Tungsten Disulfide-Based Materials and Their Conjugates for Cancer Photothermal Therapy

Abstract

Cancer remains one of the most critical global health issues. Conventional treatments, such as radiotherapy, surgery, or chemotherapy, have limitations, especially concerning side effects, resistance, and recurrence. Consequently, new innovative treatments to overcome these problems are needed. Photothermal therapy (PTT) is a promising alternative that uses photothermal agents that convert near-infrared light (NIR) into heat to kill cancer cells. Nanoparticles can be used as photothermal agents and also as drug delivery platforms, improving the drugs’ stability, allowing for targeted delivery, and reducing toxicity. Due to its broad absorption band, high surface area, and versatility for surface functionalization, tungsten disulfide (WS₂) has high potential in this context. This paper presents the state-of-the-art on the use of WS₂-based materials to achieve effective and biocompatible new anticancer treatment strategies.

1. Introduction

Cancer is one of the biggest world health issues, leading the way for a huge number of deaths worldwide. In 2020, 19.3 million new cases of cancer were reported worldwide, from which about 10 million resulted in death. The World Health Organization estimates reveal 28.9 million new cases by 2040, and 16.2 million deaths per year [1]. Radiotherapy is the most used treatment for cancer in combination with surgery, immunotherapy, and chemotherapy. However, this treatment is limited by the radiation that can be emitted, due to the need to not harm healthy cells around the cancer ones. So, there is an urgent need to find new ways to target only the tumor cells while not damaging the healthy ones around them [2].

As an alternative to more conventional treatments, photothermal and photodynamic therapy (PDT) with nanoparticles have been gaining more attention. Photothermal therapy (PTT) uses agents that are administered at the tumor site that absorb near-infrared (NIR) light from a light source (e.g., laser), converting it to heat [3]. Photodynamic therapy uses photosensitizers (PS) that, when exposed to light, produce reactive oxygen species (ROS) that will kill tumor cells [4]. Even though PTT and PDT hold great value for the treatment of cancer, there are still issues to be addressed regarding the use of laser devices that can harm normal tissues, the body absorption/distribution, toxicity, and water stability of phototherapeutic agents used, and the heat resistance of some types of cancer cells [5]. Using nanomaterials as PTT agents not only improves the photothermal efficiency, but also reduces the risks associated with the treatment. Different nanoparticles such as plasmonic gold and silver, iron (magnetic), and carbon nanomaterials, among others, have been studied for these treatments, since they possess good physicochemical and optical absorption properties. Furthermore, surface modifications can be performed with other molecules, improving their water stability, biocompatibility, and selectivity [6]. PTT and chemotherapy show great synergy. The increase in temperature caused by PTT enhances the cellular uptake and release of the chemotherapeutic drugs in the tumor. The introduction of 2D nanomaterials in PTT improves this synergy by providing a platform for drug delivery due to their large surface area and the possibility of surface modifications [7].

Several types of 2D nanomaterials have been tested for PTT, such as graphene-based materials (GBM), transition metal dichalcogenides (TMDCs), transition metal oxides (TMOs), transition metal carbides, nitrides and carbonitrides (MXenes), black phosphorus (BP), and others. These can be produced through different routes based on top-down or bottom-up approaches. The top-down methods involve the breaking of bulk materials into smaller particles through mechanical and/or liquid exfoliation methods. These methods essentially implicate the breaking of the Van der Waals interactions between the layers of the bulk material. On the other hand, the bottom-up approaches involve the synthesis of nanomaterials from the atomic or molecular level into larger structures. Chemical-based methods are often used, such as chemical vapor deposition and wet-chemical synthesis [8].

Graphene is composed of one thin layer of carbon atoms arranged in a honeycomb lattice. Graphene nanoparticles present unique properties, such as a large surface area, planar and thin shape, high thermal properties (5000 W m⁻¹ K⁻¹), and electrical conductivity. Furthermore, it has good mechanical and optical properties, and a high extinction coefficient in the near-infrared range (NIR), which makes graphene a good photothermal agent [9,10]. Different types of graphene-based materials can be obtained using different production/modification methods [11,12]. Transition metal oxides (TMOs) are composed of metals bound to oxygen atoms, in a single or multilayer arrangement, where the oxygen atom polarizes the transition metal’s electrons. Due to this strong polarization, planar TMOs show large non-linear and non-uniform distribution charges, which lead to extraordinary local surface and interfacial properties [13]. TMOs have good optical properties, and high surface-to-volume ratios. Furthermore, TMOs can interact directly with biomolecules through surface modification, making them suitable for drug delivery or cancer therapy [14]. MnO₂ is the most commonly studied TMO [15]. MXenes include transition metal carbides, nitrides, and carbonitrides. The generic formula for MXenes is M_n+1X_n, where M represents an early transition metal and X represents carbon or nitrogen [16]. MXenes’ surfaces can be modified with hydroxyls, oxygen, or fluorine, which makes them stable in aqueous media, improves biocompatibility, and allows conjugation with other molecules [17]. Another interesting 2D nanomaterial is black phosphorus (BP), a single layer of phosphorus atoms arranged in a honeycomb-like lattice structure. The tunable band gap, good thermal and optical properties, and nontoxic degradation products make this material suitable for biomedical applications [18]. Other nanomaterials, such as Palladium (Pd) nanoparticles, have been investigated for PTT applications due to their high photothermal conversion efficiency, high NIR absorption, and excellent photothermal stability and biocompatibility [19]. Layered double hydroxides (LDHs) have also gained more attention due to their biocompatibility, tunable chemical composition, and the ability to exchange intercalated anions [20].

Transition metal dichalcogenides (TMDCs) are composed of transition metals inserted into layers of chalcogen atoms. TMDCs have a generic formula of MX₂, where M represents the transition metal and X is the chalcogen. The transition metals are positioned between two chalcogen (X) atom layers, linked by weak Van der Waals interactions [21]. TMDCs can be produced through various methods, such as mechanical exfoliation, liquid phase exfoliation, chemical exfoliation, solvothermal synthesis or chemical vapor deposition [22]. TMDCs present singular properties, especially cytocompatibility, versatile surface chemistry and high photothermal conversion, making them suitable to be used as photothermal agents and in drug delivery applications [6]. TMDCs show broad absorption in the NIR region because of their small band gap, which allows them to absorb light in this region while having a high photothermal conversion efficiency, which enables the use of lower-power-density NIR sources [23]. Also, because of their versatile surface chemistry, surface functionalization can be done with polymers or surfactants to improve dispersibility, stability and biocompatibility [23]. Molybdenum disulfide (MoS₂) is the most researched TMDC, followed by WS₂ and MoSe₂. In vitro studies with MoS₂ were published for the first time in 2013, demonstrating its potential as a photothermal agent [24].

WS₂ nanoparticles consist of sheets of tungsten atoms sandwiched between layers of sulfur atoms. WS₂ can be produced via either top-down or bottom-up approaches. For the top-down methods, since the interlayer Van der Waals force of the bulk material is weak, 2D nanosheets can be produced from their initial bulk materials through various types of exfoliation, such as mechanical, liquid-based, alkalis-based, ion intercalation, or electrochemical methods. Mechanical exfoliation is based on the adhesion of adhesive tape and bulk crystal to obtain monolayer or few-layer nanosheets. As for liquid-phase exfoliation, this method is based on the ultrasonication of bulk crystals in a solvent. The waves resulting from ultrasonication propagate through the solvent, causing the weaker Van der Waals interactions to break. Chemical exfoliation methods can also be used to produce ultrathin nanosheets. In this method, species are inserted into the interlayer cavity, expanding its distance and weakening the interactions between each layer. Lithium is the most widely used species in intercalation, due to its reaction with water, which produces hydrogen gas that, through its expansion, allows the separation of the nanosheet’s layers [22,25]. On the other hand, bottom-up production can also be employed, with methods such as chemical vapor deposition (CVD) or hydro-/solvothermal synthesis being the most common [26]. CVD refers to the reaction of a metal and a chalcogen with precursors under high temperature and pressure, resulting in ultrathin nanosheets. In the solvothermal method, a precursor under specific solvent conditions and specific reaction time is used to obtain the materials [22,25]. More details on WS₂-based materials production methods can be found somewhere else [27].

The relevant features of WS₂ are that it has a large specific surface area and many active sites on the surface, which make surface functionalization easier. Furthermore, WS₂ has a big adsorption band, having good absorption from ultraviolet to infrared regions. These features enable its use for drug delivery and cancer PTT [28]. Despite all the above-mentioned advantages, WS₂ still faces some problems, such as poor stability in physiological conditions, low biocompatibility at high concentrations, and low cellular uptake efficiency. Different types of surface modification have been used to overcome this problem, such as using polyethylene glycol (PEG) or bis(trimethylsilyl)acetamide (BSA). These coatings not only enhance the stability and dispersibility of the WS₂ nanosheets in physiological environments, but can also improve cellular uptake in cancer cells, as well as improving the PTT effect and drug-loading efficiency [29].

2. Functionalization of WS₂ Nanoparticles

Surface functionalization can be performed by covalent or non-covalent interactions. In covalent functionalization, a chemical bond is formed between the nanomaterial and the functional compounds. One type of covalent functionalization is chemical oxidation, whereby oxygen groups like carboxyls (-COOH), epoxides (-C-O-C) and hydroxyls (-OH) are introduced at the nanomaterials’ surface [6]. Another example is the introduction of PEG molecules (PEGylation), from the reaction with nanomaterials’ carboxyl groups, for example, which usually results in improved stability in physiological media and biocompatibility [30]. However, this type of process can lead to changes in nanomaterials’ structure and key properties, for example, by disrupting delocalized π–π bonds, which can cause a decrease in photothermal conversion efficiency, affecting their application in PTT. Therefore, sometimes, non-covalent functionalization is more suitable. Non-covalent modifications are based on surface interactions with other molecules through Van der Waals forces, π–π stacking, hydrophobic/hydrophilic interactions, electrostatic forces, hydrogen bonding, and ionic and coordination bonds. These interactions can often be reversed and are usually weaker than covalent ones [6].

Several strategies have been successfully developed to functionalize WS₂ (Table 1). WS₂ can be conjugated with polymers, such as PEG, BSA and PVP, or other inorganic or organic molecules, for example, liposomes. PEG is one of the most used polymers for WS₂ surface modification. Xie et al. [29] proved that the photothermal properties of WS₂ were improved using the thiol chemistry to functionalize the WS₂ surface with PEG. After 5 min of NIR irradiation (808 nm laser, 0.8 W cm⁻²), a concentration-dependent increase in WS₂-PEG temperature was found. The mass extinction coefficient measured at 808 nm was 23.8 L g⁻¹ cm⁻¹, which is higher than graphene oxide (3.6 L g⁻¹ cm⁻¹) and similar to rGO (24.6 L g⁻¹ cm⁻¹) [31]. These results suggest that WS₂-PEG can be a strong photothermal agent for PTT. WS₂ functionalized with iron oxide (IO) nanoparticles at the surface, using a solvothermal method, coated with mesoporous silica (MS), and further functionalized with PEG, WS₂-IO-MS-PEG, was loaded with DOX by mixing both solutions for 24 h at room temperature in PBS (pH 8), resulting in WS₂-IO-MS-PEG/DOX. This nanoparticle demonstrated improved stability in water, saline, and serum media, without any agglomeration observed up to 24 h. TEM imaging showed a mesoporous silica shell, with a thickness of 10 nm, on the surface of WS₂-IO-MS, while BET measurements determined a high surface area of 568.03 m² g⁻¹, which favors effective drug loading and release. The mass extinction coefficient, at 800 nm, was 23.8 L g⁻¹ cm⁻¹, the same as for WS₂-PEG above mentioned [29]. WS₂-IO-MS-PEG also showed excellent photothermal stability upon five cycles of 808 nm laser irradiation (0.8 W cm⁻², 3 min each cycle) [32]. Yi et al. [33] studied the functionalization of WS₂ with BSA by electrostatic surface adsorption. This functionalization improved the stability in water, saline or DMEM media. The photothermal conversion capability of WS₂-BSA was also tested for different concentrations and laser power densities. With the increase in concentrations and laser power, the temperatures achieved also increased. As an example, when a concentration of 50 ppm WS₂-BSA, at T = 26 °C, was irradiated (1.5 W cm⁻², 808 nm), a temperature of 44.5 °C was reached within 5 min. Furthermore, WS₂-BSA showed good photothermal stability. An innovative WS₂ nanosheet derived from Fe(III) species and modified with PVP on the surface, produced by a solvothermal method, was tested for stability in different media, such as water, saline and DMEM, and did not show any meaningful changes after 24 h. In addition, the biodegradation rate was also studied, and it could be seen that it increased quickly due to the presence of the Fe(III) species. The mass extinction coefficient of Fe(III)@WS₂-PVP was calculated to be 3.4 L g⁻¹ cm⁻¹, lower than other WS₂ (only) nanoparticles but similar to graphene oxide (3.6 L g⁻¹ cm⁻¹). The photothermal conversion capability of these nanoparticles was also tested using different concentrations and power laser densities. For a concentration of 200 µg mL⁻¹, upon NIR laser irradiation (5 min, 808 nm laser, 1 W cm⁻²), a temperature of 45 °C was reached [34].

Organic molecules, such as liposomes, can also be used for surface modification. WS₂ can be conjugated with liposomes by electrostatic adsorption, which improves not only the stability in water and other media, but can also improve the drug delivery inside the cells and the biocompatibility of the nanoparticles. In a study, WS₂-lipid (200 µg mL⁻¹) reached a temperature of 60 °C after 10 min of NIR irradiation (808 nm laser, 2 W cm⁻²) [35]. Xie et al. [36] developed iron oxide-loaded WS₂ (m-WS₂) modified with lipids. Iron oxide was loaded onto the WS₂ surface by the solvothermal method, and lipids were mixed at 37 °C for 24 h, resulting in mWS₂-lipid nanoparticles. After 10 min of NIR irradiation (808 nm laser, 2 W cm⁻²), the mWS₂-lipid reached 60.7 °C. WS₂/Au-lipid, gold-doped WS₂, produced through a sodium citrate reduction method and modified with lipids by the method previously described, reached 65 °C, under the same irradiation conditions [37]. Apart from previously mentioned functionalization strategies, there are other methods to produce innovative WS₂ nanoparticles, with unique characteristics. For example, the production of 1T-WS₂ ultrathin nanosheets through a hydrothermal reaction created nanosheets with good biocompatibility and photostability, exhibiting a strong absorption in the NIR region. These NPs were highly hydrophilic and presented great dispersibility in water, with no agglomeration, even after 1 year. They also showed good photothermal conversion capabilities, with temperature increments of 34 °C within 5 min, under 808 nm laser irradiation (0.6 W cm⁻²) [38].

The doping of WS₂ nanosheets’ surfaces with elements is another strategy that can be used to improve their properties. The doping of WS₂ with gadolinium, Gd³⁺, and their further modification with PEG resulted in stable NPs in PBS, 1640-Medium, and fetal bovine serum (FBS), with improved photothermal characteristics. After 5 min of NIR irradiation (808 nm laser, 0.8 W cm⁻²), the NPs also showed a concentration-dependent temperature increase. The mass extinction coefficient of WS₂:Gd³⁺-PEG was 22.6 L g⁻¹ cm⁻¹, which is slightly lower than for WS₂-PEG and WS₂-IO-MS-PEG [39]. Ying et al. [40] developed a PEGylated WS₂ (W) delivery system of DOX (D), which was adsorbed onto the surface of the NPs in conjunction with ICG (I), a medical contrast agent that, under NIR laser irradiation, can produce ROS and hyperthermia in PDT/PTT therapy. This system was then coated with DSPE-PEG-2000-FA-modified erythrocyte membrane (M-FA) to produce WID@M-FA NPs. The modification improved the cell-targeting ability of the nanocarrier through FA receptor interactions. WID@M-FA NPs showed great photothermal properties, reaching 60 °C after 5 min of 808 nm laser irradiation (1 W cm⁻²), while showing good photothermal stability.

Table 1. Tungsten disulfide functionalization and drug conjugation strategies.

Material Produced	Drug Conjugation	Method	Physicochemical Properties’ Alteration	Reference
WS2-BSA	-	(1) WS2 production by ultrasound-assisted liquid phase exfoliation (2) BSA electrostatic adsorption on the surface by stirring	Improved stability in saline, water and DMEM Zeta potential: BSA = −27.4 mV WS2 = 20.8 mV WS2-BSA = −19.8 mV	Yi et al. [33]
Fe (III)-WS2-PVP	DOX Drug loading: 41% (Fe (III)-WS2 -PVP: DOX weight ratio: n/s) Drug release: 7.5 (pH 7.4; 6 h) 12.7% (pH 7.4; 48 h) 37% (pH 6.0; 6 h) 48.2% (pH 6.0; 48 h)	(1) Fe (III)-WS2-PVP formation by hydrothermal reaction (2) DOX adsorption on Fe (III)-WS2-PVP	Increased biodegradation rate Improved stability in saline, water and DMEM	Wu et al. [34]
WS2-PEG	-	Covalent bond formation between WS2 and PEG	Improved stability in saline Improved photothermal properties: broad NIR absorption band 700–1000 nm	Cheng et al. [29]
WID-M-FA	DOX loading: 24.2% (WID-M-FA: DOX weight ratio: n/s) DOX release: 12% (pH 7.4; 20 h) 30.9% (pH 7.4; 72 h) 24.2% (pH 5.2; 20 h) 56.4% (pH 5.2; 72 h) ICG loading: 22.9% (WID-M-FA: ICG weight ratio: n/s) ICG release: 14.3% (pH 7.4; 20 h) 19.5% (pH 7.4; 72 h) 27.1% (pH 5.2; 20 h) 35.2% (pH 5.2; 72 h)	(1) LA-PEG2000-NH2 adsorption on WS2 surface (2) WI formation through non-covalent bond formation between ICG (I) and WS2-PEG (3) WID formation through non-covalent bond formation between WI and DOX (D) (4) WID-M formation through physical extrusion of WID and erythrocyte vesicles (5) WID-M-FA formation trough DSPE- PEG2000-FA adsorption on WID-M membrane	Improved water stability and biocompatibility Improved photothermal properties: higher absorption at 808 nm. Zeta potential: WS2 = −16.5 mV WS2-PEG = −7.4 mV DOX = −2.8 mV ICG = −5.5 mV M = −9.6 mV WID = −14.2 mV WID-M = −24.9 mV WID-M-FA = −30.9 mV	Long et al. [40]
N-WS2	-	N-WS2 formation by hydrothermal reaction	Improved stability in water and biocompatibility Improved absorption in the NIR region	Liu et al. [38]
WS2-IO-MS-PEG/DOX	DOX Drug loading: 13.5% (WS2-IO-MS-PEG: DOX weight ratio: n/s, pH 8) Drug release: 10.6% (pH 7.4; 5 h) 12.5% (pH 7.4; 24 h) 11.9% (pH 5.5; 5 h) 42.5% (pH 5.5; 24 h)	(1) Covalent bond formation between DMSA-modified IONPs and WS2 (2) Covalent bond formation between SiO2 and WS2-IO (3) PEG adsorption on WS2-IO-MS surface (4) DOX adsorption on WS2-IO-MS-PEG	Improved stability in water, saline and serum	Yang et al. [32]
PEG-WS2:Gd3+	-	(1) Covalent bond formation between WS2 and Gd3+ (2) C18PMH-PEG adsorption on WS2:Gd3+ surface	Improved stability in water, saline, PBS, 1640-Medium and FBS	Cheng et al. [39]
WS2-lipid	DOX Drug loading: 87% (WS2-lipid: DOX weight ratio 1:5, pH 7.4) Drug release: 13.3% (pH 7.4; 8 h) 32.1% (pH 7.4; 168 h) 22.3% (pH 5; 8 h) 43.2% (pH 5;168 h)	(1) Liposomes formation by membrane hydration methods (2) WS2-lipid formation by liposomes adsorption on WS2 surface (3) DOX adsorption on WS2-lipid	Improved stability in distilled water, PBS and RPMI-1640 medium containing 10% fetal bovine serum Zeta potential: WS2 = −42.9 mV Liposome = −25.87 mV WS2-lipid = −33.77 mV	Xie et al. [35]
mWS2-lipid	DOX Drug loading: 179.53% (WS2-lipid: DOX weight ratio 1:2) Drug release: 12.5% (pH 7.4; 8 h) 33% (pH 7.4; 168 h) 23% (pH 5; 8 h) 48% (pH 5;168 h)	(1) mWS2 formation by solvothermal reaction (2) Liposome adsorption on mWS2 (3) DOX adsorption on mWS2-lipid	Improved stability in water, PBS and DMEM Zeta potential: WS2 = −42.96 mV mWS2 = −46.25 mV mWS2-lipid = −24.74 mV Lipid = −24.66 mV Superparamagnetic properties	Xie et al. [36]
WS2/Au-lipid-DOX	DOX Drug loading: 84.54% (WS2/Au-lipid: DOX weight ratio n/s, pH 7.4) Drug release: 12.5% (pH 7.4; 8 h) 24% (pH 7.4; 168 h) 17% (pH 5; 8 h) 42.5% (pH 5; 168 h)	(1) WS2/Au was synthesized using Na3C6H5O7 reduction method (2) WS2/Au-lipid by magnetic stirring (3) DOX adsorption on WS2/Au-lipid	Improved stability in water, PBS and DMEM Zeta potential: WS2 = −42.19 mV WS2/Au = −40.61 mV WS2/Au-lipid = −44.72 mV Lipid = −36.79 mV	Li et al. [37]

BSA, bovine serum albumin; C18PMH-PEG, poly(ethylene glycol)-grafted poly(maleic anhydride-alt-1octadecene); DMEM, dulbecco’s modified eagle’s medium; DOX, doxorubicin; DSPE-PEG2000-FA,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000]; FBS, fetal bovine serum; ICG, Indocyanine greenIndocyanine green; IO, iron oxide; MS, mesoporous; PBS, phosphate buffered saline; PEG, polyethylene glycol; PVP, poly(vinylpyrrolidone).

Table 1. Tungsten disulfide functionalization and drug conjugation strategies.

Material Produced	Drug Conjugation	Method	Physicochemical Properties’ Alteration	Reference
WS2-BSA	-	(1) WS2 production by ultrasound-assisted liquid phase exfoliation (2) BSA electrostatic adsorption on the surface by stirring	Improved stability in saline, water and DMEM Zeta potential: BSA = −27.4 mV WS2 = 20.8 mV WS2-BSA = −19.8 mV	Yi et al. [33]
Fe (III)-WS2-PVP	DOX Drug loading: 41% (Fe (III)-WS2 -PVP: DOX weight ratio: n/s) Drug release: 7.5 (pH 7.4; 6 h) 12.7% (pH 7.4; 48 h) 37% (pH 6.0; 6 h) 48.2% (pH 6.0; 48 h)	(1) Fe (III)-WS2-PVP formation by hydrothermal reaction (2) DOX adsorption on Fe (III)-WS2-PVP	Increased biodegradation rate Improved stability in saline, water and DMEM	Wu et al. [34]
WS2-PEG	-	Covalent bond formation between WS2 and PEG	Improved stability in saline Improved photothermal properties: broad NIR absorption band 700–1000 nm	Cheng et al. [29]
WID-M-FA	DOX loading: 24.2% (WID-M-FA: DOX weight ratio: n/s) DOX release: 12% (pH 7.4; 20 h) 30.9% (pH 7.4; 72 h) 24.2% (pH 5.2; 20 h) 56.4% (pH 5.2; 72 h) ICG loading: 22.9% (WID-M-FA: ICG weight ratio: n/s) ICG release: 14.3% (pH 7.4; 20 h) 19.5% (pH 7.4; 72 h) 27.1% (pH 5.2; 20 h) 35.2% (pH 5.2; 72 h)	(1) LA-PEG2000-NH2 adsorption on WS2 surface (2) WI formation through non-covalent bond formation between ICG (I) and WS2-PEG (3) WID formation through non-covalent bond formation between WI and DOX (D) (4) WID-M formation through physical extrusion of WID and erythrocyte vesicles (5) WID-M-FA formation trough DSPE- PEG2000-FA adsorption on WID-M membrane	Improved water stability and biocompatibility Improved photothermal properties: higher absorption at 808 nm. Zeta potential: WS2 = −16.5 mV WS2-PEG = −7.4 mV DOX = −2.8 mV ICG = −5.5 mV M = −9.6 mV WID = −14.2 mV WID-M = −24.9 mV WID-M-FA = −30.9 mV	Long et al. [40]
N-WS2	-	N-WS2 formation by hydrothermal reaction	Improved stability in water and biocompatibility Improved absorption in the NIR region	Liu et al. [38]
WS2-IO-MS-PEG/DOX	DOX Drug loading: 13.5% (WS2-IO-MS-PEG: DOX weight ratio: n/s, pH 8) Drug release: 10.6% (pH 7.4; 5 h) 12.5% (pH 7.4; 24 h) 11.9% (pH 5.5; 5 h) 42.5% (pH 5.5; 24 h)	(1) Covalent bond formation between DMSA-modified IONPs and WS2 (2) Covalent bond formation between SiO2 and WS2-IO (3) PEG adsorption on WS2-IO-MS surface (4) DOX adsorption on WS2-IO-MS-PEG	Improved stability in water, saline and serum	Yang et al. [32]
PEG-WS2:Gd3+	-	(1) Covalent bond formation between WS2 and Gd3+ (2) C18PMH-PEG adsorption on WS2:Gd3+ surface	Improved stability in water, saline, PBS, 1640-Medium and FBS	Cheng et al. [39]
WS2-lipid	DOX Drug loading: 87% (WS2-lipid: DOX weight ratio 1:5, pH 7.4) Drug release: 13.3% (pH 7.4; 8 h) 32.1% (pH 7.4; 168 h) 22.3% (pH 5; 8 h) 43.2% (pH 5;168 h)	(1) Liposomes formation by membrane hydration methods (2) WS2-lipid formation by liposomes adsorption on WS2 surface (3) DOX adsorption on WS2-lipid	Improved stability in distilled water, PBS and RPMI-1640 medium containing 10% fetal bovine serum Zeta potential: WS2 = −42.9 mV Liposome = −25.87 mV WS2-lipid = −33.77 mV	Xie et al. [35]
mWS2-lipid	DOX Drug loading: 179.53% (WS2-lipid: DOX weight ratio 1:2) Drug release: 12.5% (pH 7.4; 8 h) 33% (pH 7.4; 168 h) 23% (pH 5; 8 h) 48% (pH 5;168 h)	(1) mWS2 formation by solvothermal reaction (2) Liposome adsorption on mWS2 (3) DOX adsorption on mWS2-lipid	Improved stability in water, PBS and DMEM Zeta potential: WS2 = −42.96 mV mWS2 = −46.25 mV mWS2-lipid = −24.74 mV Lipid = −24.66 mV Superparamagnetic properties	Xie et al. [36]
WS2/Au-lipid-DOX	DOX Drug loading: 84.54% (WS2/Au-lipid: DOX weight ratio n/s, pH 7.4) Drug release: 12.5% (pH 7.4; 8 h) 24% (pH 7.4; 168 h) 17% (pH 5; 8 h) 42.5% (pH 5; 168 h)	(1) WS2/Au was synthesized using Na3C6H5O7 reduction method (2) WS2/Au-lipid by magnetic stirring (3) DOX adsorption on WS2/Au-lipid	Improved stability in water, PBS and DMEM Zeta potential: WS2 = −42.19 mV WS2/Au = −40.61 mV WS2/Au-lipid = −44.72 mV Lipid = −36.79 mV	Li et al. [37]

BSA, bovine serum albumin; C18PMH-PEG, poly(ethylene glycol)-grafted poly(maleic anhydride-alt-1octadecene); DMEM, dulbecco’s modified eagle’s medium; DOX, doxorubicin; DSPE-PEG2000-FA,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000]; FBS, fetal bovine serum; ICG, Indocyanine greenIndocyanine green; IO, iron oxide; MS, mesoporous; PBS, phosphate buffered saline; PEG, polyethylene glycol; PVP, poly(vinylpyrrolidone).

3. Drug Loading and Release

The large surface area of WS₂-based materials makes them great platforms for drug delivery and PTT. Their surface can be modified not only to improve biocompatibility and water stability, but also to facilitate and improve drug loading. Physical adsorption is a common approach, where the drugs are adsorbed through non-covalent interactions, such as hydrogen bonds or Van der Waals forces. Drugs can also be conjugated via covalent bonding, involving functional groups at the 2DnMat surface [41]. Drug release is usually triggered by changes in temperature, pH or light exposure. This allows release on the target site, enhancing the therapy efficacy and minimizing the size effects [41]. PTT involves the use of NIR light, which triggers drug release by temperature increase. The pH-mediated target release is also a commonly used strategy. Usually, the tumor exhibits an acidic pH, and so the drug carriers can be adapted in order to release in acidic environments [41].

The combination of WS₂ nanoparticles and drugs, such as DOX, has been studied to optimize loading and optimum release conditions. Fe(III)-WS₂-PVP nanocapsules have high DOX loading capacity and, because of the continuous redox reactions between Fe(III) and WS₂, Fe²⁺ triggers the biodegradation of the Fe(III)-WS₂ -PVP nanocapsules and the consequent release of DOX. The optimal drug loading capacity of these nanocapsules was found to be 41%, with concentrations of Fe(III)-WS₂-PVP and DOX of 500 µg mL⁻¹. The drug release profiles demonstrated the pH dependence and the increase in the efficacy of the Fe(III)-WS₂ -PVP in comparison to WS₂-PVP. After 48 h, the drug release was 12.7% at pH 7.4 and 48.2% at pH 6 [34]. Long et al. [40] investigated the possibility of using membrane systems for surface modifications to improve the drug delivery and photothermal capacity of the NPs (Figure 1a). WID-M-FA was produced first by the adsorption of DOX and ICG on the WS₂-PEG surface and then by the coating with DSPE-PEG₂₀₀₀-FA-modified erythrocyte membrane. These nanoparticles have drug loadings of 22.9% and 24.2% for ICG and DOX, respectively. After 72 h, at pH 7.4, 30.9% DOX and 19.5% ICG were released from the WID-M-FA, which represents a decrease compared to the release of the WID without coating (44.3% DOX and 35.2% ICG). For a pH of 5.8, 70.9% DOX and 38.6% ICG were released from the WID NPs, and 56.4% DOX and 35.2% ICG after the coating with the erythrocyte membrane (Figure 1b). These results suggest that the erythrocyte membrane reduces the drug release, improving the accumulation of the drug at the tumor site. With NIR irradiation (808 nm laser, 1 W cm⁻²) for 30 min, the release of DOX was 30% at pH 5.2, which is 3 times higher than that of the control.

The loading of DOX at the surface of WS₂-IO-MS-PEG/DOX reached a maximum of 13.5% for the 0.5 mg mL⁻¹ concentration. The drug release showed pH dependence, with the acidic medium being the most favorable, with 42.5% release at pH 5.5 after 24 h, versus 12.5% at pH 7.4 (Figure 1c). An NIR-triggered DOX release was also tested in PBS at pH 5.5 and 7.4, with 808 nm laser irradiation (5 min, 0.8 W cm⁻²). For pH 5.5, a release of 43% was reached, while at pH 7.4, a release of about 25% was obtained at the same time point [32]. In a different study, the DOX loading rate at the surface of WS₂ was 95%, while for WS₂-lipid, WS₂ coated with liposomes by electrostatic adsorption, it decreased to 87% at pH 7.4. The biggest difference was found regarding the stability of both dispersions. While WS₂-DOX showed strong aggregation, WS₂-lipid-DOX was well dispersed under physiological conditions. The DOX cumulative release from WS₂-lipid nanoparticles, after 168 h at pH 7.4, was 32.1%, while at pH 5 it was 43.2% [35]. DOX was also conjugated with lipid-modified WS₂ loaded with a magnetic nanoparticle (m-WS₂), iron oxide, by a solvothermal method. The IONPs have high targeting ability due to their magnetic targeting properties, which can be used for the targeted release of the drug at the tumor site. The drug loading was about 95% for WS₂ alone. After magnetization, drug loading increased about 2 times to 180% (Figure 1d). After lipid modification, drug loading stayed the same. This indicates that the liposomes did not affect the loading of DOX, which was also proven by fluorescence scanning, suggesting that the DOX was loaded on the m-WS₂ surface and, therefore, was not adsorbed onto the lipids. The drug release curves suggest a pH dependence. After 168 h, about 24% of DOX was released at pH 7.4 and 42.5% at pH 5 for the m-WS₂-lipid NPs. Also, the m-WS₂-lipid-DOX and m-WS₂-DOX release curves were similar, suggesting that the lipid modification did not influence the drug release [36]. DOX was also loaded in WS₂/Au-lipid nanoparticles. WS₂/Au was produced using the sodium citrate reduction method, where WS₂ is magnetically stirred for 30 min with a solution of sodium citrate (C₆H₅Na₃O₇) and chloroauric acid (HAuCl₄). Then, liposomes were produced through thin film dispersion, and were mixed with the previous solution, resulting in WS₂/Au-lipid nanoparticles. Their loading with DOX was tested and reached a loading rate of about 85%, which is slightly worse than WS₂ and WS₂/Au, both of which were about 90%, similar to WS₂-lipid, itself about 2 times worse than m-WS₂-lipid [35,36]. The difference resides in the stability that the lipid modification provides, having better stability than the other conjugates. Drug release was about 20% higher in an acidic environment (pH 5.0) than in a neutral one (pH 7.4). At pH 5.0, DOX release from WS₂/Au was 48%, while DOX release from WS₂/Au-lipid was 42%. This indicates that the lipid modification reduced the DOX release [37].

4. In Vitro Biocompatibility Studies

A biocompatible nanomaterial should be able to interact with cells or organisms without causing them harm [42]. Therefore, it is important to determine parameters such as material size, structural morphology, hydrophilicity, dispersibility in water, surface chemistry, and concentration, which are directly related to their biocompatibility. In order to access materials’ biocompatibility, in vitro and in vivo studies should be performed before their use in biomedicine [8].

WS₂ biocompatibility has been explored in several studies.

Table 2. In vitro biocompatibility studies for tungsten disulfide-based materials.

2D Material	Particle Size (nm)	Culture Conditions and Cell Viability	2DnMat Location	Additional Outcomes	Reference
WS2-BSA	150–200	WS2-BSA (HeLa1, 2, 24, 36, 48 h incubation) >80% (6.25 ppm) >70% (12.5 ppm) >70% (25 ppm) >80% (50 ppm) Concentrations tested: 6.25–50 ppm	-	-	Yi et al. [33]
Fe (III)-WS2-PVP	108	Fe (III)-WS2-PVP (HT29, 24 h incubation) >80% (100 µg mL−1)	-	-	Wu et al. [34]
WS2-PEG	94	WS2-PEG (4T1, 24 h incubation) >90% (0.1 mg mL−1) WS2-PEG (HeLa, 24 h incubation) >90% (0.1 mg mL−1) WS2-PEG (293T, 24 h incubation) >90% (0.1 mg mL−1) Concentrations tested: 0.006–0.1 mg mL−1	-	LDH release (4T1, HeLa, 293T, 24 h incubation): 0% (100 μg mL−1) ROS generation (4T1, HeLa, 293T, 24 h incubation): 7% (50, 100 μg mL−1) Concentrations tested: 13–100 μg mL−1	Cheng et al. [29]
WS2-IO-MS-PEG/DOX	90	WS2-IO-MS-PEG/DOX (4T1, 24 h incubation) >70% (0.781 µg mL−1) >60% (1.563 µg mL−1) >60% (3.125 µg mL−1) >60% (6.25 µg mL−1) >30% (12.5 µg mL−1) >20% (25 µg mL−1) Concentrations tested:0.781–0.25 µg mL−1	-	-	Yang et al. [32]
WID-M-FA	162	WI-M-FA (HeLa, 24 h incubation) 89% (12.5 µg mL−1) 89% (25 µg mL−1) 75.9% (50 µg mL−1) 68.5% (100 µg mL−1) Concentrations tested: 12.5–100 µg mL−1	Observed in lysosomes (4 h incubation)	1% hemolysis (100 μg mL−1, 4 h incubation with RBCs)	Long et al. [40]
N-WS2	150	N-WS2 (Hela, 24 h incubation) >90% (15 µg mL−1) >88% (120 µg mL−1) N-WS2 (MDA-MB-231, 24 h incubation) >90% (15 µg mL−1) >90% (120 µg mL−1) N-WS2 (HepG2, 24 h incubation) >90% (15 µg mL−1) >90% (120 µg mL−1) Concentrations tested: 0.469–120 µg mL−1	-	-	Liu et al. [38]
PEG-WS2:Gd3+	90–100	PEG-WS2:Gd3+ (4T1, 24, 48 h incubation) >90% (50 µg mL−1) Concentrations tested: 1.56–100 µg mL−1	-	-	Cheng et al. [39]
WS2-lipid-DOX	222.58	WS2-lipid (MCF-7, 24 h incubation) >90% (100 µg mL−1) Concentrations tested: 2.5–200 µg mL−1 WS2-lipid-DOX (MCF-7, 24 h incubation) >80% (1, 2.5, 5 µg mL−1) >40% (12. 5 µg mL−1) >30% (25 µg mL−1) >20% (50 µg mL−1) Concentrations tested: 1–50 µg mL−1	Observed in cytoplasm (MCF-7, 4 h incubation)	-	Xie et al. [35]
mWS2-lipid	318.07	mWS2-lipid (MCF-7, 4 h incubation) >90% Concentrations tested: 1–50 µg mL−1 mWS2-lipid-DOX (MCF-7, 4 h incubation) 50% (2.5 µg mL−1) 34% (5 µg mL−1) 22% (12.5 µg mL−1) 17% (25 µg mL−1) 14% (50 µg mL−1) Concentrations tested: 2.5–50 µg mL−1	Observed in cytoplasm (MCF-7, 24 h incubation)	-	Xie et al. [36]
WS2/Au-lipid-DOX	196	WS2/Au-lipid (MCF-7, 4 h incubation) >90% (50 µg mL−1) Concentrations tested: 2–50 µg mL−1 WS2/Au-lipid-DOX (MCF-7, 4 h incubation) >68% (2.5 µg mL−1) >50% (5, 12.5 µg mL−1) >41% (25 µg mL−1) >33% (50 µg mL−1) Concentrations tested: 2.5–50 µg mL−1	Observed in cytoplasm (MCF-7, 4 h incubation)	-	Li et al. [37]

BSA, bovine serum albumin; DOX, doxorubicin; IO, iron oxide; LDH, lactate dehydrogenase; MS, mesoporous; PEG, polyethylene glycol; PVP, poly(vinylpyrrolidone); RBCs, red blood cells; ROS, reactive oxygen species.

compiles the current literature on WS₂-based materials tested with cell lines and the impacts of the different materials on cell viability and particle internalization, among others.

WS₂-BSA nanosheets were tested for cytotoxicity following the MTT assay with HeLa cells, with incubation times of 2, 24, 36, and 48 h. The overall cell viability was found to always be above 70% for concentrations between 6.25 ppm and 50 ppm. For a concentration of 50 ppm, the cell survival was above 80% [33]. The cytotoxicity of WS₂-PEG was also evaluated through this assay, using 4T1, HeLa, and 239T cells. After 24 h, no signs of toxicity were observed, even for concentrations as high as 0.1 mg mL⁻¹, with cell viability remaining higher than 90%. A lactate dehydrogenase (LDH) study was also performed to examine any potential damage to the cells caused by WS₂-PEG, and the results show that the level of LDH release was normal, indicating that the nanoparticles caused no damage to the cells [29].

WI NPs were produced by mixing WS₂-PEG and Indocyanine green (ICG). From WI NPs, WI-M-FA NPs were produced by the sonication of an erythrocyte vesicle (M) with WI NPs, resulting in WI-M NPs. Finally, the addition of DSPE-PEG₂₀₀₀-FA to this dispersion yielded WI-M-FA NPs. Through the MTT assay, the cytotoxicity of these NPs was studied with concentrations ranging from 12.5 to 100 µg mL⁻¹ incubated with HeLa cells for 24 h. The cell viability after incubation with WI-M-FA for 24 h was above 90%, for all concentrations, in contrast to WI, which killed the cells in a concentration-dependent manner. The cell viability of these NPs decreased with the increase in concentrations, from 89% (12.5 µg mL⁻¹) to 68.5% (100 µg mL⁻¹) [40]. The uptake by the macrophages was also studied by incubating RAW264.7 macrophages with WID-M-FA and WID NPs. Through the CLSM images, it is possible to observe that the intracellular fluorescence signal of the WID NPs increased by more 70% compared to the WID-M-FA NPs signal, which indicates that the addition of the erythrocyte membrane reduced the uptake of the NPs by the macrophages, which can improve the uptake of the nanocarrier in the tumor, by prolonging the blood circulation time of the NPs. A live/dead staining revealed that the combination of WID-M-FA with NIR laser irradiation (808 nm, 1 W cm⁻², 5 min) killed more than 70% of the tumor cells (Figure 2a) [40].

WS₂ nanosheets functionalized with iron oxide nanoparticles, coated with mesoporous silica, and then functionalized with polyethylene glycol (PEG), WS₂-IO-MS-PEG, were produced to improve the drug loading and release capacity of WS₂ for cancer therapy applications. According to the MTT assay, these nanomaterials showed no sign of toxicity towards different types of cells, namely, 4T1 murine breast cancer cells, HeLa human cervical cancer cells, and 293T human embryonic kidney cells, even for concentrations as high as 200 µg mL⁻¹ (Figure 2b). The conjugation of WS₂-IO-MS-PEG and DOX was also tested for impact on cell viability. For a concentration of 25 µg mL⁻¹, the viability of 4T1 cells was 20% after 24 h [32]. WS₂/Au-lipid, gold-doped WS₂ nanosheets modified with lipids, showed no toxicity when incubated with MCF-7 cells, but after 4 h cell viabilities above 90% were obtained. The same test was performed for WS₂-lipid and m-WS₂-lipid (Figure 2c), with similar results being obtained. However, after DOX loading, the conjugate (WS₂/Au-lipid-DOX) decreased cell viability up to a minimum of 33%, at a concentration of 50 µg mL⁻¹ [37]. The drug-loaded complex of WS₂-lipid-DOX and m-WS₂-lipid-DOX also caused concentration-dependent cell viability decreases, reaching even lower cell viability values of 20% and 14%, respectively (50 µg mL⁻¹) [35,36,37].

Human colorectal carcinoma (HT29) cells were incubated for 24 h with Fe(III)-derived WS₂ nanosheets coated with PVP, Fe(III)-WS₂-PVP nanocapsules (100 µg mL⁻¹), resulting in a cell viability of 85.6%. Furthermore, the potential influence of the degradation products on cell viability was also studied. Fe(III)@WS₂-PVP nanocapsules were degraded in PBS for 7 days, and the viability of HT29 cells was not influenced (98.3%) [34].

For N-WS₂, an ammonia-intercalated WS₂ ultrathin nanosheet, the cell viability was higher than 90%, after 24 h of incubation with HeLa cells, MDA-MB-231 cells, and HepG2 cells, at concentrations up to 120 µg mL⁻¹. Also, there was no sign of cell proliferation decrease [38].

The cytotoxicity of PEG-WS₂:Gd³⁺ was also studied based on the MTT assay in 4T1 murine breast cancer cells incubated with different concentrations for 24 h and 48 h. It was observed that cell viability was above 90%, for concentrations as high as 50 µg mL⁻¹ [39].

Most WS₂-based nanoparticles had cell viability in the range of 80–90%, which is above the limit toxicity reported in ISO10993-5:2009(E) [43]. These NPs did not show a concentration-dependent viability, except for WI-M-FA, which showed a decrease in cell viability as the concentration increased. Regarding nanoparticle–drug conjugates, cell viability follows a clear concentration dependence. The cell viability of MCF-7 incubated with m-WS₂-lipid-DOX decreased from 50% to 15% when increasing concentrations from 2.5 µg mL⁻¹ to 50 µg mL⁻¹. When the cells were incubated with WS₂-Lipid DOX, the cell viability decreased from 100% to 20%, with concentrations of 1 µg mL⁻¹ and 50 µg mL⁻¹. WS₂-IO-MS-PEG/DOX reached a 20% viability for only 25 µg mL⁻¹, while WS₂-lipid, m-WS₂-lipid and WS₂/Au/lipid reached 30%, 17% and 41%, respectively, at the same concentration.

5. In Vivo Biocompatibility Studies

Biological models are often used to test the biocompatibility of new nanomaterials.

Table 3. In vivo biocompatibility studies for tungsten disulfide-based materials.

2D Material	Animal Model	Animal Survival	Treatment Conditions	Main Results	Reference
WS2-BSA	Zebrafish embryos	120 h: >80%	Zebrafish embryos were incubated in E3 medium with WS2-BSA Concentrations tested: 0–50 ppm	120 h: Hatching rate: 50% (0 ppm) 27.5% (6.25 ppm) 32.5% (12.5 ppm) 45% (25 ppm) 48.5% (50 ppm)	Yi et al. [33]
Fe (III)- WS2-PVP	HT29 colorectal carcinoma bearing KM mice	-	i.v. administration (100 µg mL−1) Heart: 1 day—0.03 µg g−1 7 days—0.11 µg g−1 Liver: 1 day—0.52 µg g−1 7 days—0.25 µg g−1 Spleen: 1 day—0.77 µg g−1 7 days—0.18 µg g−1 Lung: 1 day—0.22 µg g−1 7 days—0.07 µg g−1 Kidney: 1 day—0.07 µg g−1 7 days—0.02 µg g−1	28 days: no changes on heart, liver, spleen, lung and kidney tissues	Wu et al. [34]
WS2-PEG	4T1 tumor-bearing Balb/C mice	-	i.t administration (2 mg kg−1, 30 min) i.v. administration (20 mg kg−1, 24 h) No changes on body weight 28 days: serum biochemistry markers on normal variation ranges (ALT, ALP, AST, BUN levels, WBC, RBC, HCT, Hgb, MCV, MCH, MCHC, and platelets)	45 days: no changes on liver, spleen, kidney, heart and lung	Cheng et al. [29]
WS2-IO-MS-PEG/DOX	4T1 tumor bearing Balb/C mice	-	i.v. administration (WS2 = 8.4 mg kg−1, DOX = 7 mg kg−1, 24 h) Circulation half-life = 4.77 h Heart: 24 h—1.18% ID g−1 Liver: 24 h—25.88% ID g−1 Spleen: 24 h—38.82% ID g−1 Lung: 24 h—5.29% ID g−1 Kidney: 24 h—4.71% ID g−1 Stomach: 24 h—2.35% ID g−1 Intestine: 24 h—1.76% ID g−1 Muscle: 24 h—1.76% ID g−1 Tumor: 24 h—8.24% ID g−1	24 h: high accumulation on liver spleen and tumor	Yang et al. [32]
WID-M-FA	HeLA tumor-bearing Balb/C mice	-	i.v. administration (DOX = 2 mg kg−1, ICG = 5 mg kg−1, 24 h) 18 days: serum biochemistry markers on normal variation ranges (RBC, RDW, MCHC, MCV, PLT, WBC, ALT, AST, CRE, BUN)	18 days: no changes on liver, spleen, kidney, heart and lung	Long et al. [40]
N-WS2	HeLa tumor-bearing female NOD/SCID mice	-	i.t administration (1.2 mg mL−1, 40 µL, 24 h) 16 days: serum biochemistry markers on normal variation ranges (WBC, RBC, Hb, HCT, PLT, MCV, MCHC, MCH)	48 h: no changes in liver and lung tissues	Liu et al. [38]
PEG-WS2:Gd3+	4T1 tumor-bearing Balb/C mice	-	i.v. administration (2 mg mL−1, 200 µL, 24 h) Tumor: 24 h: 11.8% ID g−1 Liver: 24 h: 50.9% ID g−1 Spleen: 24 h: 94.5% ID g−1	T1-MR signal (a.u.): 200 (before i.v. injection) 500 (after i.v. injection)	Cheng et al. [39]

ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BSA, bovine serum albumin; BUN, blood urea nitrogen; CRE, creatinine; DOX, doxorubicin; HCT, hematocrit; Hgb, hemoglobin; ICG, indocyanine green; ID, injected dose; IO, iron oxide; i.t., intratumoral; i.v., intravenous; KM, Kunming mouse; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; MS, mesoporous; NOD/SCID, nonobese diabetic/severe combined immunodeficiency; PEG, polyethylene glycol; PLT, platelet; PVP, poly(vinylpyrrolidone); RBC, red blood cells; RDW, red blood cell distribution width; WBC, white blood cells.

presents current studies on tungsten disulfide-based materials’ in vivo biocompatibility.

Zebrafish embryos were used to study the influence of WS₂-BSA nanosheets on the survival rate of the fertilized embryos. The survival rate for different concentrations was found to be above 80% for incubation times as high as 120 h, and followed the same trend as the control group. This indicates that the WS₂-BSA nanosheets presented low toxicity. With time, the embryos started to hatch, and the hatching rate was affected by increasing concentrations of WS₂-BSA NPs with which they were incubated. WS₂-BSA NPs delayed the hatching time of the embryo groups in the two first incubation stages, 72 h and 96 h. After 120 h, the incubation rate of the embryos incubated with WS₂-BSA started to catch up with the control group, suggesting that the delay in hatching caused by the NPs was overcome with time, especially for high concentrations [33].

Fe(III)-WS₂-PVP was intravenously administrated to HT29 colorectal carcinoma-bearing KM mice, and their biodegradation profiles were studied by inductively coupled plasma atomic emission spectroscopy (ICP-AES). It was found that there was an accumulation of the W element in the liver and spleen, organs responsible for the excretion of particles from the blood, on the first day, which ended up decreasing over the course of 28 days (Figure 3a). The overall distribution of W in the organs was very low after 7 days of administration. From the hematoxylin and eosin (H&E) staining tests, there was no sign of changes on the major organs during the 28 days due to the fast in vivo biodegradation, proving the good biocompatibility of these nanomaterials (Figure 3b) [34]. For WID-M-FA NPs, a dose of 2 mg kg⁻¹ DOX and 5 mg kg⁻¹ ICG was administered intravenously to HeLa tumor-bearing Balb/C mice. After 24 h, the results reveal that the nanoparticles did not affect the blood parameters tested (Table 3), in contrast with what occurred with just DOX administration. These results suggest that the modifications reduce DOX leakage in the blood circulation. Furthermore, the side effects of the WID@M-FA NPs on the liver and kidney were studied. Histological analyses of kidney, lung, spleen, liver and heart showed no effects of the NPs on renal and hepatic functions, in contrast with the DOX control injection, which induced hepatotoxicity and nephrotoxicity [40]. HeLa tumor-bearing female NOD/SCID mice intratumorously administered with 1.2 mg mL⁻¹ N-WS₂ also displayed normal blood parameters (Figure 3c) [38].

To evaluate the WS₂-PEG toxicological profile, a serum biochemistry assay and complete blood tests were performed on Balb/C mice intravenously injected (2 mg kg⁻¹) or intratumorously injected (2 mg kg⁻¹) with WS₂-PEG, after 1, 14, and 28 days. All parameters were within standard limits, with WS₂-PEG NPs causing no toxicity to the animals [29]. Similar studies were conducted with WS₂-IO-MS-PEG/DOX, a WS₂ nanosheet functionalized with iron oxide, coated with mesoporous silica, and functionalized with PEG. Doses of 8.4 mg kg⁻¹ WS₂ and 7 mg kg⁻¹ DOX were intravenously injected into tumor-bearing Balb/C mice. After 24 h, the inductively-coupled plasma atomic emission spectroscopy (ICO-AES) results showed high retention in the liver and spleen. Apart from this, a relativity high amount of W was found in the tumor after 24 h. This high tumor retention is directly related to the long blood circulation half-life, 4.77 h, which enhances the permeation and retention of the nanomaterials in the tumors. The tumor uptake was 8.24% of the injected dose per gram of tissue (% ID/g) [32]. The same trend can be seen in the analysis of PEG-WS₂:Gd³⁺, where after the intravenous administration of 2 mg mL⁻¹ to 4T1 tumor-bearing Balb/C mice, high accumulations in the liver and spleen, followed by increases in tumor concentration, were found. In this case, the tumor uptake, measured by ICP, was 11.8% ID/g [39].

Several studies report histology results that show no signs of toxicity in the liver, spleen, kidney, heart and lungs after 48 h to 45 days of WS₂-based materials administration, even for doses as high as 20 mg kg⁻¹. However, other studies report a high accumulation of nanoparticles, especially in the spleen, liver, and tumor. Accumulation in the spleen and liver deserves further study, since it has not been discussed in any of the works abovementioned. However, in all cases, no toxicity has been found, and tumor accumulation was identified.

6. In Vitro Photothermal Therapy Studies

Different variables must be considered when doing PTT studies, such as the light source type and features, time of irradiation, and concentration of the photothermal agent, among others.

Table 4. In vitro photothermal therapy studies using tungsten disulfide-based materials.

2D Material	Irradiation Method	Energy (W cm−2)	Time of Irradiation (min)	Culture Conditions and Cell Viability	Reference
WS2-BSA	Laser (808 nm)	1.5	5	Tmax = 44.5 °C (1.5 W cm−2, 5 min) WS2-BSA (HeLa, 24 h incubation) 65% (6.25, 12.5 ppm) 40% (25 ppm) 35% (50 ppm)	Yi et al. [33]
Fe (III)-WS2-PVP	Laser (808 nm)	1	5	Tmax = 46 °C (1 W cm−2, 5 min) DOX-Fe (III)-WS2-PVP (HT29, 24 h incubation) 5.2% (250 µg mL−1)	Wu et al. [34]
WS2-PEG	Laser (808 nm)	0.1, 0.3, 0.5, 0.8	5	WS2-PEG (4T1, 6 h incubation) 100% (0.1 W cm−2, 0.1 mg mL−1) 72.7% (0.3 W cm−2, 0.1 mg mL−1) 45.5% (0.5 W cm−2, 0.1 mg mL−1) 7.3% (0.8 W cm−2, 0.1 mg mL−1)	Cheng et al. [29]
WS2-IO-MS-PEG/DOX	Laser (808 nm)	0.1, 0.3, 0.8	20	WS2-IO-MS-PEG/DOX (4T1, 24 h incubation) 41.3% (0.1 W cm−2, 50 µg mL−1 DOX) 16% (0.3 W cm−2, 50 µg mL−1 DOX) 14.7% (0.8 W cm−2, 50 µg mL−1 DOX)	Yang et al. [32]
WID-M-FA	Laser (808 nm)	1	5	Tmax = 60 °C (1 W cm−2, 5 min) WID-M-FA (HeLa, 24 h incubation) 18.5% (1 µg mL−1 DOX and 10 µg mL−1 ICG)	Long et al. [40]
N-WS2	Laser (808 nm)	0.3, 0.45, 0.6, 0.75	10	Tmax = 50 °C (0.6 W cm−2, 4 min) N- WS2 (HeLa, 6 h incubation) 89.4% (0.3 W cm−2, 120 µg mL−1) 71.2% (0.45 W cm−2, 120 µg mL−1) 40.4% (0.6 W cm−2, 120 µg mL−1) 30.8% (0.75 W cm−2, 120 µg mL−1) Concentrations tested: 15–120 µg mL−1	Liu et al. [38]
PEG-WS2:Gd3+	Laser (808 nm)	0.8	5	PEG-WS2:Gd3+ (4T1,12 h incubation) 75.5% (6.25 µg mL−1) 51.1% (12.5 µg mL−1) 28.9% (25 µg mL−1) 4.4% (50 µg mL−1)	Cheng et al. [39]
WS2-lipid	Laser (808 nm)	2	10	Tmax = 60 °C (2 W cm−2, 10 min) WS2-lipid (MCF-7, 24 h incubation). 78% (25 µg mL−1) 43% (50 µg mL−1) 18% (100 µg mL−1) 9% (200 µg mL−1) WS2-lipid-DOX (MCF-7, 24 h incubation) 20% (50 µg mL−1 DOX)	Xie et al. [35]
mWS2-lipid	Laser (808 nm)	2	10	Tmax = 68.1 °C (1.5 W cm−2, 10 min) mWS2-lipid (MCF-7, 24 h incubation). 77% (12.5 µg mL−1) 58% (25 µg mL−1) 30% (50 µg mL−1) 16% (100 µg mL−1) mWS2-lipid-DOX (MCF-7, 24 h incubation) 27% (50 μg mL−1 DOX)	Xie et al. [36]
WS2/Au-lipid-DOX	Laser (808 nm)	2	10	Tmax = 75 °C (1.5 W cm−2, 10 min) WS2/Au-lipid (MCF-7, 24 h incubation). 54% (12.5 µg mL−1) 29% (25 µg mL−1) 18% (50 µg mL−1) 7% (100 µg mL−1) WS2/Au-lipid-DOX (MCF-7, 4 h incubation) 30% (12.5 μg mL−1 DOX)	Li et al. [37]

BSA, bovine serum albumin; DOX, doxorubicin; IO, iron oxide; MS, mesoporous; PEG, polyethylene glycol; PVP, poly(vinylpyrrolidone).

congregates the results of in vitro PTT studies using tungsten disulfide-based materials available.

WS_2-PEG (0.1 mg mL⁻¹) was incubated with 4T1 cells for 6 h, followed by irradiation with an 808 nm laser at irradiances of 0.1, 0.3, 0.5, 0.8 W cm⁻², for 5 min, resulting in cell viabilities of 100%, 72.7%, 45.5% and 7.3%, respectively [29]. WS₂-IO-MS-PEG/DOX (50 µg mL⁻¹ DOX), under the same laser conditions and with a time of irradiation of 20 min, showed decreased 4T1 cell viability to 41.4% (0.8 W cm⁻²), 16% (0.3 W cm⁻²), and 14.7% (0.8 W cm⁻²) [32]. N-WS₂ also caused a similar effect on HeLa cells, with cell viabilities of 89.4%, 71.2%, 40.4%, and 30.8% being obtained for irradiances of 0.3, 0.45, 0.6 and 0.75 W cm⁻². A N-WS₂ concentration of 120 µg mL⁻¹ was used, and irradiation was performed with an 808 nm laser for 10 min [38]. The temperature of the tumors incubated with N-WS₂ and irradiated with the laser increased rapidly to 50 °C within 4 min, an increment of 21 °C, and remained at 50 °C for 6 min more. PEG-WS₂:Gd³⁺ and PEGylated-WS₂, doped with the metal gadolinium (Gd³⁺), were incubated for 4 h with 4T1 cells at concentrations of 6.25, 12.5, 25 and 50 µg mL⁻¹. When irradiated with an 808 nm laser with a power of 0.8 W cm⁻² for 5 min, the cell viabilities decreased with the increase in the concentration, reaching 75.5%, 51.1%, 29.9% and 4.4%, respectively [39]. A similar trend was seen for WS₂-BSA, incubated with HeLa cells with a gradient of concentration between 6.25 and 50 ppm, where cell viability decreased from 65% to 35%. Furthermore, photothermal tests (50 ppm, 1.5 W cm⁻²) revealed a temperature increase from 26 °C to 44.5 °C, within 5 min. These tests suggest that the materials have the potential to be used for cancer PTT [33].

Studies concerning the cell viability of HT29 cells incubated with Fe(III)-doped WS₂ modified with PVP and/or DOX, Fe(III)-WS₂-PVP and DOX-Fe(III)-WS₂-PVP for PTT treatment were conducted. The viability of HT29 cells treated with Fe(III)-WS₂-PVP (250 µg mL⁻¹) and DOX-Fe(III)-WS₂-PVP (150 µg mL⁻¹ WS₂ and 100 µg mL⁻¹ DOX) decreased to 37.7% and 5.2%, respectively, upon 808 nm laser irradiation (1 W cm⁻², 5 min). As for the heating assays, the Fe(III)-WS₂-PVP nanocapsules under 808 nm laser irradiation (1 W cm⁻², 5 min) registered an increment of approximately 14.6, 24.3, and 45.0 °C for concentrations of 50,100 and 200 50 µg mL⁻¹, respectively (Figure 4a) [34].

Lipid-modified WS₂ NPs also showed the same trend when incubated with MCF-7 cells for 24 h and irradiated with an 808 nm laser (2 W cm⁻², 10 min). Post-treatment cell viabilities were of 43% and 9%, for concentrations of 50 and 200 µg mL⁻¹. After conjugation with DOX, WS₂-lipid-DOX (50 µg mL⁻¹ DOX), cell viability decreased to 20% under the same conditions (Figure 4c) [35]. Magnetic WS₂ functionalized with lipids, mWS₂-lipid, resulted in lower cell viability (30%, 50 µg mL⁻¹). However, DOX conjugation (50 µg mL⁻¹ DOX) only slightly further reduced viability in this case (27%) [36]. Gold-doped WS₂/Au-lipid was revealed to achieve higher cell viability without DOX (>90%). However, when DOX was loaded (50 µg mL⁻¹), cell viability decreased to 30% (Figure 4d) [37]. Furthermore, photothermal tests under 808 nm laser (2 W cm⁻², 10 min) irradiation increased the temperature of WS₂-lipid (200 µg mL⁻¹), mWS₂-lipid (200 µg mL⁻¹) and WS₂/Au-lipid (200 µg mL⁻¹) to 60 °C, 68.1 °C and 75 °C (Figure 4b).

WID-M-FA, PEGylated WS₂ loaded with DOX and ICG, and coated with an erythrocyte membrane, was incubated with HeLa cells at a concentration of 1 μg mL⁻¹ DOX and 10 μg mL⁻¹ ICG. After 24 h, irradiation was performed with an 808 nm laser (1 W cm⁻², 5 min), with a decrease of about 30% being obtained in cell viability. A live/dead assay revealed that about 70% of the tumor cells were killed after laser irradiation [40].

In sum, the abovementioned studies generally revealed that cell viability decreases with WS₂-based materials’ concentrations and laser power increases. Conjugation with drugs, such as DOX, can also contribute to increase in vitro anticancer effectiveness.

7. In Vivo Photothermal Therapy Studies

In vivo studies have been performed to evaluate the possibility of clinical translation of the use of WS₂-based materials for cancer PTT. The main results are summarized in

Table 5. In vivo photothermal therapy studies using tungsten disulfide-based materials.

2D Material	Energy (W cm−2)	Time (min)	Animal Model	Tumor Growth	Additional Outcomes	Reference
Fe (III)-WS2-PVP-DOX	1	5	HT29 colorectal carcinoma-bearing Balb/C mice	i.v. administration (250 µg mL−1) Tumors volume decreased during the study (15% compared to control, 28 days after irradiation)	-	Wu et al. [34]
WS2-PEG	0.8	5	4T1 tumor-bearing Balb/C mice	i.t administration (2 mg kg−1, 30 min incubation) i.v. administration (20 mg kg−1, 24 h incubation) Tumors were eliminated after 2 days of irradiation No tumor regrowth during the study (14 days after irradiation)	-	Cheng et al. [29]
WS2-IO-MS-PEG/DOX	0.55	10	4T1 tumor-bearing Balb/C mice	i.v. administration (WS2 = 8.4 mg kg−1, DOX = 7 mg kg−1, 24 h) Tumors’ volume decreased during the study (10% compared to control, 14 days after irradiation) Tumor mass decreased after 14 days	-	Yang et al. [32]
WID-M-FA	1	5	HeLa tumor-bearing Balb/C mice	i.v. administration (DOX = 2 mg kg−1, ICG = 5 mg kg−1, 24 h) Tumor was eliminated after 18 days of irradiation No changes on body weight	-	Long et al. [40]
N-WS2	0.6	10	HeLa tumor-bearing female NOD/SCID mice	i.t administration (1.2 mg mL−1, 40 µL, 0 h incubation) Tumor was eliminated after 4 days of irradiation No tumor regrowth during the study (14 days after irradiation)	No changes in body weight (14 d after irradiation) Significant damage in tumor tissue (2 d after irradiation) Serum biochemistry markers on normal ranges (6, 16 d after irradiation, WBC, RBC, Hgb, HCT, platelets, MCV, MCH, MCHC)	Liu et al. [38]
PEG-WS2:Gd3+	0.5	10	4T1 tumor-bearing Balb/C mice	i.v. administration (20 mg kg−1, 24 h) Tumor was eliminated 12 days after irradiation	-	Cheng et al. [39]
WS2-lipid	2	2	4T1 breast tumor-bearing ICR mice	i.v. administration (1 mg mL−1, 24 h)	-	Xie et al. [35]
mWS2-lipid	1.5	5	4T1 tumor-bearing Balb/C mice	i.v. administration (2 mg mL−1, 24 h) Tumors volume decreased during the study (50% compared to control, 7 days after irradiation)	No changes in body weight	Xie et al. [36]
WS2/Au-lipid	1.5	5	4T1 breast tumor-bearing female Balb/C mice	i.v. administration (DOX = 150 μg), 24 h) Tumor was eliminated after 11 days of irradiation	No changes in body weight	Li et al. [37]

.

The 4T1 tumor-bearing Balb/C mice model was administrated with 2 mg kg⁻¹ WS₂-PEG intratumorously or 20 mg kg⁻¹ intravenously. After 30 min, 5 min of NIR laser irradiation (808 nm, 0.8 W cm⁻²) was performed. In both cases, the tumor was eliminated 2 days after treatment, with no sign of regrowth after 14 days (Figure 5a) [29]. HeLa tumor-bearing female NOD/SCID mice were intratumorously injected with N-WS₂ (1.2 mg mL⁻¹, 40 µL), and tumors were eliminated 4 days after 5 min laser irradiation (808 nm, 0.6 W cm⁻²), with no changes in the liver, lung, and other organs being found [38]. Blood tests presented normal values for all parameters, except for white blood cells (WBC), which slightly decreased 6 days after the treatment, which could be related to the inflammation after the photothermal therapy, even though after 16 days, the values recovered to the normal range. HeLA tumor-bearing Balb/C mice were intravenously injected with WID-M-FA NPs, erythrocyte vesicle-containing WS₂ loaded with 2 mg kg⁻¹ DOX and 5 mg kg⁻¹ ICG. After 24 h, NIR laser irradiation (808 nm, 1 W cm⁻²) was performed for 5 min. After 18 days, the tumor was eliminated, and no body weight changes were recorded during the experiment. Biodistribution studies revealed the high tumor accumulation of WID-M-FA NPs when laser irradiation was applied (Figure 5c) [40]. For the 4T1 tumor-bearing Balb/C mice treated with PEG-WS₂:Gd³⁺ (i.v. injection: 20 mg kg⁻¹, 24 h) and irradiated with NIR light (808 nm, 0.5 W cm⁻²) for 10 min, the tumor was eliminated after 12 days [39]. Fe(III)-WS₂-PVP-DOX NPs (250 µg mL⁻¹) were intravenously injected in HT29 colorectal carcinoma-bearing Balb/C mice, followed by NIR laser irradiation (808 nm, 1 W cm⁻²) for 5 min. After 28 days, tumor volume decreased by about 15%, compared to the control group [34]. For 4T1 tumor-bearing Balb/C mice treated with WS₂-IO-MS-PEG/DOX (WS₂ = 8.4 mg kg⁻¹, DOX = 7 mg kg⁻¹, 24 h) and irradiated for 5 min with a laser (808 nm, 0.55 W cm⁻²), tumor volume decreased 10% compared to control, 14 days post-treatment (Figure 5d) [32]. The 4T1 tumor-bearing Balb/C mice model was intravenously injected with m-WS₂-lipid, followed by NIR laser irradiation (808 nm, 1.5 W cm⁻²) for 5 min every 24 h. After 7 days, tumor volume growth was reduced by 50% compared to the control group (PBS), with no changes in body weight being found. For WS₂/Au-lipid-conjugated with DOX (150 µg) and exposed to 5 min NIR irradiation (808 nm, 1.5 W cm⁻²), the tumor was eliminated after 11 days of treatment (Figure 5b). Overall, lipid-modified nanoparticles were more stable in water, allowing better permeation and retention in the tumor, and also increasing their photothermal conversion capacity. The combination of PTT with chemotherapy with DOX increased the anticancer effectiveness [35,36,37].

In sum, doses of WS₂-based materials from 1 to 20 mg kg⁻¹ have been used. The laser wavelength was 808 nm, the irradiance varied from 0.55 to 2 W cm⁻², and irradiation times ranged 2–10 min. Many studies report a substantial tumor volume reduction or even complete tumor elimination without toxicity being found. The most successful irradiation conditions were using irradiances from 0.5 to 1 W cm⁻² for 5–10 min, and material concentrations of 1.2–20 mg kg⁻¹.

8. Conclusions

For biomedical applications, WS₂ is usually produced by ultrasound-assisted liquid phase exfoliation in water. Stability in physiological conditions is improved by surface adsorption or covalent functionalization with different molecules, such as PEG, BSA, or liposomes. Surface adsorption of the anti-cancer drug doxorubicin or gold nanoparticles has been reported to increase therapeutic effects. Usually, acidic pH and NIR laser irradiation increase drug release.

WS₂-based materials revealed to be generally biocompatible in vitro and in vivo. In vitro exposure to WS₂-based nanoparticles resulted in cell viability in the range of 80–90%. Regarding nanoparticle–drug conjugates, cell viability follows a clear concentration dependence. For example, the cell viability of MCF-7 incubated with m-WS₂-lipid-doxorubicin decreased from 50% to 15% when increasing concentrations from 2.5 µg mL⁻¹ to 50 µg mL⁻¹. In vivo, a high accumulation was observed in the spleen, liver, and tumor. Accumulation in the spleen and liver deserves further study, even though histology results show no signs of toxicity in the liver, spleen, kidney, heart, and lungs after 48 h to 45 days of WS₂-based materials administration, even for doses as high as 20 mg kg⁻¹.

WS₂-based materials proved to be effective for cancer PTT both in vitro and in vivo. In vitro, cancer cell viability decreases with WS₂-based materials concentration and laser power increase, reaching values as low as 5–30%. Conjugation with drugs, such as doxorubicin, contributed to a further increase in in vitro anticancer effectiveness. In vivo, many studies report a substantial tumor volume reduction or even complete tumor elimination without toxicity being found. The most successful irradiation conditions were using irradiances from 0.5 to 1 W cm⁻² for 5–10 min, and material concentrations of 1.2–20 mg kg⁻¹.

Despite the promising results obtained, WS₂-based materials’ long-term toxicity effects and biodegradation still need to be studied before clinical trials can be outlined. There is also a lack of understanding of the detailed interaction of WS₂-based materials with normal and cancer cells, for example, studying by which mechanisms they are internalized. The impacts of those materials on the immune system are also a relevant point to be assessed. To assure safety and to comply with the requirements necessary for clinical translation, a deep study of the production methods and standardization will be required, as well as the development of suitable sterilization and preservation methods for WS₂-based biomaterials. Despite the previous findings, these new exciting nanomaterials hold a great potential to be used for cancer PTT.