(thiophen-2-yl)prop-2-en-1-one (29) (Figure 15), have exhibited the strongest inhibitory effect (IC_50s of 10.25, 9.70, 9.55, 9.39 μM respectively), approximately three times stronger than that of doxorubicin, showing great promise for a future potential drug against human breast cancer [59].

Also noteworthy, regarding sulfur-based heterocycles, are thiadiazole and thiazole complexes owing to their reactivity within the biological context. Indeed, these compounds have been found to be present in drug development for the treatment of many pathologies such as cancer, allergies, infection diseases, neurological disorders, chronic pain, fungal complications and many others [60,61,62,63]. Several new thiazole-based nitrogen mustard heterocycles have recently proven to possess strong inhibitory effects towards a panel of human cancer cell lines (MV4-11, A549, HCT116 and MCF-7). Among the several derivatives, the compound (E)-N-(4-(2-(2-(4-(bis(2-chloroethyl)amino)benzylidene)hydrazinyl)thiazol-4-yl)phenyl)methanesulfonamide, 30 (Figure 16) elicited a strong inhibitory effect in human leukemia HCT116 and MCF7 cells with reported IC_50s of 5.48 μM and 4.53 μM. In this study, particular emphasis was directed towards the evaluation of the cytotoxicity of these derivatives in healthy cells. Furthermore, quantum chemical calculations and binding interaction studies showed that the stronger interaction with DNA is observed for compound 30 compared with the other compounds. Moreover, in silico analysis demonstrated the potential of the compounds to exert their action through the inhibition of human topoisomerase II [64].

Benzothiophene acrylonitrile derivatives, similar in structure to the natural combretastatins, have equally proven to be interesting scaffolds towards the synthesis of novel anticancer compounds with improved pharmacological profiles, as Peter Crooks has elegantly demonstrated [65]. Analog screening against 60 human cancer cell lines revealed three potent anticancer compounds ([Z-3-(benzo[b]thiophen-2-yl)-2-(3,4-dimethoxyphenyl)acrylonitrile] (31), [Z-3-(benzo[b]thiophen-2-yl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile] (32) and [E-3-(benzo[b]thiophen-2-yl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile]) (33) (Figure 17) with GI₅₀ ranging from <10 to >100 nM [65]. In addition, long term acquired resistance commonly associated with many forms of tumor progression due to the overexpression P-glycoprotein (P-gp) efflux pumps seems to have been surpassed by the use of these analogues [65].

Liposome is a phospholid system comprising a bilayer structure, and since its early studies has been described as a potential drug delivery system [78]. Since its discovery, the synthesis method has seen much improvement, allowing a more large scale production and smaller sizes, from 20–50 nm. Liposomal doxorubicin (Doxil^®) was the first nanodevice approved by FDA for cancer treatment [79,80], and an additional five formulations have already attained approval—DaunoXome^®, Myocet^®, Marqibo^®, Lipo-Dox^® and DepoCyt^® [80,81].

In terms of drug loading, liposomes not only encapsulate water soluble drugs but can also encapsulate hydrophobic compounds inside their membranes [82]. However, the latter, due to the small amount of room available between their bilayer structure, and the destabilization effect in the outer membrane, is limited [81]. It is possible to increase drug loading in response to pH gradients that favors weak bases drugs encapsulation, which represents the majority of cancer drugs. For the remaining drugs, that do not precipitate inside liposome and are difficult to retain, one surpasses the limit of solubility to force precipitation, or turning such drugs, for example Paclitaxel, into a weak base drug [80]. Liposomes are mainly considered for water soluble drugs [81]. Most liposome formulations are biocompatible and almost biologically inert, with great circulation time, but intravenous injection seems to be associated to complement activation-related pseudo-allergy [81,83]. To increase the circulation time in blood, liposomes may be conjugated with PEG, allowing to bypass the immune system, primarily evading opsonization. Liposomes may be conjugated with targeting moieties, e.g., antibodies, forming immunoliposomes. Despite the potential advantages of active targeting, thus far, no immunoliposome has attained FDA approval, and only a few are in clinical trials—MM-302, a ErbB2/ErbB3-targeted liposomal doxorubicin, and, MBP-436, a transferrin-targeted liposomal oxaliplatin [80].

Polymeric nanocarriers are polymer-based structures, such as polymeric micelles, dendrimers and polymeric nanoparticles, acting as an alternative to liposomes, but with more in vivo stability, higher drug circulation time and loading, and a more controlled release profile [81].

Polymeric micelles are composed by amphilic block copolymers, forming a sphere-like structure with micelles and a hydrophobic core [84]. The core can contain drugs with poor water solubility and the micelles allow inclusion of hydrophilic drugs, which provides stability to the micelles themselves [85]. Directed targeting of tumors and stimuli-responsive approaches, drug entrapment into the core and functionalization with targeting moieties are also being developed. Currently, there are only eight polymeric micelle formulations in ongoing clinical trials [81].

Dendrimers are hyperbranched polymeric molecules ranging from 10–100 nm [85,86]. Dendrimer syntheses are performed from a central core followed by consecutive controlled polymeric reactions yielding a highly controlled architecture and tuning of pharmacokinetic parameters [85,86,87]. Anticancer drugs may be non-covalent encapsulated into the core or covalent conjugated to the dendrimer surface, modulating the drug release profile [85,86]. Non-covalent encapsulation in the core is reported to cause toxicity and an uncontrolled release profile, and is only used in intratumor administration [81].

Due to the amino and carboxyl groups present in albumin, it is capable of forming covalent bonds with drugs and encapsulating them [77,78,90]. In addition, albumin is biodegradable, biocompatible, and considered non-toxic and non-immunogenic, which is optimal for synthesis of nanoparticles [78]. Albumin bound nanoparticles’ (nab) technology is an ideal platform to encapsulate lipophilic drugs. Despite the advantages, only one nab has been approved by FDA and is currently in clinical practice—nab-paclitaxel (Abraxane^®) [78].

Among all nanoparticles systems used in cancer research, these may be the more versatile model, showing applicability in, from enhanced radiotherapy, tumor imaging and drug delivery [77]. The most widely used metallic nanoparticles are superparamagnetic ion oxide nanoparticles (SPIONs) and gold nanoparticles.

SPIONs are mainly used for imaging (magnetic resonance imaging, MRI) and diagnostic purposes. The only metallic nanoparticle approved for cancer therapy, NanoTherm^®, is a good example of a SPION [77].

Gold nanoparticles (AuNPs) have become an efficient platform for diagnosis and imaging (CT scanning), selective drug delivery and for direct treatment by hyperthermia. One of the greatest features of AuNPs is the diversity available in shape and size that may be easily tuned for conjugation to biomolecules of interest. Their capability to generate heat upon near-infrared excitation is a promising approach for directed hyperthermia [91]. To date, no metallic nanoparticle for drug delivery has been approved. Some studies show an efficient binding capacity of drugs, for example paclitaxel has been known to form conjugates with gold nanorods acting as a drug nanocarrier and as a photothermal agent [92].

Drug conjugates, nanosized carriers in cancer therapy and diagnosis, comprehend both polymer-based and antibody-drug conjugates, however, here we focus mainly on the latter. The antibody-drug conjugate (ADC) concept dates back to the 1980s [89,93], where a drug was combined directly to a targeting agent such as an antibody. A second-generation ADC emerged with improved linker technology and better targeting knowledge [94]. The primary feature of these nanostructures is the reduced side effects as an intrinsic characteristic. The mechanism of action comprises the targeting of cancer cells with a certain biomarker by a corresponding antibody, and, consequently, internalization of the nanoconjugate. An expanded therapeutic window from its specific targeting explains the reduced side effects of these nano-systems [95].

Several antibody-drug conjugates have been and are continuing to be approved by FDA. The last entities recognized by this organization as drugs with anticancer properties were Dinutuximab (Unituxin^®) in 2015 and Nivolumab (Opdivo^®) in 2014. Dinutuximab is indicated for pediatric high-risk neuroblastoma and targets GD2, a glycolipid overexpressed on the surface of neuroblastoma cancer cells [96]. Nivolumab was approved to treat patients with unresectable or metastatic melanoma [97]. Studies addressed by Caroline Robert and coworkers reported enhanced survival ratios in overall patients with metastatic melanoma without BRAF mutation [98].

Antibody-drug conjugates are a thriving area of investigation and surely are attracting pharmaceutical companies with flourishing ideas in order to improve linker technologies or antibody stability. In combination with clinical results, ADC technology is a growing field with a promising future [93]. The scope of this review does not focus on this particular nano-scale concept, and so, for further insights readers are advised to view the following literature [95,99,100,101].

Either considering the application of nanomedicine’s toolbox for heterocyclic compound vectorization in cancer, or any other application of nanoscale resources suitable for biomedicine (e.g., targeted therapy, molecular diagnostics, theranostics), one has to keep in mind that the theoretical and experimental research that has taken place over the last decades related to nanoscience and nanobiotechnology has raised several biological questions intimately connected with public health issues. Perhaps at the forefront of these biological questions is how such nanoparticulate formulations interact with the human body based on the role of the particle size, surface area, material composition, shape, surface chemistry and overall charge [113,114]. All of these inherent characteristics will eventually determine how the nanoparticulate formulations cross barriers, interact with biological fluids, and how and where these will interfere with protein interactions and other fundamental molecular players that comprehend natural entities in biological systems. These will influence the delivery efficiency of the drug payload into the target cell/tissue and the overall effect of the nanoformulation itself (benign or toxic). Similarly with many toxicological agents, several nanoparticle systems are prone to interact adversely with cellular metabolism; however, some reports are inconsistent or conflicting. For instance, while several authors report the biocompatibility of gold nanoparticles [115,116], others seem to contradict these findings [117,118]. This situation is also evident for other types of nanoparticles such as carbon nanotubes, for instance [119,120].

Nonetheless, considering the full spectrum of physicochemical properties that a single nanoparticle might encompass in its formulation, it is not yet known exactly how these fully interact with cell/tissue or, for that matter with the plethora of molecular factors at work at a the cellular scale. It is suggested though, that nanoparticles when in contact with physiological conditions, due to their small size and chemistry, interact with opsonins and may act like haptens by altering proteins at the surface of different cells, facilitating their uptake by mammalian cells. In particular, authors have attempted to correlate the size dependence of nanoparticles and uptake kinetics into mammalian cells. Several reports state that maximum uptake by cells occurs at a (gold)nanoparticle size of around 50 nm [121,122,123], while other studies have reported that this effect might equally be dependent on the composition of the nanoparticle itself. For instance, a study conducted using polystyrene nanoparticles to determine their uptake efficiency in Caco-2, a human colon adenocarcinoma cell line, contradicted the idea that 50 nm is the ideal size for internalization. The authors concluded that in fact the efficiency of internalization for 50 nm particles was the lowest out of a range of different sizes. Interestingly, here nanoparticles two times larger were significantly more internalized by cells [124]. Following the same rationale basis, it is easy to understand that depending on the different moieties functionalized onto the nanoparticles surface, the uptake and intracellular fate of such nanoformulations will be conditioned. Literature reports demonstrate that endosomal route of uptake can be conditioned by surface modification of nanoparticles with different biological moieties such as penetrating peptides [125]. Moreover, the specific moieties functionalized onto a nanocarrier to target a subset of cells towards a select route may actually have different uptake mechanisms as compared to the moiety alone [126].

The advent of nanoparticles as drug carriers has led without doubt to an increasing need to understand nanoparticle endocytosis mechanisms. The main energy dependent uptake processes involves phagocytosis, pinocytosis, and caveolae-dependent or clathrin-mediated endocytosis, and the activation of such methods is not yet clear although it is known to be dependent on the overall shape, size, and physicochemical properties [113].

The success of an effective drug delivery system is size and shape dependent. Both characteristics influence blood circulation time and biodistribution when administrated to a patient [127,128].

Shaping nanoparticles may prove to be a useful tool in cancer targeting. When comparing spherical nanoparticles to oblate-shaped ones, the latter are subjected to torques resulting in lateral drift due to tumbling and rotation. This phenomenon directs particles towards the blood vessel walls [127,129].

Nanoparticles bigger than a few nanometers, e.g., 7 nm, and smaller than 400 nm are more likely to accumulate in tumors due to the leakiness of tumor vessels caused by fenestrations between defective endothelial cells [128,130].

Relatively large nanoparticles, in order to reach tumor cells, must pass through a highly interconnected network of collagen fibers. This barrier may significantly reduce the amount of nanoparticles reaching the tumor, by nonspecific interactions or simply by hindering nanoparticle movement, thus, leading to the nanoparticles being concentrated in the vicinity of blood vessels that they leaked from [126,129,130,131].

To be effective, nanoparticles must avoid recognition by the reticulo-endothelial system (RES) composed of macrophages. Generally, particles larger than 100 nm are rapidly eliminated from the circulation by the RES [128].

In addition, macrophages recognize nanoparticles as foreign bodies which results in a rapid uptake and elimination [128,132]. In order to evade these specialized cells of the RES, and increase the circulation time, nanoparticles may be coated with several polymer moieties to avoid opsonization and phagocytosis. Among these polymer moieties, PEG is the most commonly used to this end [128,132].

The loading ratio of drug into the nanoparticle may lead to challenges in achieving the required dose to be delivered into the tumor. Lower ratios of drug loading lead to insufficient accumulation in the tumor. Another limiting factor is the fast release rate in circulation.

Conjugates of nanoparticles with drugs may be synthetized by either covalent binding of the drug to the nanoparticle surface, or, entrapment by either non-covalent or covalent bound. In reality, various chemical bonds (amide, ester, azide, etc.) are used to design nanoconjugates. The release of drugs from nanoparticles depends on the nature of these chemical bonds. The release may be dependent on either pH (usually acidic pH), temperature or on enzymatic cleavage. The nature of the chemical bound will also determine the release rate. In order for the nanocarrier to delivery its drug content into the tumor, the chemical bond should be stable upon blood circulation, preventing its rapid release and promoting the therapeutic effect in the tumor site [128,133]. A rapid release rate may produce results similar to free drugs in terms of biodistribution and toxicity [128], however, this strategy limits the interaction of nanoparticles with the tumor cell membrane and decreases uptake by tumor cells [128].