An overview on the exploring the interaction of inorganic nanoparticles with microtubules for the advancement of cancer therapeutics

MTs), dynamic and stable proteins in cells, by different ligands have been reported to be a potential strategy to combat cancer cells. Inorganic nanoparticles (NPs) have been widely used as anticancer, antibacterial and free radical scavenging agents, where they come in contact with biological macromolecules. The interaction between the NPs and biological macromolecules like MTs frequently occurs through different mechanisms. A prerequisite for a detailed exploration of MT structures and functions for biomedical applications like cancer therapy is to investigate profoundly the mechanisms involved in MT – NP interactions, for which the full explanation and characterization of the parameters that are responsible for the formation of a NP-protein complex are crucial. Therefore, in view of the fact that the goal of the rational NP-based future drug design and new therapies is to rely on the information of the structural details and protein – NPs binding mechanisms to manipulate the process of developing new potential drugs, a comprehensive investigation of the essence of the molecular recognition/interaction is also of considerable importance. In the present review, first, the microtubule (MT) structure and its binding sites upon interaction with MT stabilizing agents (MSAs) and MT destabilizing agents (MDAs) are introduced and rationalized. Next, MT targeting in cancer therapy and interaction of NPs with MTs are discussed. Furthermore, interaction of NPs with proteins and the manipulation of protein corona (PC), experimental techniques and direct interaction of NPs with MTs, are discussed, and finally the challenges and future perspective of the field are introduced. We envision this review can provide useful information on the manipulation of the MT lattice for the progress of cancer nanomedicine.


Introduction
Cancer is known as one of the most challenging diseases which still requires effective therapeutic strategies to deal with the uncontrolled proliferation of tumor cells [1].As a number of proteins are involved in cell proliferation, differentiation and metastasis of cancer cells, any compound that can potentially interact with main autophagy/cell cycle/ apoptosis/necrosis-related proteins and induce significant structural/ functional changes can be nominated as an anticancer agent.
Indeed, proteins are known as a very crucial class of biomacromolecules with unique structures, dynamics and functions.Proteins with diverse roles, including cytoskeleton (tubulin), structural (collagen, keratin, elastin), mechanical (actin/myosin), biochemical (different types of enzymes), and cell signaling (different types of hormones) functions [2,3] regulate the network dynamics and cell physiology.Also, proteins regulate their biological functions through potential interaction with other biomacromolecules, cell or organelle membrane and small molecules as ligands [4,5].Therefore, exploring thermodynamic and structural parameters of protein-ligand interaction can be used as a potential strategy to investigate the anticancer properties of different ligands.Protein-ligand interaction can occur in the living organisms with different levels of specificity and affinity to form a nonspecific or specific complex.These kinds of interactions account for the principal constituent of all processes in biological systems [6].Protein-mediated interactions can regulate different signaling pathways in biological systems [7].A comprehensive exploration of protein-ligand interactions are therefore crucial to recognize biology of cancer cells at the molecular level [8].Furthermore, detailed information regarding mechanisms involved in the protein-ligand interaction and recognition could promote discovery, synthesis and development of potential anticancer drugs [2,9,10].
It has been well-documented that microtubules (MTs) modulate different signaling pathways involved in cancer cell proliferation through mitosis and the proper functioning of the spindles [11].Tubulin-mediated pathways have been shown to be one of the main key mechanisms which stimulate cancer cell growth, proliferation and metastasis [12][13][14].Therefore, disruption of the MT organization and dynamics, by deregulation of tubulin polymerization/depolymerization balance, results in cell cycle arrest at metaphase stage.In fact, it has been shown that the variations in resistance to antimitotic drugs in different cells are associated with the alterations in the level of polymerized tubulin [15], changed tubulin isotype expression [16], tubulin modifications [17], and expression pattern of the tubulin gene superfamily [18].Also, it has been reported that cells through tubulin diversity, acts as code, are able to develop effective cellular MT mosaics [19] with multiple dynamic statuses [20] and functions [21].
Thus, targeting tubulin is a promising approach to inhibit proliferation of cancer cells.For example, it was reported that an anti-tubulin compound like vinorelbine could inhibit metastatic potential of tumor cells by controlling the epithelial-mesenchymal transition [12].Moreover, it was found that Ferulin C ligand could trigger potent anticancer effects in breast cancer through control of tubulin polymerization [22].Furthermore, it was discussed that 2-methoxyestradiol derivatives could inhibit angiogenesis and tubulin polymerization in vitro [23].Therefore, it seems that regulation of MT dynamics can be considered as a potential strategy in inhibition of proliferation of human cancer cells [24][25][26].
Generally, the use of inorganic nanoparticles (NPs)-based platforms for cancer therapy is one of the important objectives of increasingly expanding research [27].As a consequence, there is a significant increase in the number of published papers on the interaction of proteins and NPs in recent years [28][29][30].A profound detailed assessment of protein-NPs is necessary as the current level of our understanding about interactions of NPs with proteins is still incomplete, mainly because of a lack of mechanistic information.Even in the case of therapeutic utilizations of NPs, knowledge of the potential protein-NPs interaction is still inadequate.
As NPs are in a comparable size range as proteins, NPs may potentially interact with proteins and induce some (un)wanted structural/ functional changes and underlying different cell responses.The utilization of descent techniques applied in cellular and molecular biophysics may facilitate the exploration of the biomolecular interactions between proteins and NPs and disclose the crucial factors involved in the biological impacts of NPs.
It has been indicated that nano-based compounds are highly successful among anticancer drugs to potentially interact with tubulin.So far, it has been shown that many different inorganic NPs or nanocarriers have the ability to interact with MTs or carry tubulin polymerization inhibitors [31][32][33][34][35].
One of the main possible anticancer mechanisms mediated by NPs is perturbation of tubulin structure to induce cell cycle arrest and apoptosis [32,36].Indeed, NPs due to their high surface to volume ratio and corresponding highly reactive surface area can cause disruption of the MT dynamics during the polymerization process [37].For example, it has been shown that several types of inorganic NPs including titanium oxide (TiO 2 ) NPs [37,38], magnetite NPs [39,40], selenium NPs [33], and bare or functionalized gold (Au) NPs [32,34,[41][42][43] can stimulate disruption of tubulin structure and associated cytotoxicity against cancer cells.
There are already a number of review reports in the literature, which survey the interaction of different NPs with proteins for the advancement of cancer therapy.A comprehensive review paper discussing the perturbation of tubulin structure and dynamics directed by inorganic NPs triggering cell cycle arrest and apoptosis underscores the developments of MT-targeted NPs.Lopus et al., surveyed the application of noscapinoids as potential candidates in MT-targeted cancer therapeutics [44].In another study, Banerjee et al., reviewed several approaches in NP-based drug delivery for tubulin inhibitors along with their antitumor activities mediated by vascular disruption [31].Moreover, Čermák et al., outlined MT-targeting small molecules and effect in cancer therapy [45].Borys et al. have also surveyed the intrinsic and extrinsic factors influencing MT structure and dynamics in different noncancerous and cancer cells [46].In addition, Kanagathara et al., reviewed the βIII-tubulin expression-based potential therapy for cancer [47].Although different papers have reviewed various aspects of drugs/ nanomaterials in control of MT dynamics for improved cancer therapy, to the best of our knowledge, there is no comprehensive review selectively discussing inorganic NPs.Accordingly, in the present review we tried to outline the advances in the area of NP-MT interaction for exploring one of the main mechanisms involved in cancer therapy.In the present paper, we aimed to provide a brief description of the studies, exploring the interaction of several inorganic NPs with MTs, over the last decade to reveal the MT dynamics and relevant inhibition of cancer cell proliferation.

Microtubule structure
MTs as rigid hollow cylindrical tubes (25 nm in diameter and up to 20 μm in length) with 13 linear protofilaments arranged in a shape to form the MT wall are known as ubiquitous cytoskeletal structures composed of self-assembly tubulin heterodimers (α/β, 450 amino acid each, 41% identical) [48][49][50][51].α (N-site) and β (E-site) tubulin binds nonexchangeable and exchangeable guanine nucleotides, respectively [Fig.1a(i)] [52].Although MT polymerization is performed along with GTP (cap) hydrolysis, the GDP formation leads to MT depolymerization [20,54,55] [Fig.1a(ii)] [52].In other worlds, it can be indicated that MTs show a stable-unstable transition upon GTP hydrolysis and ligand binding [Fig.1a(iii)] [52].It has been also reported that MT assembly/ disassembly is the interplay of biochemistry and mechanics, as longitudinal and lateral interactions between tubulin dimers result in the formation of MT lattice as tubulin adopt three potential conformations including curved, straight expanded, straight compacted conformations [56].Indeed, the model of curved filaments could indicate that MT dynamics is controlled by associated proteins and mechanical force [57].In other words, it has been indicated that some proteins directly interact with the tubulin dimer via its tumor-overexpressed gene (TOG) domains to enhance MT polymerization at protofilament tips and endbinding (EB) proteins mediate the introduction of lateral bonds to form straight protofilaments [58].Then, curved protofilaments upon interaction with MT associated proteins (MAPs), such as kinesin-related protein MCAK or stathmin, results in MT destabilization [57].Also, it should be noted that nucleotide state along with protofilament number can change the skew angle of protofilaments, which could significantly alter the lateral interactions that stabilize the lattice against mechanical stress (rearrangement of H-bonds at the E-site) and influence the overall stability of the MT (Fig. 1b) [53].
It seems that γ, δ and ε tubulin have a key role in the MT selfassembly [63,64].Indeed, it has been reported that when the MT W. Zhang et al. polymerization/depolymerization balance is distorted, cells can use different isotypes to restore the normal equilibria [65].

Binding sites
MTs are known as one the potential dynamic targets in cancer therapy [66,67].MTs as important components of the cytoskeleton are involved in several important cellular processes, such as movement, division along with differentiation and cell signaling.Tubulin, a soluble heterodimer exists in the cytoplasm and MTs as dynamic polymers show a reversible exchange between the polymerization and depolymerization phases mediated by GTP-GDP transition.These unstable dynamics of the MT can be disrupted by various chemical agents and cause inhibition of cell division.MT stabilizing agents (MSAs) could promote lateral and/or longitudinal tubulin interaction, whereas MT destabilizing agents (MDAs) are able to inhibit either the interaction of tubulin or promote the conformational change of tubulin (curved-straight transition).Indeed, six different drug-binding sites on tubulin has been identified and two sites (Taxane and Laulimalide/peloruside sites) have a potential affinity with MSAs, whereas four sites (Colchicine, Vinca, Maytansine, Pironetin sites) are potentially targeted by MDAs (Fig. 2a) (Table 1) [68].
It has been found that some other enzyme inhibitors can also target MTs [75][76][77].For example, it has been shown that BKM120 (buparlisib, NVP-BKM120) as one of the most studied drugs which acts as a phosphoinositide 3-kinase (PI3K) inhibitor (ATP site), can potentially bind to tubulin (colchicine site) and destabilize MTs (Fig. 2b) [69].
So far, various small molecules and drugs derived from different sources have shown a wide range of antitumor effects through antimitotic activity [68,78], which has attracted the attention of many researchers to develop small molecules-based antitumor agents.The unique chemical structures of small molecule compounds can destabilize MT structure and dynamics by different mechanisms resulting in cell cycle arrest, apoptosis and mitigated metastasis of cancer cells.Studies to date have led to the discovery of many drugs from different resources that are currently under clinical trials by commercial companies.For example, it has been shown that heterocyclic scaffolds can play a key role in targeting MTs or kinases for cancer drug development [79].

MT targeting in cancer therapy
It has been shown that disruption of MT dynamics by MT targeting agents (MTAs) can lead to mitotic arrest, overexpression of proapoptotic proteins (p53) and induction of apoptosis signaling pathways (Fig. 3a) [80].Also, it has been indicated that MTAs can trigger autophagy-mediated apoptosis [84], cell ferroptosis [85], disturbance of cellular morphology and function [86].
In addition, it has been shown that MTAs can induce anticancer effects through disruption of the interphase MT function, antivascular performance and MT interactions with actin and intermediate filaments.For example, it has been found that disruption of the MT cytoskeleton by Taxol and 2-methoxyestradiol (2ME2) leads to a significant tumor angiogenesis impairment mediated by inhibiting the cytoplasmic hypoxia-inducible factor (HIF-1α) in PC3 cells (Fig. 3b) [81].In fact, HIF-1α protein potentially interacts with polymerized MTs and results in dynein motor protein-mediated nucleus trafficking [81].
MT targeting agents (MTAs) are typically recruited as clinically potent chemotherapies for different types of cancers.However, their widespread applications might be hindered by the acquired or intrinsic resistance of cancer cells to MTAs-induced cytotoxicity mediated by MT lattice destabilization.The combination of MTAs with some other potential anticancer agents like actin depolymerization agents could provide a promising alternative way to overcome drug resistance.
It has also been reported that a synergetic effect can be seen after addition of actin depolymerization agent [methyl-β-cyclodextrin (MCD)] along with some MTAs (Vinblastine, Taxol and Crocin).For

Table 1
Binding site of different MT stabilizing/destabilizing agents determined either by X-ray crystallography or cryo-EM.

Agents
Site Residues Kind of interaction Ref.

MT stabilizing agents
Taxane-site ligands Helix H7, strand S7, loops H6-H7, S7-H9 (the M-loop) and S9-S10 of β-tubulin Hydrophobic and polar contacts  example, it was shown that pre-treatment of HeLa cells with MCD (1 mM, 4 h) could augment the impacts of vinblastine, crocin and Taxol on MT of both interphase and mitotic cells [Fig.3c] [82].Therefore, it can be indicated that concurrent targeting actin dynamics and disturbance of MT stability can play a key role in synergistic enhancement of anticancer effects on cancer cells [87].In general, it has been widely reported that MTAs induce their anticancer effect though cell cycle arrest mediated by MT-related cellular disassembly (Fig. 3d) [83].

Interaction of nanoparticles with microtubules in the cell
Conventional drug treatment of cancer is rarely successful and makes the tumor resistant to the drug, so the use of potential alternative compounds to treat cancer has widely received interested.NPs, due to their direct or indirect interaction with MTs and preventing or promoting MT depolymerization and subsequent inhibition of mitotic cell division or changing the cell morphology, respectively prevent the development and progression of cancer.Therefore, among different strategies for combating cancer, NP-based therapeutics are considered as one of the most potential approaches due to their unique properties.
NPs could interact with tubulin subunits to induce MT selfdisassembly-mediated chromosome missegregation and aneuploidy in dividing cells [88][89][90][91].Therefore, NPs are known to induce MT destabilization and in relatively low concentrations inhibit tubulin selfassembly in vitro, and at higher concentrations, interact directly with MTs and result in dissociation of protofilaments from the MT walls [92].
In addition to their key role in the formation of mitotic spindles, MTs participate in other cellular functions such as motility and transport [93].Anticancer effects of NPs may be due to the deactivation of these functions.
Conventional drugs, especially small molecules, could only interact with MTs or some other targets like kinases, whereas NPs in addition to interacting with MTs and other proteins can be recruited in the NPsmediated enhanced hyperthermia, photodynamic therapy (PDT) and photothermal therapy (PTT) as a promising horizon on effective cancer treatment.Also, the size of NPs enables potential cell uptake and apparent interaction with cell components such as membrane, organelles and biomolecules.Elevated apoptosis rate of cancer cells after interaction of NPs with MTs suggests that tubulin and MAPs-targeted NPs can be potentially developed for cancer therapy [41].
Although engineered NPs are being widely used for the development of biomedical settings, to date little is revealed concerning the possible mechanisms of their translocation into targeted tissues from blood circulation (vascular transport), as well as the consequent implications of unwanted side effects.One possible reason for enhanced cellular uptake of NPs in tumor cells is a significant increase in endothelial cell dysfunction through oxidative stress-mediated MT remodeling.For example, Apopa and coworkers showed that iron oxide (IO) NPs can induce MT remodeling [Fig.4a(i)] and overexpression of tubulin [Fig.4a(ii)] in human microvascular endothelial cells [94].Therefore, it can be indicated that magnetic NPs can control the spatiotemporal assembly of MTs [96].Furthermore, it has been also reported that titanium oxide (TiO 2 ) NPs (100 μg ml − 1 ) could potentially interact with tubule heterodimers, MTs and associated tau proteins and result in the low stability of MT.Also, it has been shown that Withania somnifera (WS)-synthesized AuNPs (25 nm) could show anticancer effects through MT disassembly dynamicsmediated apoptosis (Fig. 4b) [95].
IONPs can be also used as therapeutic agents for combined thermochemotherapy treatment of cancer through potentiating the chemotherapeutic effect of drugs mediated by further stress stimulated by nanoscale heating to cell organelles and MTs.Therefore, modulation of paclitaxel (PTX)-stimulated mitotic block and triggered mitotic exit mediated by hyperthermia could be used in the development of effective combination therapy approaches for overcoming drug resistance [97].Therefore, it can be deduced that NPs mitigate the proliferation of cancer cells-mediated by perturbation of MTs (Fig. 4c) [36].

Interaction of nanoparticles with blood proteins and formation of protein corona
Upon interaction of NPs with body fluids, different physical and chemical interactions between blood proteins and NPs result in the formation of protein corona (PC) at the bio-nano interface, which changes their subsequent interactions with cells [98].Therefore, it has been well-documented that PC could heavily change the physicochemical properties of NPs and associated cytotoxicity.For example, it has been reported that interaction of NPs with blood proteins and formation of PC usually results in opsonization, phagocytosis by mononuclear phagocytic cells (MPCs), decreased circulation time, colloidal destabilization, and aggregation, which reduce their targeting efficacy and underlying accumulation in cancer cells [99].Therefore, exploring the direct interaction of bare NPs with different proteins except blood proteins like tubulin has been mostly done in vitro.Indeed, due to the possible interaction of NPs with blood proteins and the formation of hard and soft PC around NPs in vivo, the direct interaction of bare NPs with tubulin is not physiologically possible.
It has been suggested that the stealth coating of NPs may result in the limited interaction of NPs with blood proteins and associated formation of PC.For example, modification of NPs with tunable surfactants (C 18 -PEEP 21 and C 18 -PEEP 78 ) has been shown to be a potential strategy to inhibit the formation of PC around NPs [100].Based on this approach, it was found that by variation in the length and composition of surfactants, the total amount of PC around NPs could be modulated and reduced to around 5 times lower than that of bare NPs [100].
PEGylation of NPs can also reduce the interaction of NPs with unspecific blood proteins and results in modulation of PC.In this case, the thermodynamic parameters of NP surfaces can be adjusted by tuning the block lengths of PEG to form different hydrophilic surfaces.Consequently, the unspecific protein interaction and self-aggregated properties of NPs mediated by PC can be manipulated to regulate cell uptake and recognition state of NPs.In other words, it can be indicated that the PC of PEGylated NPs could be potentially enriched with specific blood proteins which are able to regulate a specific biological function (Fig. 5a) [101].Therefore, it was found that non-covalent interaction between protein and NP surfaces could be regulated through PEGylation of NPs to facilitate the engineering of PC at the nano-bio interface.Another possible strategy to modulate the formation of PC around NPs is pre-coating of NPs with proteins like albumin and transferrin.Indeed, pre-coating of NPs with IgG depleted plasma in vitro was found to result in the induction of a stealth effect (artificial PC) to avoid the potential interaction of some nonspecific serum proteins (IgG) with NPs and resultant reduction of the macrophage uptake (RAW 264.7) (Fig. 5b) [102].Also, it should be noted the physicochemical properties of NPs such as the size and hydrophobicity (Fig. 5c), composition (Fig. 5d) and surface charge (Fig. 5e) could play a key role in the regulation and amount of adsorbed proteins on the NP surface.
Therefore, exploring the properties of PC can be considered as a complex phenomenon and of great importance for the biological assessment of NPs, especially in cancer therapy.The formation of PC can be regulated by acceleration of the interaction of specific protein against non-specific binding mediated by surface modification of NPs along with tuning their physicochemical properties.
Another crucial characteristic of protein interaction with NP is the possible structural changes of the protein and subsequent loss of function [105,106].Indeed, conformational changes of proteins following interaction with NPs can result in the exposure of new antigenic as well as hydrophobic residues, which may trigger immune responses and protein stability, respectively [107,108].In general, it should be noted the conformational changes of proteins after interaction with NPs depend on several parameters including the intrinsic stability of NPs, physicochemical properties of NPs and the pH of the medium [106,109].For example, it has been shown that the smaller sized AgNPs have a stronger interaction with BSA relative to that of larger-sized NPs [110], and the rod-like AuNPs induce more structural changes in albumin in comparison with spherical-shaped counterparts due to higher surface energy [111].In another study, it was shown that the interaction of albumin with magnetic NPs (MNPs) reduced as the pH of the medium increased from 6 to 7.5 due to possible variation in the charge distribution of the system [112].Zaheer and co-workers also indicated that the binding affinity of NPs with proteins is lower than that of ions due to the different coordination affinities of NPs with their ionic counterparts [113].

Different experimental techniques on the interaction between microtubules and nanoparticles
Several experimental techniques are available for exploring the structure and thermodynamics of protein upon interaction with ligands/ NPs.Table 2 summarizes some experimental techniques with relevant information used for study of protein-NP interaction.

Direct interaction of inorganic NPs with microtubules
Inorganic NPs can be made useful for potential application in cancer therapy due to their unique properties along with tunable structural characteristics.Indeed, it has been indicated that inorganic NPs upon interaction with cells can result in the MT disorganization, defective MT reassembly, induction of MT acetylation, and cell cycle arrest [42].In fact, it has been shown that NP-induced MT acetylation could significantly potentiate anticancer effects of T-cells [33].Therefore, exploring the thermodynamic/structural and binding parameters of protein-NP complexes can provide useful information about the interactions involved in the targeted therapeutic effects of NPs on different cancer cells.These investigations also put forward the field with fundamental details on alterations in protein structure, protein stabilization/destabilization mechanisms and approaches to clear up the character and fates of NPs after biomedical applications.
Cytoskeletal proteins like tubulin and actin can interact with the surface of NPs through different functional groups and undergo slight or substantial structural changes and different characteristics of NPs can play a key role in the mechanism of interaction between NPs and proteins.It has been reported that actin has a higher affinity than tubulin for silver (Ag) NPs, while both proteins underwent substantial structural changes upon interaction [124].
Also, Zhou and co-workers by using SERS and X-ray photoelectron spectroscopy (XPS) explored the nature of the binding sites of tubulin upon interaction with AuNPs [125].Several kinds of functional groups were detected by SERS to have a potential interaction with the AuNPs such as imidazole, SH, aromatic rings, NH 2 , and COOH [125].However, the imidazole ring derived from histidine was shown to be the most potential functional moiety for AuNP binding.The results from these studies provide better understanding of the binding between Au and the biotemplate and give insight concerning methods to improve Au coverage for MT-templated Au nanowires.Also, it has been shown that fullerene derivative, C 60 (OH) 20 , is able to inhibit MT polymerization at very low concentration (1 mg mL − 1 ) with 8 binding sites (n), which were mainly derived from formed hydrogen bonding between the NPs and the tubulin, as explored by theoretical analyses [126].Circular dichroism spectroscopy study showed a significant alteration in the secondary structures of building blocks of MTs, while ITC determined that fullerene NPs bind to tubulin with a great affinity (K b = 1.3 × 10 6 M − 1 ) through an exothermic process (ΔH = − 2.648 kcal/mol) with a ΔG = − 6.343 kcal/ mol.Theoretical analyses further indicated that NPs bind to longitudinal interfaces which influence the polymerization phase of MTs [126].Moreover, Gheshlaghi and co-workers indicated that tubulin underwent an apparent conformational change with TiO 2 NPs and the NPs could result in the tubulin depolymerization [38].
Furthermore, Dadras and co-workers indicated that MTs underwent significant conformational changes on the surface of magnetite IONPs (50-100 nm) at both the secondary [Fig.6a(i)] and tertiary conformer [Fig.6a(ii)] levels, as evidenced by circular dichroism and ANS fluorescence spectroscopy analyses, respectively [39].They reported that the static IONP-tubulin complex with a single binding site had several tubulin isomeric forms at different concentrations of NPs.The structural changes of tubulin were mainly associated with a decrease in the α-helical structure, whereas the proportion of β-sheet structure increased [Fig.6a(i)] [39].Increasing the concentration of IONPs in the complex led to an enhancement in ANS fluorescence intensity as a marker of hydrophobic forces.Therefore, this phenomenon could explain loss of α-helical conformers owing to interaction with IONPs and the probable structural transition from α-helix to β-sheet in the complex.Also, based on the positive value of ΔH and ΔS, it was assumed that hydrophobic forces play a key role in the interaction of IONPs and tubulin.
However, it should be noted the aggregation of NPs is a critical factor that should be modulated in spectroscopic studies.For this reason, modification of NPs can be a potential strategy to mitigate the aggregation tendency of NPs.For example, citrate functionalized AuNPs [32] or polyphenol-modified AuNPs [43] have shown a great colloidal stability.Protein aggregation can be also considered as one of the side effects of NPs after interaction with proteins.It was seen that citrate-modified AuNPs could result in tubulin aggregation (Fig. 6b) [32].Indeed, Raman and FTIR spectroscopies analyses revealed apparent structural alteration of tubulin and MTs, thus indicating that AuNP-triggered conformational change is the one of the main driving forces behind the protein aggregation and cytotoxicity against A549 cells [32].
Nirmala and co-workers also aimed to synthesize modified AuNPs stabilized by utilization of polyphenolic compounds, where they showed high stability against aggregation [43].Furthermore, it was shown that the intrinsic fluorescence [Fig.6c It has been also reported that engulfed zinc oxide (ZnO) NPs stimulating actin filament bundling and conformational changes in MTs through transition from a dynamic structure to rigid MTs, heavily influenced cell proliferation and viability [128].
Moreover, Bera and co-workers using different biophysical and cellular assays explored the effect of graphene oxide (GO) on tubulin structure and found that how the NPs-induced structural changes of proteins affect the tubulin/MT dynamics and resultant cytotoxicity [Fig.6d(i)] [127].It was seen that GO led to an apparent disruption in the structural integrity of the tubulin, with consequent inhibition of tubulin polymerization as evidenced by UV-Vis [Fig.6d(ii)] and fluorescence [Fig.6d(iii)] spectroscopy analyses.The authors also investigated the anticancer effect of GO and they found that this compound induced a significant anticancer effect against human colon cancer cells Reprinted with permission from Ref. [39].Copyright 2013 Springer.(b) Tubulin aggregation in the presence of citrate-AuNPs [32].Reprinted with permission from Ref. [32].Copyright 2013 Royal Society of Chemistry.(c) Intrinsic fluorescence (i), ANS fluorescence (ii) and circular dichroism (iii) of tubulin structure upon interaction with increasing concentrations of Triphala (Trl) polyphenols-modified AuNPs, interaction of MDA-MB-231 cells with increasing concentrations of Trl-AuNPs (iv) [43].Reprinted with permission from Ref. [43].Copyright 2021 Elsevier.(d) The anticancer effect of graphene oxide (GO) NPs against human colon cancer cells (HCT116) mediated by destabilization of the tubulin assembly and oxidative stress (i), UV-Vis (ii) and fluorescence spectroscopy (iii) analyses of tubulin polymerization with increasing concentrations of GO [127].Reprinted with permission from Ref. [127].Copyright 2021 Elsevier.(HCT116) through destabilization of the tubulin assembly and oxidative stress [127].

Challenges and future perspectives
Structural changes triggered by the binding of different NPs to proteins can lead to significant changes in the function of proteins.Along with their potential applications in cancer treatment, NPs can cause some changes in the structure and function of various proteins in the body, including blood proteins, structural proteins and cytoskeleton proteins.In addition to the industrial aspect of the interaction of proteins with NPs, the medical approach and harmful effects of NPs on proteins and changing their structural and functional properties is also a very important issue.As NPs first come into contact with proteins after entering a physiological environment, different researches have been done in this field to explore the mechanisms associated with the formation of protein-NP complexes.
Among NPs, inorganic NPs have the highest degree of commercialization in the medical sector.The unique properties of these NPs are remarkable even at very low concentrations.Surface modified inorganic NPs show stronger colloidal stability than uncoated counterparts and may show potential interaction with tubulin.Also, various studies have been performed on the interaction of different kinds of inorganic NPs with tubulin, including AuNPs, AgNPs and TiO 2 NPs.The interaction of inorganic NPs with tubulin indicates that these NPs reduce the helix structures of tubulin and induce remodeling, depolymerization and destabilization of MTs.However, these studies did not precisely explore that if the coating of NPs can further modulate the induced structural changes of tubulin and triggered anticancer effects, relative to bare NPs?
For the future perspective, the interaction of coated NPs with different functional moieties with tubulin can be compared with the bare counterparts to explore the effect of the surface modification of NPs on induced structural changes of tubulin and resultant anticancer effects.Also, the depolymerization of MTs induced by NPs in off-targeted cells should be regulated through modification of NPs.Furthermore, it is suggested to explore the effect of physicochemical properties of NPs on MT lattice through possible interactions and relevant anticancer effects.
Indeed, as inorganic NPs are widely used as promising platforms for potential cancer therapy, tuning their physical properties such as size, shape and surface charge can be very important in determining the fate of these unique particles in vivo.Particle size is one of the most important factors in determining the type of interaction between NPs and proteins because dispersion, efficiency, clearance, and pharmacodynamics of the NPs are affected by particle size.In general, the NP size must be within a certain limit in order to be thermodynamically favor for interaction with proteins.Also, the physicochemical properties of NPs could play an important role in their induced cytotoxicity and biocompatibility.
Also, it should be noted that NPs with various surface charges possibly change MT polymerization by interacting with the tubulin dimer and modulating the negative C-terminal repulsion of the tubulin.Also, increasing the concentration of charged NPs can result in nonspecific interaction of NPs with proteins and resultant MT destabilization.Therefore, based on the concentration, NPs can establish a charge-charge interaction with MTs and alter their stability.
In addition to the anticancer effects of NPs induced by MT destabilization, there are also some serious concerns about degradation, internalization by biological systems, formation of PC, and unwanted side effects.Due to the small size of NPs, they can show nonspecific interactions with off-target proteins and cells.They also can interact with intracellular structures and large molecules over a long period of time, so it is necessary to carefully assess their biocompatibility before biomedical applications.
There are several studies on the fabrication of inorganic NPs as common particles for the interaction with MTs.The effect of fabrication variables such as precursor concentration, solution pH and reaction temperature on the properties of synthesized NPs and subsequent type of interaction between NPs and proteins should be considered.
Also, MAPs are important auxiliary proteins involved in the regulation of MT stability and dynamics.For this reason, changes in the structure of MAPs should be considered when exploring the organization of MTs in the presence of NPs.Binding of NPs to the MAPs increases the structural changes of these proteins, which result in subsequent depolymerization of MTs.However, as binding of NPs with MTs and MAPs is mediated nonspecifically and multiple target proteins can interact with NPs, it is complicated to precisely determine the role of NPs-induced structural change of MTs in cancer therapy in vivo.

Conclusion
Exploring the interaction of proteins and NPs is known as the first step towards manipulating the biological effects of NPs.Different researches thus far have indicated that physicochemical properties of NPs considerably determine the amount of protein adsorption and also play a key role in modification of the conformation of the adsorbed proteins.This can apparently alter the biological behavior of the NPs and regulate the anticancer efficacy of NPs.Generally, based on this review, it can be indicated that the NP-mediated changes in the conformational and functional properties of MTs can be one of the main mechanisms involved in the anticancer effects of NPs.Therefore, these data can provide useful information for design and development of potential NPs showing less cytotoxicity against off-targeted cells while retaining significant therapeutic performances.It can be deduced that the binding of inorganic NPs to tubulin/MTs could disrupt the mechanistic basis of MT growth through changing the tubulin structure and dynamics.Therefore, cell cycle arrest and cell death signaling pathways are upregulated in response to loss of MT function.However, it should be noted that most studies reported in this field focus on in vitro assay platforms.Therefore, extrapolation of these details in predicting the fate of NP-MT complex in vivo remains a challenging area and demands further exploration.
Systematic evaluation of binding features of potential NPs with MTs having different physiological properties can advance our existing information regarding NP-MT interactions.Thorough analysis of NP-MT interactions could likely indicate potential manipulation possibilities of NPs with unique properties to interact with MTs or adsorbed small drug molecules intended for perturbation of tubulin and subsequent suppression of MT dynamics in vivo.Furthermore, this information might be helpful in regulation of nanotoxicity concerns.Also, PC changes the overall biological behavior of the NPs and exploring the characteristics of this complex interaction can thus bestow applicable discernment into compatibility, cellular uptake, therapeutic potency, and other key features of NPs that can be prospected for advancing safe and potential NP-based platforms for future directions of cancer therapeutics.

Declaration of competing interest
None.

Fig. 5 .
Fig. 5. (a) PEGylation of NPs for specific PC adsorption [101].Reprinted with permission from Ref. [101].Copyright 2017 American Chemistry Society.(b) Precoating of NPs with IgG depleted plasma to form artificial (i) and reduction of macrophage uptake as determined by flow cytometry (ii) and confocal laser scanning microscopy (iii) [102].Reprinted with permission from Ref. [102].Copyright 2018 Royal Society of Chemistry.(c) Some crucial factors contributing to the PC composition of PLGA and PLA NPs [103].Reprinted with permission from Ref. [103].Copyright 2021 Elsevier.(d) Impact of NP types on the PC formation [104].Reprinted with permission from Ref. [104].Copyright 2017 Elsevier.(e) Impact of surface charge of NPs on the PC formation [102].Reprinted with permission from Ref. [102].Copyright 2018 Royal Society of Chemistry.

W
.Zhang et al.

Table 2
Different techniques used for analysis of protein-ligand/NP interaction.