Titanium Dioxide Nanoparticles: Prospects and Applications in Medicine

Metallic and metal oxide nanoparticles (NPs), including titanium dioxide NPs, among polymeric NPs, liposomes, micelles, quantum dots, dendrimers, or fullerenes, are becoming more and more important due to their potential use in novel medical therapies. Titanium dioxide (titanium(IV) oxide, titania, TiO2) is an inorganic compound that owes its recent rise in scientific interest to photoactivity. After the illumination in aqueous media with UV light, TiO2 produces an array of reactive oxygen species (ROS). The capability to produce ROS and thus induce cell death has found application in the photodynamic therapy (PDT) for the treatment of a wide range of maladies, from psoriasis to cancer. Titanium dioxide NPs were studied as photosensitizing agents in the treatment of malignant tumors as well as in photodynamic inactivation of antibiotic-resistant bacteria. Both TiO2 NPs themselves, as well as their composites and combinations with other molecules or biomolecules, can be successfully used as photosensitizers in PDT. Moreover, various organic compounds can be grafted on TiO2 nanoparticles, leading to hybrid materials. These nanostructures can reveal increased light absorption, allowing their further use in targeted therapy in medicine. In order to improve efficient anticancer and antimicrobial therapies, many approaches utilizing titanium dioxide were tested. Results of selected studies presenting the scope of potential uses are discussed in this review.


Introduction
The intensive development of photodynamic therapy (PDT) in recent years has involved the search for new photosensitizers and specific carriers for their delivery. Among many promising approaches to be noted for photodynamic research, those in which dyes and nanoparticles (NPs) were combined led to an increase in the selectivity of the photosensitizer (PS) and/or efficacy of the therapy.
At the very beginning, it should be explained that NPs constitute a particular type of particles of the size between 1 and 100 nm (with the surrounding interfacial layer) [1]. The exact definition given in ISO technical specification 80004 states that NPs are "nano-objects with all three external dimensions in the nanoscale, whose longest and shortest axes do not differ significantly". It should be noted that in attention should be paid to the last mentioned, but exceptional category containing metallic and    of metal NPs, including TiO2, depends on many factors, including particle rface coating, size, dose, and exposure route [8,9]. can generally enter the body in three ways: orally, transdermally, or by discussion on the bioavailability of TiO2 from the gastrointestinal tract are here are many indications that titania does not penetrate the gastrointestinal al extent. Animal studies have shown that 24 hours after oral administration e of 100 mg per kilogram of body weight, no significant increase in NP f the tested tissues was detected [10]. Analogous studies using even higher ilar results confirming that orally administered TiO2 does not penetrate the d that penetration is medically insignificant [11]. However, studies using the harmacokinetic modeling technique have indicated that high concentrations to their agglomeration and thus increase their uptake by macrophages. As al. [12], the biodistribution of TiO2 NPs can proceed via two kinetic processes penetrate through the blood vessels to the organs and by phagocytosis of NPs agocyte system. However, it should be emphasized that the pharmacokinetics s administration is different [12]. As in such case, the bioavailability of NPs is ution in the body should be carefully considered. In a study performed by ere administered intravenously with 5 mg TiO2 NPs per kg of body weight 28 days. The animals were healthy and behaved normally throughout the test cal study revealed that TiO2 did not accumulate at detectable levels in blood lymph nodes. However, titania levels were the highest in the liver, while concentrations were observed in the spleen, lungs, and kidneys [13]. rvation concerning TiO2 NPs excretion by the kidneys in rats was made by y noticed that TiO2 is eliminated from the body slowly, which indicates its lation. This issue is not severe considering PDT because the photosensitizer ce or several times during the photodynamic therapy [14]. Besides, the study [15] on rats showed that the TiO2 NPs level in urine was higher than in feces, on as the primary route of TiO2 NP elimination [15].

 To sum up
inetics of TiO2 NPs depends on many factors, including particle type, surface coating, size, dose, and exposure route. t penetrate the gastrointestinal tract at all or to a minimal extent. ical study indicates that after intravenous administration TiO2 NPs accumulate iver, and to some extent in the spleen, lungs and kidneys.
is the primary route of TiO2 NPs elimination. inetics and bioavailability of TiO2 NPs require further and intensive research.

patibility-in vitro and in vivo evaluation of the toxicity of titanium dioxide
ion of titanium dioxide is related to its low toxicity. Many studies with TiO2 of nd microparticle sizes and crystal forms were performed to assess skin, lung, hematotoxicity. Although titania is a quite common ingredient of many especially sunscreens, powders, and eyeshadows, it seems that its size and nd rutile) influence the safety of its usage. vivo studies concerning the skin-related toxicity of TiO2 NPs raised two issues, self and systemic toxicity associated with skin permeation. Wu et al. studied ation of TiO2 NPs in hairless mice and porcine skin after subchronic dermal g to the presented findings, the researchers concluded that the nanosized TiO2 sk to humans after chronic dermal exposure over a relatively long period, tissue distribution. In another study, Crosera et al. studied both TiO2 NPs' ells for 24 h using intact and needle-abraded human skin as well as evaluated keratinocytes. The study demonstrated that the presence of TiO2 NPs was

To sum up
The pharmacokinetics of TiO 2 NPs depends on many factors, including particle type, surface charge, surface coating, size, dose, and exposure route. Titania does not penetrate the gastrointestinal tract at all or to a minimal extent. Histopathological study indicates that after intravenous administration TiO 2 NPs accumulate mainly in the liver, and to some extent in the spleen, lungs and kidneys. Renal excretion is the primary route of TiO 2 NPs elimination. The pharmacokinetics and bioavailability of TiO 2 NPs require further and intensive research.

Toxicity and Biocompatibility-In Vitro and In Vivo Evaluation of the Toxicity of Titanium Dioxide
The wide application of titanium dioxide is related to its low toxicity. Many studies with TiO 2 of different nanoparticle and microparticle sizes and crystal forms were performed to assess skin, lung, immune system, and hematotoxicity. Although titania is a quite common ingredient of many cosmetic formulations, especially sunscreens, powders, and eyeshadows, it seems that its size and crystal forms (anatase and rutile) influence the safety of its usage.
The in vitro and in vivo studies concerning the skin-related toxicity of TiO 2 NPs raised two issues, namely skin toxicity itself and systemic toxicity associated with skin permeation. Wu et al. studied the toxicity and penetration of TiO 2 NPs in hairless mice and porcine skin after subchronic dermal exposure [16]. According to the presented findings, the researchers concluded that the nanosized TiO 2 might pose a health risk to humans after chronic dermal exposure over a relatively long period, mainly due to deeper tissue distribution. In another study, Crosera et al. studied both TiO 2 NPs' penetration Nanomaterials 2020, 10, 387 4 of 31 on Franz cells for 24 h using intact and needle-abraded human skin as well as evaluated cytotoxicity on HaCaT keratinocytes. The study demonstrated that the presence of TiO 2 NPs was limited to the epidermal layer, whereas in the dermal layer, their concentration was below the limit of detection. A slight cytotoxic effect on human HaCaT keratinocytes was noted suggesting the potential TiO 2 NPs related risk only after long-term exposure [17]. In a related study, Yin et al. analyzed the phototoxicity of TiO 2 NPs with different molecular sizes and crystal forms (anatase and rutile) in HaCaT human skin keratinocytes [18]. The outcomes indicated that TiO 2 NPs are phototoxic to human skin keratinocytes as the result of emergence of reactive oxygen species (ROS) generated during UVA irradiation. It is important to note that the rutile form of nano-TiO 2 revealed less phototoxicity than anatase [18].
The potential risk related to TiO 2 inhalation exposure has been the subject of many studies. The toxicity study revealed mainly some adverse effects related to titania, also in experiments that could indicate significant "overload". A study conducted by Lee et al. can be given as an example [19]. It was found that long-term inhalation exposure of rats to bulk TiO 2 dust at high concentration (up to 250 mg/m 3 for 2 years, 6 h/day for 5 days/week) caused bronchioloalveolar adenomas and cystic keratinizing squamous cell carcinomas. Due to the specific nature of the relevant pre-malignant tumors, atypical for human lung cancer, and lack of tumor metastases to other organs, the researchers concluded that the observed tumors arose from the excessive dust loading in the lungs, so-called TiO 2 "overload" [19]. A very interesting research related to the potential risk of inhalation exposure has been recently reported by Vandebriel et al. [20], who studied TiO 2 NPs, which are also a common material applied in paints during their production or applications. Researchers studied TiO 2 NPs in terms of their immune activity in vitro and in vivo. The first section of the study was concentrated on the in vitro assessment of TiO 2 NPs on the maturation of dendritic cells, which form an important part of the lung immune system, whereas the second section was related to the research performed on their adjuvant activity in vivo on mice. For the study, a series of fourteen TiO 2 NPs were chosen, differentiated in terms of crystal structures and coatings. Rutile form of TiO 2 NPs was found to be safer than anatase NPs as in vitro anatase and anatase/rutile TiO 2 NPs induced a higher expression of CD83 and CD86 and a higher production of IL-12p40, than rutile NPs. In this way, the maturation of dendritic cells was induced to a greater extent by anatase and anatase/rutile NPs than by rutile NPs. This finding is important in terms of further choice of titanium dioxide crystal structure for the applications in industry, especially in the areas, where the inhalation exposure during production or application of the product should be considered [20]. It is known that the stimulation of dendritic cell maturation can lead directly to a whole cascade of physiological phenomena, including a specific immune response, and indirectly to inflammation [21]. Continuous exposure to TiO 2 can therefore potentially lead to an excessive immune response and chronic inflammation. Chronic inflammation is considered a harmful state, responsible for the destruction of the body's tissues and the development of other diseases [22]. Complementary research presenting the role of inflammatory processes was conducted by Madhubala et al., who studied in vitro cytotoxic and immunomodulatory effects of the low concentration of TiO 2 NPs on various human cell lines [23]. The immunomodulatory effects of TiO 2 NPs were tested on human monocytic leukemia (THP-1) and human mast (HMC-1) cell lines in a dose-dependent manner. The viability of THP-1 cells treated with titania NPs was significantly reduced at higher doses as indicated in MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. What is interesting, the secretion of cytokines (IL-6 and IL-10) by human cell lines was significantly correlated with the concentration of TiO 2 NPs. Titania NPs at lower concentrations induced inflammatory responses in studied cells through cytokine secretion [23].
A very interesting problem, vitally important in this review, is related to the issue of whether titanium dioxide toxicity can be modified by the combination of these NPs with porphyrinoids. This question cannot be unambiguously answered because further research is needed in the coming years. Nevertheless, some studies, like that performed by Rehman et al. can be considered at a very initial stage as promising [24]. The authors assessed the importance of TiO 2 nanowhiskers in combination with 5,10,15,20-tetra(4-sulfonatophenyl)porphyrin (TSPP) in vivo on rats. What is Nanomaterials 2020, 10, 387 5 of 31 essential, TSPP applied in the above-discussed experiment is considered as a photosensitizer, which is not free of side effects. In the study, different concentrations of either TSPP, TiO 2 , or hybrid TSPP-TiO 2 material were used. Toxic properties were assessed based on fluorescent microscopy, complete blood cells count, and serum enzymes, which allowed the evaluation of the effect of excretory and circulatory systems. The TSPP and TSPP-TiO 2 -treated rat groups were also illuminated with visible light (500-550 nm). Based on all the above-mentioned tests, it turned out that the combination of TiO 2 NPs with the porphyrin significantly reduced TSPP toxicity, especially at high concentrations. It was clearly demonstrated that TSPP (0.1 mM) combined with TiO 2 nanowhiskers (0.6 mM) was safer than TSPP (0.1 mM) alone. According to the MTT assay, TSPP combined with TiO 2 nanowhiskers revealed minimized cytotoxic effects on the normal cells in terms of increased viability. This protection of the TiO 2 nanowhiskers was attributed to their porous nature allowing a slow release of the adsorbed TSPP into the surrounding environment, thus helping in lowering adverse effects without compromising the theranostic properties [24]. d that penetration is medically insignificant [11]. However, studies using the harmacokinetic modeling technique have indicated that high concentrations to their agglomeration and thus increase their uptake by macrophages. As al. [12], the biodistribution of TiO2 NPs can proceed via two kinetic processes penetrate through the blood vessels to the organs and by phagocytosis of NPs agocyte system. However, it should be emphasized that the pharmacokinetics s administration is different [12]. As in such case, the bioavailability of NPs is ution in the body should be carefully considered. In a study performed by ere administered intravenously with 5 mg TiO2 NPs per kg of body weight 28 days. The animals were healthy and behaved normally throughout the test cal study revealed that TiO2 did not accumulate at detectable levels in blood lymph nodes. However, titania levels were the highest in the liver, while concentrations were observed in the spleen, lungs, and kidneys [13]. rvation concerning TiO2 NPs excretion by the kidneys in rats was made by y noticed that TiO2 is eliminated from the body slowly, which indicates its lation. This issue is not severe considering PDT because the photosensitizer ce or several times during the photodynamic therapy [14]. Besides, the study [15] on rats showed that the TiO2 NPs level in urine was higher than in feces, on as the primary route of TiO2 NP elimination [15].

 To sum up
inetics of TiO2 NPs depends on many factors, including particle type, surface coating, size, dose, and exposure route. t penetrate the gastrointestinal tract at all or to a minimal extent. ical study indicates that after intravenous administration TiO2 NPs accumulate iver, and to some extent in the spleen, lungs and kidneys.
is the primary route of TiO2 NPs elimination. inetics and bioavailability of TiO2 NPs require further and intensive research.

patibility-in vitro and in vivo evaluation of the toxicity of titanium dioxide
ion of titanium dioxide is related to its low toxicity. Many studies with TiO2 of nd microparticle sizes and crystal forms were performed to assess skin, lung, hematotoxicity. Although titania is a quite common ingredient of many especially sunscreens, powders, and eyeshadows, it seems that its size and nd rutile) influence the safety of its usage. vivo studies concerning the skin-related toxicity of TiO2 NPs raised two issues, self and systemic toxicity associated with skin permeation. Wu et al. studied ation of TiO2 NPs in hairless mice and porcine skin after subchronic dermal g to the presented findings, the researchers concluded that the nanosized TiO2 sk to humans after chronic dermal exposure over a relatively long period, tissue distribution. In another study, Crosera et al. studied both TiO2 NPs' ells for 24 h using intact and needle-abraded human skin as well as evaluated keratinocytes. The study demonstrated that the presence of TiO2 NPs was

To sum up
The toxicity of titanium dioxide is low. Various studies consider this material as safe or unsafe, depending on the size and crystal form, which strongly determines TiO 2 NPs' potential toxicity. The in vitro and in vivo studies concerning the skin-related toxicity of TiO 2 NPs raise both skin toxicity itself and skin permeation related systemic toxicity. The potential TiO 2 NPs related risk on skin after long-term exposure cannot be neglected. The harmful effects of TiO 2 inhalation exposure are associated with the so-called TiO 2 "overload", which is rare in everyday life. Some immunomodulation effects related to the stimulation of dendritic cell maturation by TiO 2 presented in recent studies cannot be omitted. It seems that TiO 2 toxicity can be modified by combining it with photosensitizers.

Design of Titanium Dioxide Nanoparticles-Synthesis and Stabilization Procedures, Physicochemical Properties, and Characterization
Titanium dioxide is a metal oxide that naturally occurs in nature [25]. Named after two of the most abundant minerals, the two most common tetragonal crystallographic polymorphs of TiO 2 take their name from-anatase and rutile. The third, rarer orthorhombic crystal structure, belongs to brookite. Titanium(IV) oxide is mostly produced by the purification of rutile mineral, or by subjecting ilmenite (FeTiO 3 ) to either so-called chloride or sulfate processes, which both finally yield pure titania. When the thermal treatment is applied, the amorphous TiO 2 may be transformed into anatase or brookite in a process called calcination, which takes place at around 400 • C [25][26][27]. However, these polymorphs, when heated to temperatures above 600 • C, are converted to rutile.
The synthetic methods leading to TiO 2 in general as well as to titania NPs include a series of techniques, with sol-gel synthesis and hydrothermal methods being the most frequently applied [26,27], green chemistry and microwave methods are on the rise [28,29]. By careful design and modification of the process parameters (i.e., substrates used, ratio of solvents, temperature, process time), it is possible to obtain the desired materials with varying specific physicochemical properties, such as surface area, NP morphology and form, NP size and uniformity in size distribution, crystallinity and crystal phase, photoactivity, and many others. These properties can be additionally modified during the synthesis of NPs by the addition of various surfactants or dopants or by post-synthetic modifications, such as doping, surface functionalization, or binding with organic molecules (Figure 2) [25,27].

225
Despite the promising properties of this material for photodynamic therapy, research is still 226 underway to modify the NPs' surface in order to increase the efficiency of ROS generation and to 227 improve the physicochemical properties, including the visible light absorption. Surface-modified

228
TiO2 NPs with photosensitizing properties create a potential for PDT [30]. Effective 229 photosensitization with the use of a wide band-gap semiconductor TiO2 has appeared in many 230 studies aiming to extend its spectral response. Therefore, titanium dioxide can be doped with various 231 metal ions and non-metal dopants [31,32] or combined with various dyes [33][34][35]. Surface complexes 232 acting as TiO2 photosensitizers usually include transition metal ion with inorganic or organic ligands.

233
The processes. In some cases, the complexes formed at titanium dioxide surface can be obtained upon 239 chemisorption due to the presence of anchoring groups in the structure of organic molecules [30].

240
The relevant titanium dioxide is a semiconductor-based material with an energy gap of 3.23 eV for 241 anatase and 3.06 eV for rutile polymorph [2,6]. If the molecule absorbs a photon with energy higher death. It has also been proven that TiO2 NPs inhibit efflux-mediated multidrug resistance [36]. Despite the promising properties of this material for photodynamic therapy, research is still underway to modify the NPs' surface in order to increase the efficiency of ROS generation and to improve the physicochemical properties, including the visible light absorption. Surface-modified TiO 2 NPs with photosensitizing properties create a potential for PDT [30]. Effective photosensitization with the use of a wide band-gap semiconductor TiO 2 has appeared in many studies aiming to extend its spectral response. Therefore, titanium dioxide can be doped with various metal ions and non-metal dopants [31,32] or combined with various dyes [33][34][35]. Surface complexes acting as TiO 2 photosensitizers usually include transition metal ion with inorganic or organic ligands. The organic ligands are coordinatively bound to the central ion and covalently linked to the titanium dioxide surface. Inorganic ligands, such as CN -, F -, PO 4 3can also link surface titanium with metal centers.
The photosensitization is the effect either of the photoinduced electron injection from the surface of the complex to the conduction band of the semiconducting support or of a hole injection to the valence band. Photoinduced charge injection can base on direct or indirect photosensitization processes. In some cases, the complexes formed at titanium dioxide surface can be obtained upon chemisorption due to the presence of anchoring groups in the structure of organic molecules [30]. The relevant titanium dioxide is a semiconductor-based material with an energy gap of 3.23 eV for anatase and 3.06 eV for rutile polymorph [2,6]. If the molecule absorbs a photon with energy higher or equal to that value, it passes to an excited state and can produce negative electrons in the conduction band, leaving positively charged holes in the valence band. Free electrons may attack surrounding oxygen and water molecules to form ROS, including superoxide (O 2 •-), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical ( • OH) ( Figure 3) [36]. These forms of oxygen are highly unstable in biological systems and react with the cell components causing apoptotic or necrotic cell death. It has also been proven that TiO 2 NPs inhibit efflux-mediated multidrug resistance [36]. Nanomaterials 2020 7 of 32 249 Figure 3. Simplified mechanism of reactive oxygen species generation by TiO2 (based on [37]).

250
Due to the nature of titania NPs when dispersed in aqueous solutions, in most cases, they tend 251 to form agglomerates [38,39]. These forms have a decreased surface area and thus reveal also lower 252 photoactivity. In addition to the biological activity of TiO2 NPs, sedimentation may lower their 253 concentration and interfere with the reproducibility of the results, as well as prevent the steady 254 dosage. Therefore, stable formulations of NPs functionalized on their surface to prevent or eliminate 255 this unwelcome property were developed. For example, the modifications of NPs rely on applying a 256 charge for electrostatic repulsion or adsorption of stabilizers that provide a steric hindrance [40].

257
The TiO2 NPs and their aggregates can be analyzed using microscopic methods as well as size 258 distribution techniques, such as light scattering, particle tracking analysis, or others. The techniques 259 indispensable for the characterization of TiO2 nanomaterials are X-ray powder diffraction that allows 260 studying the crystalline phase of titania and infrared spectroscopy that allows for analysis of the 261 chemical groups present on the NP surface [40]. Another method, diffuse reflectance UV-Vis (UV-Vis 262 DRS) spectroscopy is a useful tool for determining the light absorption spectrum of the functionalized 263 materials. It provides the light range that can be applied to excite the NPs, thus allowing the study of 264 their bandgap and assess their usefulness for phototherapy [29,39].     The introduction of neat titania NPs to photodynamic therapy is significantly limited by many 277 issues related to tissue overheating under the influence of light, low tissue penetration by ultra-violet 278 light, and harmful impact of UV radiation on the human body [7]. Neat TiO2 NPs and in combination 279 Figure 3. Simplified mechanism of reactive oxygen species generation by TiO 2 (based on [37]).
Due to the nature of titania NPs when dispersed in aqueous solutions, in most cases, they tend to form agglomerates [38,39]. These forms have a decreased surface area and thus reveal also lower photoactivity. In addition to the biological activity of TiO 2 NPs, sedimentation may lower their concentration and interfere with the reproducibility of the results, as well as prevent the steady dosage. Therefore, stable formulations of NPs functionalized on their surface to prevent or eliminate this unwelcome property were developed. For example, the modifications of NPs rely on applying a charge for electrostatic repulsion or adsorption of stabilizers that provide a steric hindrance [40].
The TiO 2 NPs and their aggregates can be analyzed using microscopic methods as well as size distribution techniques, such as light scattering, particle tracking analysis, or others. The techniques indispensable for the characterization of TiO 2 nanomaterials are X-ray powder diffraction that allows studying the crystalline phase of titania and infrared spectroscopy that allows for analysis of the chemical groups present on the NP surface [40]. Another method, diffuse reflectance UV-Vis (UV-Vis DRS) spectroscopy is a useful tool for determining the light absorption spectrum of the functionalized materials. It provides the light range that can be applied to excite the NPs, thus allowing the study of their bandgap and assess their usefulness for phototherapy [29,39]. of metal NPs, including TiO2, depends on many factors, including particle rface coating, size, dose, and exposure route [8,9]. can generally enter the body in three ways: orally, transdermally, or by discussion on the bioavailability of TiO2 from the gastrointestinal tract are here are many indications that titania does not penetrate the gastrointestinal al extent. Animal studies have shown that 24 hours after oral administration e of 100 mg per kilogram of body weight, no significant increase in NP f the tested tissues was detected [10]. Analogous studies using even higher ilar results confirming that orally administered TiO2 does not penetrate the d that penetration is medically insignificant [11]. However, studies using the harmacokinetic modeling technique have indicated that high concentrations to their agglomeration and thus increase their uptake by macrophages. As al. [12], the biodistribution of TiO2 NPs can proceed via two kinetic processes penetrate through the blood vessels to the organs and by phagocytosis of NPs agocyte system. However, it should be emphasized that the pharmacokinetics s administration is different [12]. As in such case, the bioavailability of NPs is ution in the body should be carefully considered. In a study performed by ere administered intravenously with 5 mg TiO2 NPs per kg of body weight 28 days. The animals were healthy and behaved normally throughout the test cal study revealed that TiO2 did not accumulate at detectable levels in blood lymph nodes. However, titania levels were the highest in the liver, while concentrations were observed in the spleen, lungs, and kidneys [13]. rvation concerning TiO2 NPs excretion by the kidneys in rats was made by y noticed that TiO2 is eliminated from the body slowly, which indicates its lation. This issue is not severe considering PDT because the photosensitizer ce or several times during the photodynamic therapy [14]. Besides, the study [15] on rats showed that the TiO2 NPs level in urine was higher than in feces, on as the primary route of TiO2 NP elimination [15].

 To sum up
inetics of TiO2 NPs depends on many factors, including particle type, surface coating, size, dose, and exposure route. t penetrate the gastrointestinal tract at all or to a minimal extent. ical study indicates that after intravenous administration TiO2 NPs accumulate iver, and to some extent in the spleen, lungs and kidneys.
is the primary route of TiO2 NPs elimination. inetics and bioavailability of TiO2 NPs require further and intensive research.

patibility-in vitro and in vivo evaluation of the toxicity of titanium dioxide
ion of titanium dioxide is related to its low toxicity. Many studies with TiO2 of nd microparticle sizes and crystal forms were performed to assess skin, lung, hematotoxicity. Although titania is a quite common ingredient of many especially sunscreens, powders, and eyeshadows, it seems that its size and nd rutile) influence the safety of its usage. vivo studies concerning the skin-related toxicity of TiO2 NPs raised two issues, self and systemic toxicity associated with skin permeation. Wu et al. studied ation of TiO2 NPs in hairless mice and porcine skin after subchronic dermal g to the presented findings, the researchers concluded that the nanosized TiO2 sk to humans after chronic dermal exposure over a relatively long period, tissue distribution. In another study, Crosera et al. studied both TiO2 NPs' ells for 24 h using intact and needle-abraded human skin as well as evaluated keratinocytes. The study demonstrated that the presence of TiO2 NPs was

To sum up
TiO 2 occurs naturally in three polymorphic forms: rutile and anatase with a tetragonal structure, and rhombic brookite. Synthetic TiO 2 is obtained by sol-gel synthesis, hydrothermal methods, green chemistry, microwave methods, and others. The TiO 2 particles can be modified by the addition of various surfactants or dopants or by post-synthetic modifications, such as doping, surface functionalization, or binding with organic molecules. Titania NPs, when dispersed tend to form agglomerates. The TiO 2 NPs functionalized on their surface can form stable, non-aggregating formulations in aqueous solutions.

Photodynamic Activity of Neat TiO 2 Nanoparticles and in Drug Delivery Systems
The introduction of neat titania NPs to photodynamic therapy is significantly limited by many issues related to tissue overheating under the influence of light, low tissue penetration by ultra-violet light, and harmful impact of UV radiation on the human body [7]. Neat TiO 2 NPs and in combination with various molecules, antibodies, or polymers revealed interesting photocytotoxicity against cancer cells and microbes, thus unveiling potential for photodynamic therapy.
Although TiO 2 is a potent oxygen radical generator, it can be excited in its pure form only by UV light. Lagopati et al. investigated the photo-induced bioactivity of titanium dioxide against cancer cells and the mechanism of action of TiO 2 NPs [41]. The studies were conducted on the MCF-7 and MDA-MB-468 breast epithelial cancer cell lines. The aqueous dispersions of nanostructured titania were irradiated with UVA (wavelength 350 nm) for 20 min. The nanostructured TiO 2 photosensitizer dispersions were prepared using the sol-gel technique. It is worth noting that in the TiO 2 sols the presence of photocatalyst in the form of anatase NPs was confirmed. According to the results of the study, the applied modification revealed strong efficacy against the highly malignant MDA-MB-468 cells, which underwent apoptotic cell death. It is important to notice that the use of UV light alone caused only a 10% decrease in MDA-MB-468 cell viability, whereas non-irradiated TiO 2 NPs at 16 µM concentration decreased the cell viability by 50%. Moreover, MCF-7 cell line was found to be resistant to this therapy under identical conditions. The observed apoptotic cell death was induced indirectly by the increase of caspase-3-mediated poly (ADP-ribose) polymerase (PARP) cleavage [41]. Non-modified titania NPs were also the subject of research of Wang et al., who investigated the effect of TiO 2 NPs in vitro on glioblastoma multiforme cells upon 365 nm light irradiation and then in vivo on glioma-bearing mice [42]. On the one hand, it was found that the performed UV-PDT protocol resulted in higher mice survivability along with tumor growth suppression. On the other hand, despite the statistically significant effects, some critical drawbacks of UV PDT protocol with neat titania NPs were found, mostly related to the limited penetration of UV light through tissues.
It seems that an increase of the therapeutic efficiency and a reduction of drug side effects can be achieved using modern medical and pharmaceutical approaches, including the so-called smart drug delivery or targeted drug delivery systems. Such an approach improving the selectivity of NPs was proposed by Xu et al., who applied TiO 2 NPs conjugated with a specific monoclonal antibody against the carcinoembryonic antigen of human metastatic colon adenocarcinoma (LoVo) cancer cells [6]. The obtained combination improved hybrid NPs distribution and increased the accumulation of the drug in the pathological tissues. Additionally, they used electroporation -a technique that induces the formation of micropores in biological membranes, thus increasing the membrane permeability. In these experiments, the application of a novel approach significantly enhanced the internalization of the materials, which resulted in a 100% decrease in viable LoVo cells after irradiation with ultraviolet 365 nm light, as compared to the death of 44% of the cell population after irradiation alone. Noteworthy, the particularly beneficial effects of electroporation with the electronic pulses at 500 V/cm for 100 µs of increased efficiency and specificity were noted at the very low concentration of antibody-TiO 2 bioconjugate (even up to 3.12 µg/mL). The proposed approach can be used in the therapy of other cancer types if appropriate antibodies are matched [6]. Another way to increase the selectivity of NPs is to combine them with folic acid, which allows achieving high selectivity for some cancers. Similarly to antibodies, folic acid raises the affinity of particles to pathological tissues, thus increasing their accumulation in the target area. Due to the augmented expression of the folate receptor in the cancer cells, folic acid conjugates penetrate more easily through the cell membrane of folate-overexpressing cells. On this basis, Feng et al. designed a new photosensitizer-folic acid-conjugated silica-coated titanium dioxide [36]. The biocompatibility of the conjugate system was assessed in two cell lines: fibroblast cells (L929) and the human nasopharyngeal epidermoid cancer (KB) cells. After 24 h incubation, significantly better permeability of folic acid conjugated silica-coated TiO 2 to L929 and KB cells was observed. Firstly, the effect of UV (365 nm) radiation on cells was tested and found non-toxic. The photosensitizer was applied at the concentration range from 12.5 up to 100 µg/mL, with the best effect on the KB cell viability reduction up to 57% at the highest concentration applied. Fluorescence tests revealed that cells absorbed significantly less neat TiO 2 NPs (P25) than the conjugated system. Also, higher mortality of cells treated with the conjugated system than neat TiO 2 alone showed the contribution of folate receptors in its internalization [36]. More insight into the influence of SiO 2 shell on the activity of TiO 2 NPs gave the study conducted by the same group. In the paper, there were presented data on the silica coating thickness on TiO 2 NPs for effective photodynamic therapy [43]. The effect of the thickness of the silica shell on the photodynamic activity of TiO 2 NPs, cytotoxicity, and photo-killing ability was unambiguously confirmed. On the one hand, it was found that the increase in the thickness of the silica shell allows better penetration of NPs through the cell membrane, reducing significantly, on the other hand, the photoactivity of the photosensitizer. Researchers were looking for the most optimal silica shell thickness guaranteeing maximum photo-killing efficiency while ensuring the best possible cytocompatibility. After a series of experiments on L929 cells, it turned out that the 5.5 nm SiO 2 -layer thickness seems to be optimal for the complete preservation of the photodynamic properties of TiO 2 NPs and the improvement of their biocompatibility.
The application of TiO 2 in PDT concerns its various forms, especially composites and hybrids, with some perspectives to use also in the area of wound healing application and management [44]. Archana et al. obtained and characterized blends of chitosan, poly(N-vinylpyrrolidone) and TiO 2 by infrared spectroscopy, thermogravimetric analysis, transmission electron microscopy, and scanning electron microscopy [45]. The mechanical properties of composite material indicated that the addition of TiO 2 NPs increases the strength of nanocomposite. The nanocomposite dressing revealed excellent antimicrobial efficacy and good biocompatibility against NIH3T3 and L929 fibroblast cells. Also, the material triggered accelerated healing of open excision type wounds in an albino rat model [45]. ilar results confirming that orally administered TiO2 does not penetrate the d that penetration is medically insignificant [11]. However, studies using the harmacokinetic modeling technique have indicated that high concentrations to their agglomeration and thus increase their uptake by macrophages. As al. [12], the biodistribution of TiO2 NPs can proceed via two kinetic processes penetrate through the blood vessels to the organs and by phagocytosis of NPs agocyte system. However, it should be emphasized that the pharmacokinetics s administration is different [12]. As in such case, the bioavailability of NPs is ution in the body should be carefully considered. In a study performed by ere administered intravenously with 5 mg TiO2 NPs per kg of body weight 28 days. The animals were healthy and behaved normally throughout the test cal study revealed that TiO2 did not accumulate at detectable levels in blood lymph nodes. However, titania levels were the highest in the liver, while concentrations were observed in the spleen, lungs, and kidneys [13]. rvation concerning TiO2 NPs excretion by the kidneys in rats was made by y noticed that TiO2 is eliminated from the body slowly, which indicates its lation. This issue is not severe considering PDT because the photosensitizer ce or several times during the photodynamic therapy [14]. Besides, the study [15] on rats showed that the TiO2 NPs level in urine was higher than in feces, on as the primary route of TiO2 NP elimination [15].

 To sum up
inetics of TiO2 NPs depends on many factors, including particle type, surface coating, size, dose, and exposure route. t penetrate the gastrointestinal tract at all or to a minimal extent. ical study indicates that after intravenous administration TiO2 NPs accumulate iver, and to some extent in the spleen, lungs and kidneys.
is the primary route of TiO2 NPs elimination. inetics and bioavailability of TiO2 NPs require further and intensive research.

patibility-in vitro and in vivo evaluation of the toxicity of titanium dioxide
ion of titanium dioxide is related to its low toxicity. Many studies with TiO2 of nd microparticle sizes and crystal forms were performed to assess skin, lung, hematotoxicity. Although titania is a quite common ingredient of many especially sunscreens, powders, and eyeshadows, it seems that its size and nd rutile) influence the safety of its usage. vivo studies concerning the skin-related toxicity of TiO2 NPs raised two issues, self and systemic toxicity associated with skin permeation. Wu et al. studied ation of TiO2 NPs in hairless mice and porcine skin after subchronic dermal g to the presented findings, the researchers concluded that the nanosized TiO2 sk to humans after chronic dermal exposure over a relatively long period, tissue distribution. In another study, Crosera et al. studied both TiO2 NPs' ells for 24 h using intact and needle-abraded human skin as well as evaluated keratinocytes. The study demonstrated that the presence of TiO2 NPs was

To sum up
The applications of neat titania NPs in photodynamic therapy are limited by the necessity to use UV light of very low tissue penetration, and harmful impact on the human body. Neat TiO 2 NPs and in combination with various molecules, antibodies, or polymers revealed interesting photocytotoxicity against cancer cells and microbes, thus unveiling potential for PDT. The SiO 2 shell influences the activity of TiO 2 NPs in photodynamic therapy. Only the optimal SiO 2 -layer thickness guarantees optimal preservation of the photodynamic properties of TiO 2 NPs as well as the improvement of their biocompatibility. TiO 2 and its composites with chitosan, poly(N-vinylpyrrolidone) can broaden the current PDT applications towards the area of wound healing management.

Doping of TiO 2 Nanoparticles with Inorganic Compounds and Carbon-Based Nanomaterials
The energy necessary for titanium dioxide NPs excitation is high, which is the result of a wide bandgap. Therefore, only UV light bears sufficient energy to excite titania [37]. Serious problem related to colloidal TiO 2 NPs is their pH-dependent tendency to form agglomerates, which reduces their photoreactivity and decreases the functional surface area [46]. An additional limitation for the broader use of neat TiO 2 NPs is their insufficient selectivity and the lack of cell-specific accumulation. The addition of inorganic compounds to the TiO 2 NPs structure during their preparation or creation of defects in the structure of already prepared TiO 2 NPs is defined as doping. This process narrows the bandgap in the TiO 2 NPs structure and lowers the activation energy. Titania may be doped with a series of molecules, including organic, inorganic-both metals and nonmetals. Many studies with TiO 2 NPs have been performed so far to maximize their visible light absorption. For example, it was found that doping or modification of the TiO 2 NP surface leads to a shift of the absorption maxima towards longer wavelengths, thus increasing the depth of tissue penetration [43].
One way to improve the effectiveness of TiO 2 -based photodynamic therapy is modifying titania to the so-called black TiO 2 NPs by reduction of the particles and inducing the formation of Ti 3+ ions on the TiO 2 surface. Such black TiO 2 NPs were obtained by Ni et al., starting from the commercially available titanium dioxide (P25, 71% anatase and 29% rutile) powder and following a facile calcination method combined with an in situ controllable solid-state reaction method [7]. In this process, according to X-ray photoelectron spectroscopy (XPS) and UV-Vis DRS measurements, the existence of Ti 3+ defects and oxygen vacancies in the black TiO 2 was confirmed. Researchers used black TiO 2 NPs as a near-infrared light-triggered PDT photosensitizer with a maximum absorbance of 808 nm on human bladder cancer cell line (T24). Bladder cancer cells were incubated with the photosensitizing NPs and then irradiated with laser at 808 nm for 0-7 min. As expected, an increase in the concentration of the photosensitizer correlated with an increase in anticancer activity. Minimal cell viability (54.32%) was observed at a concentration of 500 µg/mL, and the exposure time of 7 min. As noted by the authors, the applied black TiO 2 NPs can be considered as an excellent photosensitizer due to their flexible-dose and very good anti-cancer effect. What is more, black TiO 2 NPs on the contrary to non-doped TiO 2 NPs were the most active in visible light and NIR.
Intensive work is currently underway on various combinations of TiO 2 NPs with other inorganic elements and compounds to improve their photochemical properties. Kayani et al. developed cerium-doped (Ce-doped) TiO 2 thin films synthesized by the sol-gel dip-coating route [47]. The band gap of the Ce doped TiO 2 slightly decreased from~3.93 eV to~3.79 eV with an increase in Ce doping percentage. The obtained NPs showed favorable changes in the area of ferromagnetic sensitivity that can be correlated with the increase in cerium concentration. Unfortunately, these changes did not translate into any biological activity of the formulation. Cerium-doped TiO 2 NPs were tested on Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. Following the results, the NPs did not reveal any photodynamic activity even when the amount of the compound increased (16 mg/mL) in agar medium. This can be also associated with the problems that appeared during the measurements as the tested NPs precipitated in the agar and did not mix properly with the agar medium [47]. The anti-cancer activity of modified and unmodified titanium(IV) oxide NPs has also recently been studied by Shah et al. [48]. Their research clearly indicated that TiO 2 nanoparticles stabilized with PEG (polyethylene glycol) reveal better photodynamic activity than the non-stabilized NPs. Their antitumor activity was assessed in vitro on human cervical cells (HeLa) and human skin cancer cells (HT144). The reported results support the further development of nanomaterials based on the combination of titanium dioxide with polymers. The authors also extended the research by analyzing the activity of modified TiO 2 NPs. What is essential, doping of the metal (cobalt) and non-metal (nitrogen) onto TiO 2 nanocrystals allowed the photoactivation of doped-TiO 2 NPs in the visible/near-infrared region. Paradoxically, however, despite an improvement in their photochemical parameters, anti-tumor activity declined. The authors associate it with the so-called downregulated ROS production or reduced uptake of conjugates by cancer cells [48]. In another study, performed by Zeni et al. [49], nitrogen-doped TiO 2 NPs were applied in vitro on murine melanoma cell line (B16-F10) and fibroblasts (NIH 3T3). The nitrogen-doped TiO 2 NPs were prepared following a modified hydrogen peroxide sol-gel process with triethylamine as a nitrogen precursor. Moreover, X-ray diffraction (XRD) measurements confirmed that all TiO 2 and N-TiO 2 samples consisted of an anatase crystalline phase without any trace of rutile. The nitrogen-doped TiO 2 NPs revealed higher absorbance in the visible region than the neat ones. The use of modified titanium(IV) oxide at a concentration of 0.5 mg/mL resulted in the death of up to 93% melanoma cells under UV irradiation treatment and caused an increase in expression of the pro-apoptotic BAX gene. A comparison of both results may indicate that the sensitivity of cancer cells to modified NPs may vary significantly depending on the type of cancer [49].
An example of an interesting composite obtained by doping of the TiO 2 NPs with carbon-based nanomaterials was reported by Shang et al. [50]. They used TiO 2 NPs doped with reduced graphene oxide (RGO-TiO 2 ) in 10%-50% m/m ratio. The novel composite was obtained by the hydrothermal reduction method and then characterized by XRD, infrared spectroscopy, transmission electron microscopy, Brunauer-Emmett-Teller (BET) surface area analysis, UV-Vis spectroscopy, and XPS. Comparative analysis proved that RGO-containing NPs, when the modified proportion was 0.2 (RGO:TiO 2 ), were more active against human hepatocellular carcinoma (HepG2) cancer cells than neat TiO 2 NPs. In the study, either UVA (365 nm) or visible light (420 nm) were used. On the one hand, only at a long-term incubation with RGO-TiO 2 NPs, the material revealed dark toxicity in concentration range 0-500 µg/mL which resulted in up to 25% viability decrease, but on the other hand, it was less toxic in the dark than TiO 2 NPs alone. The treated cells died turning on apoptosis pathway as a result of increased oxidative stress. The effects observed in tested cells were as follows: a marked decrease in the ratio of the super-coiled DNA indicating DNA oxidative damage, mitochondrial membrane potential disruption, as well as an increased intracellular calcium concentration. Very similar data in terms of observed cytotoxicity of the carbon material were obtained in the study performed by Ismail et al. [51]. They explored the antiproliferative activity of -among others-TiO 2 -NPs (P25), titanium dioxide nanotubes (TiO 2 -NTs), and ZnO-NPs/TiO 2 -NTs nanocomposite under UV irradiation and received completely different results to the above-described. In this study, human liver adenocarcinoma cells HepG2 were incubated for 48 h with the plethora of various zinc oxide NPs, metal-doped zinc oxide NPs, silica-coated zinc oxide NPs, as well as the above-mentioned titania-containing materials at five different concentrations (6.25, 12.5, 25, 50 and 100 µg/mL). The plates were then irradiated for 3 min with light 320-400 nm (UV light). Interestingly, it was found that the metal-doped ZnO-NPs can induce an antiproliferative effect on HepG2 cells under UV-irradiation due to the generation of ROS. Surprisingly, none of the tested NPs containing TiO 2 showed statistically significant anti-proliferative activity. The authors indicate that the problem may result from the limited exposure time of NPs to UV light. Compared to the study mentioned earlier, the low activity of NPs with TiO 2 might result from their low accumulation in tissues due to the unmodified surface of the NPs and thus their higher agglomeration [51]. d that penetration is medically insignificant [11]. However, studies using the harmacokinetic modeling technique have indicated that high concentrations to their agglomeration and thus increase their uptake by macrophages. As al. [12], the biodistribution of TiO2 NPs can proceed via two kinetic processes penetrate through the blood vessels to the organs and by phagocytosis of NPs agocyte system. However, it should be emphasized that the pharmacokinetics s administration is different [12]. As in such case, the bioavailability of NPs is ution in the body should be carefully considered. In a study performed by ere administered intravenously with 5 mg TiO2 NPs per kg of body weight 28 days. The animals were healthy and behaved normally throughout the test cal study revealed that TiO2 did not accumulate at detectable levels in blood lymph nodes. However, titania levels were the highest in the liver, while concentrations were observed in the spleen, lungs, and kidneys [13]. rvation concerning TiO2 NPs excretion by the kidneys in rats was made by y noticed that TiO2 is eliminated from the body slowly, which indicates its lation. This issue is not severe considering PDT because the photosensitizer ce or several times during the photodynamic therapy [14]. Besides, the study [15] on rats showed that the TiO2 NPs level in urine was higher than in feces, on as the primary route of TiO2 NP elimination [15].

 To sum up
inetics of TiO2 NPs depends on many factors, including particle type, surface coating, size, dose, and exposure route. t penetrate the gastrointestinal tract at all or to a minimal extent. ical study indicates that after intravenous administration TiO2 NPs accumulate iver, and to some extent in the spleen, lungs and kidneys.
is the primary route of TiO2 NPs elimination. inetics and bioavailability of TiO2 NPs require further and intensive research.

patibility-in vitro and in vivo evaluation of the toxicity of titanium dioxide
ion of titanium dioxide is related to its low toxicity. Many studies with TiO2 of nd microparticle sizes and crystal forms were performed to assess skin, lung, hematotoxicity. Although titania is a quite common ingredient of many especially sunscreens, powders, and eyeshadows, it seems that its size and nd rutile) influence the safety of its usage. vivo studies concerning the skin-related toxicity of TiO2 NPs raised two issues, self and systemic toxicity associated with skin permeation. Wu et al. studied ation of TiO2 NPs in hairless mice and porcine skin after subchronic dermal g to the presented findings, the researchers concluded that the nanosized TiO2 sk to humans after chronic dermal exposure over a relatively long period, tissue distribution. In another study, Crosera et al. studied both TiO2 NPs' ells for 24 h using intact and needle-abraded human skin as well as evaluated keratinocytes. The study demonstrated that the presence of TiO2 NPs was

To sum up
Doping or modification of TiO 2 NPs "turns on" their excitation possibilities by visible light and increases their activity in photodynamic activity study. The combinations of TiO 2 with inorganic dopants and carbon-based nanomaterials modifying its photochemical properties seem to be an alternative not only to neat TiO 2, but also to conventional photosensitizers in PDT. The combinations of TiO 2 with inorganic dopants and carbon-based nanomaterials were studied towards antimicrobial and anticancer PDT.

Modifications of TiO 2 Nanoparticles with Photosensitizers Aiming to Improve Their Optical and Biological Properties
Metallic nanoparticles, based on gold, silver, and titanium, reveal their photodynamic activity in many in vitro and in vivo biological studies. The development of PDT studies based on the combination of nanoparticles or quantum dots with commonly used photosensitizers, such as phthalocyanines, porphyrins, and other dyes, is becoming more popular. NPs appear to be also suitable carriers for targeted therapy. The use of proper drug delivery systems for photosensitizers allows performing PDT in specific tissues [52,53].
Because of the shortcomings of neat titania NPs, mostly due to their absorption of only short UV wavelengths and aggregation in water media, they have been modified with a plethora of inorganic and organic dopants. Among organic dyes most often utilized for combining with TiO 2, are porphyrins and phthalocyanines. Such hybrid materials were numerously tested for their use as catalysts for visible-light biomedical and environmental photocatalysis in photovoltaics for the preparation of dye-sensitized solar cells (DSSC) as well as photosensitizers for PDT [54][55][56][57]. The selected TiO 2 NPs combined with photosensitizers discussed in this chapter are summarized in Table 1.

TiO 2 Nanoparticles Combined with Phthalocyanines
An excellent example of a combination of TiO 2 NPs and phthalocyanine was presented by Pan et al., who linked aluminum(III) tetrasulfonatedphthalocyanine chloride (TSAlClPc) to nitrogen-doped anatase TiO 2 NPs by electrostatic interactions (Figure 4) [58]. The hybrid material was characterized by Zeta potential measurements, transmission electron microscopy, and UV-Vis absorption spectroscopy.
The cellular uptake, intracellular distribution, cytotoxicity and the photokilling effect of the NPs were studied on human epithelial cervical cancer cells (HeLa) and human nasopharyngeal carcinoma cells (KB). On the one hand, the phthalocyanine selected for the study is known to present high absorption in the red light region, but on the other hand, its use is limited due to reduced cell permeability. The absorption spectrum of hybrid material demonstrated features of both components with absorption maxima in the red region of the spectrum and UV region, which resulted in higher production of reactive oxygen species under visible light irradiation. Moreover, both cellular uptake and intracellular distribution of phthalocyanine were remarkably improved by its nitrogen-doped TiO 2 carrier. In the biological study, HeLa or KB cancer cells were incubated for 1 h with the photosensitizer at the concentrations from the range 4.7 to 37.6 µg/mL for one hour and then immediately irradiated with a light 420-800 nm (15 J/cm 2 ). It turned out that hybrid material at the concentration of 21.88 µg/mL killed up to 86% of tumor cells. It means that the photokilling efficiency of hybrid material was over ten times higher and ROS production was 2.6-fold better than that measured for the studied phthalocyanine alone [58]. Also, in 2017 Pan et al. published the results of a very similar study, in which they tested the activity of TSAlClPc, non-doped and nitrogen-doped TiO 2 NPs as well as their conjugates [59]. Photosensitizers were applied at concentrations in the range of 5 up to 20 mg/mL for the HeLa tumor cells study. The absorbance and photokilling effect on HeLa cells were studied upon visible light irradiation of different regions of 420-800 nm (15.9 J/cm 2 ) and 420-575 nm (7.5 J/cm 2 ). The best photocytotoxic activity of the nitrogen-doped TiO 2 -TSAlClPc system was measured under irradiation of 420-800 nm when over 85% of the cancer cells were killed. Non-doped TiO 2 -TSAlClPc NPs were found less active as they killed slightly more than 70% of the HeLa cells, while TSAlClPc alone presented only weak photokilling effect with more than 83% of cells surviving. Moreover, it was noticed that the nitrogen-doping of NPs can significantly increase photodynamic activity, as it greatly enhances the formation of singlet oxygen ( 1 O 2 ) and superoxide anion radicals, whereas it suppresses the generation of hydroxyl radicals [59].  A new developing part of medicine is the so-called theranostics-a combination of diagnosis and therapy. Theranostics uses specific pathways in the body to achieve a specific molecular target, e.g., a specific receptor on cancer cells. Also, theranostics is the basis for targeted therapy, the part of which may be anti-cancer PDT [70]. Yurt et al. used in their study an unsymmetrical monocarboxylic derivative of zinc(II) phthalocyanine (MCZnPc) and MCZnPc anchored onto TiO 2 NPs-labeled with 131 I to assess their potential in therapy and diagnosis of selected cancers. MCZnPc and MCZnPc-TiO 2 NPs were tested for their antitumor activity against mouse mammary carcinoma (EMT6) and human cervical adenocarcinoma (HeLa) cells [60]. After a three-hour incubation period in the dark, the cell cultures with photosensitizers were irradiated with light at a 684 nm wavelength. This study has also focused on the cellular uptake of the radiolabeled MCZnPc and radiolabeled MCZnPc-TiO 2 . The results demonstrated higher cellular uptake for the labeled MCPc-TiO 2 NPs. Therefore, they can be considered good candidates for nuclear imaging and hence for theranostic applications against breast and cervical tumors. It was found that the photokilling effect of MCZnPc and MCZnPc-TiO 2 conjugates against both EMT6 and HeLa cells increases proportionally to the concentration of photosensitizer and light intensity topping at over a 80% decrease in cell viabilities. MCZnPc-TiO 2 caused significant phototoxicity in EMT6 cell lines at 1.57 and 3.13 µM with 30 J/cm 2 and 6.25 µM at 60 J/cm 2 . The photocytotoxicity in HeLa cell lines was the highest for MCZnPc-TiO 2 at 6.25 µM with 60 J/cm 2 , but MCZnPc was more photocytotoxic at 3.13 µM with 90 J/cm 2 [60].
The limitation of the possible side effects and the increase of tissue specificity of phthalocyanines, which are generally perceived as relatively non-toxic compounds, has been considered as a goal of many studies [71]. For this purpose Yurt et al. anchored MCZnPc with pure TiO 2 NPs [72]. The obtained material was tested as a potential agent for photodynamic therapy/photodynamic diagnosis on human healthy lung fibroblast cells (WI38) as well as selected cancer cell lines-hepatocellular carcinoma (HepG2) and colorectal adenocarcinoma (HT29). The MCZnPc solution or MCZnPc-TiO 2 dispersion in DMSO were added to the tumor cell suspension and incubated in the dark for 3 h, after which the cells were irradiated. The study confirmed the assumption that such a combination of MCZnPc and TiO 2 significantly reduces the toxicity of the photosensitizer. The hybrid ZnPc-TiO 2 material revealed higher phototoxicity than the studied phthalocyanine alone in colon tumor treatment. Parallel, the results of the study proved that MCZnPc-TiO 2 showed a stronger photokilling effect on the HepG2 than MCZnPc alone. It was also found that the photokilling effect increased with the concentration of the photosensitizer and the light intensity. The maximum effect was achieved for MCZnPc-TiO 2 at a concentration of 6.25 mM and a light intensity of 90 J/cm 2 with killing efficiency over 73% on the HepG2 cells. Also, MCZnPc-TiO 2 was radiolabeled with 131 I radioisotope, and the uptake of 131 IMCZnPc-TiO 2 in the cell lines was studied for potential applications as a bifunctional agent in nuclear imaging and PDT. According to the results of intracellular uptake, the high target/non-target tissue ratio of 131 I labeled MCZnPc-TiO 2 can be applied as a nuclear imaging agent for hepatocellular cancer [72].
The modified TiO 2 NPs also reveal an interesting potential for antimicrobial photodynamic chemotherapy (PACT). Many of the metals, including gold or silver, have been used so far for the preparation of NPs, and demonstrate relatively strong antibacterial properties. Titanium compounds, especially titanium dioxide, were also used as a bactericide, as evidenced by numerous in vitro studies on various bacteria including both Gram-positive and Gram-negative strains [56,[73][74][75]. The antibacterial properties of titanium dioxide can be enhanced by exposure to UV light. Currently, there are several ongoing studies focused on photocytotoxicity of hybrid materials composed of phthalocyanines, porphyrazines or chlorines bound to titanium dioxide. Two recently published studies indicate that the combination of TiO 2 with phthalocyanines and subphthalocyanines raises the effectiveness of antimicrobial photodynamic therapy. On the one hand, Tunçel et al. showed that the application of Zn(II) phthalocyanine with (4-carboxyphenyl)ethynyl moieties alone or after integration with TiO 2 NPs leads to very similar photocytotoxicity results [61]. On the other hand, the cellular uptake of these molecules was substantially lower when phthalocyanine was combined with titanium dioxide. The authors associate this fact with the increase of molecular weight decreasing the efficiency of photosensitizer penetration through the bacterial wall. In the same research group, Ozturk et al. proved that the combination of subphthalocyanine with titanium NPs increases their activity against S. aureus and E. coli [62]. It should be emphasized that in both cases the conjugates required lower light intensity to achieve a bactericidal effect. The viability of S. aureus and E. coli strains after irradiation was dependent upon both light doses and the compounds used in the treatment. Here, however, the cell uptake of the compounds was higher in the case of pure phthalocyanine. It is worth noting that a bactericidal effect against S. aureus of subphthalocyanine was observed at 24 J/cm 2 , whereas the subphthalocyanine-TiO 2 hybrid material was active at 16 and 24 J/cm 2 of light doses, which suggests the increased photoactivity of the material as compared to its components alone. Moreover, both subphthalocyanine and subphthalocyanine-TiO 2 hybrid material were active against Gram-negative strain E. coli at 30 J/cm 2 of light dose [61,62]. Mantareva et al. studied the photodynamic activity of Zn(II) phthalocyanine salt containing 3-dodecylpyridyloxy moieties (ZnPcDo) adsorbed on TiO 2 anatase NPs in vivo against methicillin-resistant Staphylococcus aureus (MRSA) and Salmonella enteritidis [63]. The authors assessed the use of 346 nm light, 643 nm light, or both light sources simultaneously. The ZnPcDo-TiO 2 hybrid material demonstrated high activity against MRSA bacteria after exposure to red light (2 logs inactivation), but no antimicrobial activity was observed after irradiation with UVA alone. Tests on S. enteritidis strain revealed that only the ZnPcDo-TiO 2 combination was active after UVA and LED exposure. As pointed out by authors, irradiation with two sources at the same time does not significantly increase the photodynamic activity of the photosensitizer. The practical application of the novel hybrid material was related to photoinactivation of pathogenic bacteria in wastewater [63].
Antimicrobial photodynamic therapy can be used not only against bacteria or fungi, but also against parasites. Lopez et al. studied the photocytotoxicity of zinc(II) phthalocyanine (ZnPc), nano-TiO 2 , and ZnPc-TiO 2 composite, against a panel of tumor and non-tumor mammalian cells, including the African green monkey epithelial cells (Vero cells, ATCC), human hepatocellular liver carcinoma cells (HepG2, ATCC), human acute monocytic leukemia cell line THP-1 (ATCC), and a primary culture of human-derived fibroblasts (HDFs) and on promastigote forms of Leishmania parasites [64]. Neat TiO 2 NPs under visible light irradiation were not phototoxic for the cells, whereas ZnPc was photocytotoxic for all the studied cells and Leishmania parasites. Mammalian cells were incubated with TiO 2 , ZnPc-TiO 2 , or ZnPc for 24 h and then illuminated with light (either 670 nm or in the 597-752 nm range). The growth was microscopically assessed by counting parasite numbers. Unfortunately, contrary to expectations, neither TiO 2 NPs nor ZnPc-TiO 2 caused phototoxic effect against tested L. chagasi or L. panamensis promastigotes. However, ZnPc-TiO 2 was active against tumor and non-tumor mammalian cells, but less than the pure ZnPc. Moreover, ZnPc-TiO 2 was internalized by the cells at a lower level than ZnPc. Both ZnPc-TiO 2 and ZnPc were localized in mitochondrial cytoplasm [64].
A study in which phthalocyanine was deposited on TiO 2 nanopore thin films, can be indicated as the further development of TiO 2 applications in photodynamic therapy. Perillo et al. used this method to prepare a potential photosensitizer containing copper tetracarboxyphthalocyanines (TcPcCu) active against MRSA [65]. The suspension of bacteria and photosensitizer was irradiated with visible light. A sample containing only TiO 2 thin film showed no differences as compared to the control. However, the TiO 2 /PcTcCu thin film sample reduced the development of MRSA by 81.5% [65].

TiO 2 Nanoparticles Combined with Porphyrins, Chlorins and Methylene Blue
Porphyrins doped on TiO 2 NPs were studied in terms of their potential applications in photodynamic antimicrobial therapy by Sułek et al. (Figure 5) [57]. They received very promising results combining titanium dioxide with fluorinated porphyrins, 5,10,15,20-tetrakis(2,6-difluoro-3sulfophenyl)porphyrin (FTSPP) and its zinc(II) derivative [57]. The combination of TiO 2 NPs with the obtained FTSPP halogen derivatives improved the overall properties of both compounds. In the study, the influence of conjugates on representative Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria was assessed. The combination of fluorinated porphyrins and TiO 2 NPs after two hours of exposure to visible light (1 J/cm 2 at 420 nm or 10 J/cm 2 at 530 nm) led to spectacular 7 logs reduction in colony-forming units. The activity of conjugates against Gram-negative bacteria was significantly lower, which is typical for PACT. It is worth noting that the addition of KI (50 mM) transformed the hybrid materials into effective antimicrobial photosensitizers able to efficiently inactivate Gram-negative bacteria. Remarkably higher activity of hybrid material towards Gram-positive bacteria was related to the specificity of the cell wall structure of these bacteria. Unfortunately, the additional outer-membrane that contains lipopolysaccharide present in the structure of Gram-negative bacteria, decreases membrane permeability for lipophilic compounds and impedes the penetration of reactive oxygen to the target structures in the cell, thereby limiting the effectiveness of the photosensitizer [57].  The application of PDT is not only limited to the treatment of cancer and bacterial infections, but it is also becoming more popular for the treatment of other diseases. The first generation of porphyrins has been applied in clinical practice for several years. Although the quality of porphyrin photosensitizers considerably improved over the years, mainly due to the development of the secondand third-generation photosensitizers, the problem of efficient distribution and selectivity of these compounds in the body has still not been resolved. The implementation of the therapy with the use of porphyrins and derivatives, as well as various NPs, was extended and also applied in rheumatoid arthritis, atherosclerosis, macular degeneration, and diabetes mellitus [76].
Many attempts have been made to combine porphyrins with nanoparticles, including TiO 2 NPs, aiming to obtain an effective formulation. Zhao et al. designed a nanocomposite from the water-soluble TSPP and TiO 2 nanowhiskers as a potential agent in the theranostics and PDT of rheumatoid arthritis [66]. Rheumatoid arthritis is an autoimmune disease and is usually treated with TNF-α blockers, minocycline, azathioprine, and non-steroidal anti-inflammatory drugs. The hybrid material was tested on rats and mice with artificially collagen-induced arthritis. After injection of aqueous TSPP-TiO 2 suspensions, for fluorescence imaging, the animals were treated using a special device (IVIS Lumina XRMS Series III) with excitation wavelength at 520 nm and emission wavelength at 620 nm. The measurements were conducted from the start of the study until the day of the initial clinical symptoms (day 28). For the first time, very strong fluorescence was observed on day 16, when the first clear clinical symptoms had not yet occurred. Importantly, during the test, only sick joints showed a clear and intense fluorescence, while the muscles showed poor fluorescence-this indicates that the photosensitizer accumulated selectively in areas of inflammation. The properties of the photosensitizer allow it to be used as a selective bio-imaging agent at the early (sub-clinical) stage of the disease without the risk of complications. Singlet oxygen quantum yields were determined using a time-resolved Nd:YAG laser set-up with excitation at 532 nm and a liquid N 2 cooled Ge photodetector. The nanowhiskers solution in the PDT experiment on rats revealed an ameliorating effect on the rheumatoid arthritis by decreasing significantly the IL-17 and TNF-α level in blood serum. In addition, fluorescent imaging was helpful in the diagnosis of the rheumatoid arthritis disease in subclinical stages and to bio-mark the rheumatoid arthritis affected joint. These findings are of a very important value, as rheumatoid arthritis is one of the major age-related diseases with roughly 1% of the population suffering from this disease, which is predicted to rise [66,77].
In another experiment, Rehman et al. utilized photodynamic therapy in the treatment of diabetes mellitus [67]. It is a severe metabolic disease that occurs in two types: type I, so-called insulin-dependent, and type II, non-insulin-dependent. Although PDT has been used for the treatment of many cancer diseases, its use in the therapy of metabolic diseases is still considered innovative. According to the procedure, each treated mouse was injected TSPP-TiO 2 and irradiated for 1 h with visible light (500-550 nm), every day for a week. The results indicated that such an approach was effective only in the case of non-insulin-dependent diabetes mellitus, as two hours after the treatment, a reduction in sugar levels by up to 33% was observed. The type II diabetes mellitus mouse model response to photoactivated nanocomposites was observed in lowering the blood glucose level, which could be due to ROS and 1 O 2 generation during PDT. As a consequence, it could influence cellular uptake and metabolism of the glucose with the help of insulin. As the authors pointed out, this could also be related to a decrease in the number of mitochondria in visceral adipocytes, leading to a reduction of white fat content in the body and greater sensitivity to insulin.
As already mentioned above, TiO 2 NPs allow to significantly increase the selectivity of porphyrin and chlorin photosensitizers and reduce their adverse effects. The properties of TiO 2 NPs allow the use of even potentially more toxic photosensitizers. Therefore, scientists have been trying to combine them with photosensitizers belonging to other chemical groups, including chlorins and methylene blue. In one of the studies, TiO 2 NPs were conjugated with the photosensitizer Chlorin e6 (Ce6) ( Figure 5) [68]. TiO 2 NPs were modified by adding layers consisting of two silane reagents (3-aminopropyl) triethoxysilane (APTES) and tetraethyl orthosilicate (PEGylated NPs: TiO 2 @ 4 Si-Ce6-PEG). Also, NPs modified only by APTES (APTES-modified NPs: TiO 2 -APTES-Ce6) were prepared. Ce6 was covalently bound to TiO 2 NPs (P25 containing 75% anatase and 25% rutile) through an amide bond. The photocytotoxicity of hybrid NPs was assessed on the glioblastoma cells (U87), after irradiation with a 652 nm light at fluence rate of 10 J/cm 2 . Nanoparticles modified with APTES alone demonstrated higher photodynamic activity in comparison to PEGylated core-shell structured NPs. In the first case, the unique photokilling effect of the photosensitizer was observed at the highest concentration and the cancer cell viability was decreased by 89% after the visible light illumination in the presence of 200 µg/mL of TiO 2 -APTES-Ce6 NPs, which held a concentration of 0.22 µM of Ce6. A similar effect was obtained using Ce6 alone, but at a much higher concentration (10 µM). This indicated that TiO 2 -APTES-Ce6 conjugates led to an increase in cellular uptake of the photosensitizer [68].
Tuchina et al. performed the photocytotoxicity study with one of the oldest photosensitizers used in the laboratory and medical practice-methylene blue (MB) ( Figure 5) [69]. They investigated the antimicrobial photodynamic activity of a mixture of two individuals-MB and TiO 2 NPs against S. aureus, E. coli, and Candida albicans. Suspensions of bacteria or fungi together with the photosensitizer were incubated in the dark for 10 min and then irradiated with two LED lamps simultaneously (405 and 625 nm). The mixture of both PSs and irradiation with red and blue light simultaneously, reduced the number of S. aureus cells by up to 90%. Almost identical results were obtained using a combination of photosensitizers against C. albicans. Curiously, almost no activity against E. coli was observed.
3 of 32 of metal NPs, including TiO2, depends on many factors, including particle rface coating, size, dose, and exposure route [8,9]. can generally enter the body in three ways: orally, transdermally, or by discussion on the bioavailability of TiO2 from the gastrointestinal tract are here are many indications that titania does not penetrate the gastrointestinal al extent. Animal studies have shown that 24 hours after oral administration e of 100 mg per kilogram of body weight, no significant increase in NP f the tested tissues was detected [10]. Analogous studies using even higher ilar results confirming that orally administered TiO2 does not penetrate the d that penetration is medically insignificant [11]. However, studies using the harmacokinetic modeling technique have indicated that high concentrations to their agglomeration and thus increase their uptake by macrophages. As al. [12], the biodistribution of TiO2 NPs can proceed via two kinetic processes penetrate through the blood vessels to the organs and by phagocytosis of NPs agocyte system. However, it should be emphasized that the pharmacokinetics s administration is different [12]. As in such case, the bioavailability of NPs is ution in the body should be carefully considered. In a study performed by ere administered intravenously with 5 mg TiO2 NPs per kg of body weight 28 days. The animals were healthy and behaved normally throughout the test cal study revealed that TiO2 did not accumulate at detectable levels in blood lymph nodes. However, titania levels were the highest in the liver, while concentrations were observed in the spleen, lungs, and kidneys [13]. rvation concerning TiO2 NPs excretion by the kidneys in rats was made by y noticed that TiO2 is eliminated from the body slowly, which indicates its lation. This issue is not severe considering PDT because the photosensitizer ce or several times during the photodynamic therapy [14]. Besides, the study [15] on rats showed that the TiO2 NPs level in urine was higher than in feces, on as the primary route of TiO2 NP elimination [15].

 To sum up
inetics of TiO2 NPs depends on many factors, including particle type, surface coating, size, dose, and exposure route. t penetrate the gastrointestinal tract at all or to a minimal extent. ical study indicates that after intravenous administration TiO2 NPs accumulate iver, and to some extent in the spleen, lungs and kidneys.
is the primary route of TiO2 NPs elimination. inetics and bioavailability of TiO2 NPs require further and intensive research.
patibility-in vitro and in vivo evaluation of the toxicity of titanium dioxide ion of titanium dioxide is related to its low toxicity. Many studies with TiO2 of nd microparticle sizes and crystal forms were performed to assess skin, lung, hematotoxicity. Although titania is a quite common ingredient of many

To sum up
The combination of photosensitizers with TiO 2 nanoparticles can be beneficial for the effectiveness of PDT and can reduce the side effects of chemotherapy. Among organic dyes, most often utilized for combining with TiO 2 are porphyrins and phthalocyanines, which were numerously applied as photosensitizers for PDT. The nitrogen-doping of TiO 2 NPs combined with phthalocyanines can significantly increase the efficacy of photodynamic activity, as it greatly enhances the formation of singlet oxygen and superoxide anion radicals, whereas it suppresses the generation of hydroxyl radicals.
Phthalocyanines anchored onto TiO 2 NPs and labeled with 131 I were assessed for PDT diagnosis of selected cancers. Hybrid materials composed of phthalocyanines, porphyrazines, or chlorines bound to TiO 2 were studied in terms of their effectiveness in antimicrobial PDT against bacteria, fungi, and parasites. The combination of fluorinated porphyrins and TiO 2 NPs after exposure to visible light revealed 7 logs reduction in colony-forming units of Staphylococcus aureus. The addition of KI transformed the hybrid materials into effective antimicrobial photosensitizers able to efficiently inactivate Gram-negative bacteria.
The application of PDT with the use of porphyrins and their derivatives as well as various NPs was extended to rheumatoid arthritis, atherosclerosis, macular degeneration, and diabetes mellitus. The combination of methylene blue with TiO 2 NPs irradiated with light sources simultaneously (405 and 625 nm) reduced the number of S. aureus cells by up to 90%. Almost identical results were obtained using a combination of photosensitizers against C. albicans.

TiO 2 Nanoparticles As a Vehicle for Chemotherapeutics
Cancer is considered one of the most significant challenges for modern medicine. Despite the continuous development of modern cancer treatment methods, the first line of therapy is the surgical removal of tumor and/or radiotherapy. Chemotherapy is usually a complementary therapy, but it is limited by many factors. First of all, chemotherapeutics are extremely toxic to rapidly proliferating, both cancer and healthy, tissues in the human organism. Therefore, novel delivery systems that would increase the tissue specificity of the therapy and reduce the systemic effects are still being sought. Many chemotherapeutic agents are also ineffective because of the multidrug resistance (MDR) mechanism displayed by cancer cells and related to the overexpression of some members of the ABC superfamily of efflux transporters that treat the drug as a poison and remove it from the matrix [78,79]. Currently, one of the most commonly studied chemotherapeutic drug is doxorubicin (DOX). Although it provides many advantages for the therapy of various cancers, the use of doxorubicin is associated with adverse effects, out of which, the cardiotoxicity is the most severe and dangerous [80]. Potential solution to both problems may be the use of nanoparticles and the combination of chemotherapy with photodynamic therapy or photothermal therapy ( Figure 6). Titania nanoparticles offer significant advantages in this field, enabling efficient delivery of the drug molecules, and thus better pharmacokinetics, and their targeted delivery [81,82].  Inorganic photosensitizers, including TiO 2 NPs, similarly to organic photosensitizers, can be used for PDT, which was presented above. TiO 2 NPs were investigated for PDT under the excitation of UV or visible light after doping with organic photosensitizers. Considering some limitations of UV light mainly related to harmful effects for human body accompanied by low penetration to tissues, it is worth exploring NIR-triggered inorganic photosensitizers for the non-invasive PDT in deep tissues. The rare-earth-doped nanoparticles converting NIR into UV light, NIR-triggered PDT of TiO 2 inorganic photosensitizers can be obtained by coupling two different NPs, including up-conversion NPs (UC NPs) and TiO 2 NPs. UC NPs can be coated by TiO 2 inorganic photosensitizers to form a core-shell structure or combined to form UC/TiO 2 nanocomposite with a non-core-shell structure. The construction of nanocomposite seems to be more beneficial as such a system can be applied for the chemotherapy and NIR-triggered inorganic PDT. Improvement of the DOX targeting to the cancer cells may be achieved by selective release of the drug in the desired site of action. This would then result not only in lowered toxicity of the anticancer agent, but also in increased concentration of the drug in cancer cells, enabling lower doses to be used and reducing the occurrence of adverse effects. Selected TiO 2 NPs combined with doxorubicin discussed in this chapter are summarized in Table 2. The role of TiO 2 -based nanomaterials in confinement of adverse toxic effects of DOX was presented by Zeng et al. [88]. The scientists designed folic acid (FA)-targeted NaYF 4 :Yb/Tm-TiO 2 nanocomposites and loaded them with DOX (FA-NPs-DOX) for near-infrared (NIR)-triggered inorganic PDT and enhanced chemotherapy to overcome MDR of breast cancers both in vitro and in vivo. An in vivo study was performed on female Balb/c nude mice bearing DOX-sensitive human breast cancer cells MCF-7 or DOX-resistant MCF-7/ADR tumors. The FA-NPs and FA-NPs-DOX-treated groups were additionally irradiated for 10 min with laser light (980 nm) with a power density of 500 mW/cm 2 . Mice bearing both types of tumor were more sensitive to FA-NPs-DOX conjugates compared to free DOX, demonstrating the excellent efficacy of FA-NP-DOX nanocomposites for DOX-resistant tumors. It is essential that the viabilities of DOX-resistant MCF-7/ADR cells decreased from 71.1% to 17.6%, whereas the tumor inhibition rate of MCF-7/ADR tumor-bearing nude mice increased from 6.41% to 96.74%, compared with free DOX. Also, treatment with FA-NPs alone gave quite good results (inhibition at 83.62% and 76.86%). In both cases, irradiation played a significant role, substantially increasing inhibitory effect (almost doubling it in both cases). In a study by Flak et al., TiO 2 -based hybrids combined with zinc(II) phthalocyanine (ZnPc@TiO 2 ) and folic acid (FA/ZnPc@TiO 2 ) were studied as the delivery system for doxorubicin [83]. Their research confirmed that DOX could be easily applied to these structures and released due to electrostatic interactions. Furthermore, cytotoxic studies demonstrated that these nanostructures are selectively captured by cancer cells. Cellular uptake was estimated based on monitoring of the concentration by fluorescent confocal microscopy in 2D and 3D cell cultures. DOX-loaded photosensitizers accumulated more in HeLa cells than in normal fibroblasts (MSU-1.1). It was found that DOX-loaded hybrid nanostructures were significantly more cytotoxic than non-loaded ones. Moreover, the cytotoxic effect of DOX-loaded hybrid nanostructures was even more severe upon the near-visible irradiation with the viable cell fraction below 2% [83].
Similar effects confirming the decreased cytotoxicity of doxorubicin-loaded on upconverting nanoparticles TiO 2 were presented by Chen et al. [84] and Tong et al. [89]. In the experiment, Chen et al. obtained special nanoplatforms (NaYF 4 :Yb,Tm@NaYF 4 ) coated with hollow mesoporous TiO 2 (UCNPs@mHTiO 2 ). Toxicity was tested on the HeLa cell culture irradiated with NIR light at 980 nm, thus triggering the UV emission and in this way stimulating TiO 2 towards the production of ROS. Porous and cavity structure of NPs allowed obtaining interesting antitumor drug DOX acid-enhanced drug release system, which was tested for intercellular specific chemotherapy. The synergistic effect of chemotherapy and PDT was presented within the study. Moreover, the excited energy from the higher-lying energy level (visible emission) of UCNPs was transferred to DOX via the luminescence resonance energy transfer mechanism. The intracellular drug release kinetics were studied by the recovery of the visible emission from UCNPs. The results showed a negligible toxicity (determined by MTT assay) of UCNPs@mHTiO 2 on HeLa cells (the cell viability above 95.89%), while the combination of PDT with DOX-UCNPs@mHTiO 2 exerted synergistic effect, causing death of almost 90% of cancer cells (the cell viability dropped to 11.26%). The optical bioimaging experiment revealed the potential application of the DOX-UCNPs@mHTiO 2 in theranostics as it proved the recovery of the upconversion luminescence by the diffusion of most DOX molecules into the media. A different approach was utilized by Tong et al. They combined up-conversion nanoparticle core-mesoporous silica shell that was coated with titanium dioxide as a photosensitizer [89]. This system was then loaded with DOX molecules, which were trapped in silica pores by UV-sensitive linker o-nitrobenzyl derivative-linker TC. What is interesting, the UV emission induced the photodecomposition of TC linker, allowing the DOX drug release. Toxicity studies performed on HeLa cells showed slight dark toxicity of this system. In a further study, the nanocomposite mediated ROS after NIR-irradiation and released DOX. After illumination for five minutes with NIR light, cell viability decreased to 85%, 79%, and 77% after the treatment with UCNPs@mSiO 2 /TiO 2 , DOX-UCNPs@mSiO 2 -TC, and DOX-UCNPs@SiO 2 /TiO 2 -TC, respectively. Complex studies also confirmed that cells can quickly capture the prepared nano vehicle by endocytosis or macropinocytosis, which potentially increases the effectiveness of the therapy. Everything considered, DOX-UCNPs@mSiO2/TiO 2 -TC nano vehicle revealed usefulness for NIR light-sensitive chemo/photodynamic synergistic therapy.
Wang et al. used diamond-shaped TiO 2 NPs for the delivery of DOX [85]. The prepared titanium dioxide nano bricks were functionalized with PEG chains and loaded with DOX molecules. Firstly, the material was found to exhibit pH responsiveness, as DOX was almost entirely released in acidic conditions, which are associated with cancer cells. Due to the porosity of the nanoparticles, the material could contain over 10% DOX. The material was then tested for its cytotoxicity on HepG2 cells, showing its non-cytotoxic nature (cell viability >80%) for empty nano bricks, whilst the viability for DOX-loaded NPs dropped to <10%. The subsequent in vivo test on Balb/c mice bearing H-22 tumors (murine hepatocellular carcinoma), showed the high activity of the nano bricks as drug delivery materials, because the mice treated with NPs were found to exhibit smaller tumor volumes as compared to the DOX-only treated group. Similar studies were conducted by Li et al. [86]. They tested the anti-tumor activity of the combination of doxorubicin with TiO 2 NPs in human hepatocarcinoma therapy. In this case of in vitro tests, better results were obtained of synergistic action of both compounds. An increase in the biocompatibility of TiO 2 and better photocatalytic activity was also observed [86].
One of the latest discoveries in the field of mixed chemotherapy and photodynamic therapy is the encapsulation of doxorubicin into "capsules" composed of Au-TiO 2 NPs. This combination allows the drug for easy diffusion around the tumor due to the acidic environment of the tissue core. The released chemotherapeutic agent-DOX acts directly on the tumor tissue and reaches a higher concentration in it. On the other hand, the nanoparticles that form the shell take an active part in PDT by generating reactive oxygen species (under the influence of UV light). PDT based on the doped material of Au-TiO 2 and Au-TiO 2 @DOX revealed cell-killing properties with its maximum achieved via a direct excitation process of the final product by laser light with a suitable wavelength of 500 nm. The synergistic combination of these substances resulted in a photocytotoxicity study in vitro on breast carcinoma cells MCF-7 in cancer-cell viability loss of up to 70%, according to MTT assay. The synergistic response of Au-TiO 2 @DOX via PDT was also confirmed in an in vivo study on the rat model [90]. As the development of gold-titania hybrids, Xu et al. elaborated a system of TiO 2 nanotubes, that were functionalized with gold nanoparticles [87]. Ampicillin-an antibacterial drug-was attached via an alkylsilane to the system. Such hybrid system was tested in vitro on E. coli bacteria cultures. The designed activity was achieved-upon visible light irradiation. The drug molecules were cleaved without degradation and acted in an antibacterial manner. The researchers loaded antibiotic-ampicillin sodium in the lower part of the hydrophobic TiO 2 nanotube stack and subsequently triggered visible-light-induced Au/TiO 2 surface plasmon resonance release allowing the carrying out of antibacterial studies towards E. coli. When the system was not illuminated, the drug was not released and the antibacterial activity of the nanomaterial was severely decreased.
A different, yet somewhat similar use for TiO 2 NPs was tested by Bakhshizadeh et al. [91]. They constructed a nanosystem comprised of titania core coated with mitoxantrone-loaded polymer based on diacrylated polycaprolactone as a biodegradable cross-linker and methacrylic acid or 4-vinylpyridine as the functional monomer. Authors utilized the ability of TiO 2 NPs to emit visible light upon irradiation with X-rays-the so-called scintillation nanoparticles. It was established that light emission of the NPs matched the absorption spectrum of mitoxantrone. In this way, a simple but effective method for light delivery in PDT for more deeply lying tumors was developed. The nanoparticles were than tested for their cytotoxicity against HT1080 cells (fibrosarcoma). Cell viability was found to be unchanged when the NPs alone were tested, while around 70% of cells were viable after X-ray irradiation.
3 of 32 of metal NPs, including TiO2, depends on many factors, including particle rface coating, size, dose, and exposure route [8,9]. can generally enter the body in three ways: orally, transdermally, or by discussion on the bioavailability of TiO2 from the gastrointestinal tract are here are many indications that titania does not penetrate the gastrointestinal al extent. Animal studies have shown that 24 hours after oral administration e of 100 mg per kilogram of body weight, no significant increase in NP f the tested tissues was detected [10]. Analogous studies using even higher ilar results confirming that orally administered TiO2 does not penetrate the d that penetration is medically insignificant [11]. However, studies using the harmacokinetic modeling technique have indicated that high concentrations to their agglomeration and thus increase their uptake by macrophages. As al. [12], the biodistribution of TiO2 NPs can proceed via two kinetic processes penetrate through the blood vessels to the organs and by phagocytosis of NPs agocyte system. However, it should be emphasized that the pharmacokinetics s administration is different [12]. As in such case, the bioavailability of NPs is ution in the body should be carefully considered. In a study performed by ere administered intravenously with 5 mg TiO2 NPs per kg of body weight 28 days. The animals were healthy and behaved normally throughout the test cal study revealed that TiO2 did not accumulate at detectable levels in blood lymph nodes. However, titania levels were the highest in the liver, while concentrations were observed in the spleen, lungs, and kidneys [13]. rvation concerning TiO2 NPs excretion by the kidneys in rats was made by y noticed that TiO2 is eliminated from the body slowly, which indicates its lation. This issue is not severe considering PDT because the photosensitizer ce or several times during the photodynamic therapy [14]. Besides, the study [15] on rats showed that the TiO2 NPs level in urine was higher than in feces, on as the primary route of TiO2 NP elimination [15].

 To sum up
inetics of TiO2 NPs depends on many factors, including particle type, surface coating, size, dose, and exposure route. t penetrate the gastrointestinal tract at all or to a minimal extent. ical study indicates that after intravenous administration TiO2 NPs accumulate iver, and to some extent in the spleen, lungs and kidneys.
is the primary route of TiO2 NPs elimination. inetics and bioavailability of TiO2 NPs require further and intensive research.
patibility-in vitro and in vivo evaluation of the toxicity of titanium dioxide

To sum up
TiO 2 in combination with anticancer agents offers a platform for more efficient delivery of chemotherapeutics. Thanks to either release mechanism used: pH-dependent, irradiation-triggered, or simple delivery, drug release in tumor cells is much higher than in healthy cells. As a result, the amount of drug used in the treatment can be significantly lower, while the pharmacological effect is maintained and fewer potential adverse effects occur. This can still be improved by not only combining different therapies, as shown by utilization of PDT and classical chemotherapy, but also by assessment of a mixture of anticancer drugs and other anticancer therapies. Most research concerns the combination of TiO 2 NPs with doxorubicin, and the results are encouraging. The decreased cytotoxicity of doxorubicin-loaded on upconverting nanoparticles containing TiO 2 was observed in many studies. Mixed chemotherapy and PDT were studied after encapsulation of doxorubicin into "capsules" composed of Au-TiO 2 NPs. The diffusion of the drug around the tumor was noted as the result of the acidic environment of the tissue core.

Other Applications of TiO 2 Nanoparticles in Medicine
Applications of titanium dioxide in medicine are going further than the design of drug delivery systems or applications as vehicles for chemotherapeutics. Titanium dioxide NPs have been applied in pharmacy, especially in pharmaceutical chemistry and technology, as well as medicine, including growing areas related to dentistry and surgery. The selected TiO 2 forms of potential applications in dentistry, surgery, and pharmacy discussed in this chapter are summarized in Table 3. In dentistry, the photochemical activity of titanium dioxide was utilized for the improvement of tooth personal care and teeth whitening. This property was demonstrated by Cuppini et al., who studied TiO 2 gel bearing H 2 O 2 and methylene blue [97]. They noticed that the combination of TiO 2 with H 2 O 2 allows for reducing the time necessary for tooth bleaching by 30 min of gel-tooth direct contact significantly [97]. Importantly, Kurzmann et al. reported initial toxicity studies of TiO 2 based gels for tooth bleaching. They observed that diluted gel formulations tested against L-929 cells, 3T3 cells, and gingival fibroblasts did not reveal any noticeable reduction in cell viability. The authors suggested additional experiments aiming to assess the TiO 2 gel toxicity [92]. Sodagar et al. solved the problem of caries next to brackets in orthodontic treatment. They proposed the addition of TiO 2 NPs at the concentration up to 10% into the orthodontic bond. The presence of TiO 2 in the composite decreased the colony counts of Streptococcus mutans and S. sanguinis. Unfortunately, the presence of titanium dioxide NPs resulted also in loss in the shear bond strength in comparison to unmodified composite. Finally, the addition of up to 5% of TiO 2 helped to achieve a compromise between bacterial growth reduction and loss in the shear bonding strength [98]. During the orthodontic treatment with a fixed appliance, the caries lesions are commonly spotted, especially next to orthodontic brackets. It is a result of difficult access to these areas in daily personal care. Therefore, the development of a new class of orthodontic bonds with bactericidal activity is very desirable. Sharma et al. reported the necessary shear bond strength for orthodontic treatment as 5.9-8 MPa [99]. The TiO 2 modified composite developed by Sodagar et al. with shear bond strength 13.9 MPa seems to be a promising material. It should be analyzed whether it is suitable for safe debonding of orthodontic brackets. In another study, Sun et al. prepared TiO 2 nanotubes and loaded them with tetracycline [100]. They carried out several tests against Porphyromonas gingivalis. Pure TiO 2 and loaded nanotubes showed great adhesion potential and antibacterial properties. Also, tetracycline was released quickly within 15 min of the experiment and the material was stabilized in 90 min [100]. TiO 2 nanoparticles have not only antibacterial but also antifungal properties. Huang et al. prepared Co-Cr alloy, on which they deposited a thin layer of TiO 2 . Several tests under UV-irradiation revealed that the prepared material exhibited significant antifungal activity and that it can be considered in the future against denture stomatitis [101].
Another significant area of titanium dioxide application is tooth hypersensitivity treatment, which is described as "sharp pain arising from exposed dentine in response to stimuli". Hypersensitivity is stimulated by warm, tactile, chemicals, osmotic cause, and others. It is estimated that 15% of the population suffers from tooth hypersensitivity. The majority of them are at the age between 20 and 40. Dentin is the main tooth structure that provides a skeleton, which is covered with a hard structure-an enamel. In dentine, there are located dentinal tubules, small channels through which the dentinal fluid is flowing from pulp to outside. In an insensitive tooth, only a few channels are opened. Due to the abrasion processes and gingival recession, more and more dentinal tubules are exposed to external factors. A hydrodynamic mechanism of tooth hypersensitivity is considered as the most accurate. According to this mechanism, mentioned factors cause an increase in the outward flow of dentine fluid. This phenomenon is a reason for the growth pressure, which directly triggers a mechanoreceptor response [102]. Commercial toothpaste containing TiO 2 is available on the market. Despite this fact, there is still interest in a new form of TiO 2 in order to achieve improved or better-occluding properties. For example, Onwubu et al. prepared eggshell-TiO 2 composite for occluding opened dentine tubules [93]. The authors noticed that the developed nanomaterial provided efficient dentine occlusion and allowed the covering of a large area of dentine. They compared their dentine occluding capability with commercially available toothpaste. Moreover, it was proved that presented eggshell-TiO 2 was resistant to acidic conditions. This property is highly relevant because of a decreased pH in the oral cavity after sugar consumption [93]. In another study, Sereda et al. functionalized titanium dioxide with chondroitin sulfate to increase the dentine adhesiveness property of nanomaterial. This TiO 2 -based nanomaterial deposited on dentine hampered the adhesion of acidogenic bacteria [103], which play a crucial role in caries formation.
Titanium dioxide scaffolds have been considered as interesting materials for medical applications, including the preparation of implants for surgery in bone tissue engineering [104]. Coating the surface of titanium endoprostheses with a bioactive titanium dioxide layer was also studied in terms of the efficiency of fibrointegration [94]. Atomic layer deposition coating of TiO 2 nano-thin films on magnesium-zinc alloys was found to enhance cytocompatibility for bioresorbable vascular stents [105].
In pharmaceutical sciences, titanium dioxide has been used as a pharmaceutical excipient in the manufacture of tablets as well as catalytic systems able to eliminate dangerous chemical and pharmaceutical pollutants. Lately, Hautala et al. have developed an ultrathin coating of minitablets by atomic layer deposition [106]. The obtained coat provides the tablets with improved properties related to the accelerated disintegration in the in vitro study [106]. TiO 2 nanoparticles can also be used for masking the bitter taste of drugs, which was presented by Amin et al. when the addition of TiO 2 NPs to azithromycin resulted in an improvement of taste and prolonged physicochemical stability of particles for 90 days [107]. Undoubtedly, a very evolving research field related to the tremendous amount of pharmaceutical pollutants in the environment is related to the development of photocatalytic systems based on TiO 2 NPs aiming to degrade and eliminate them from aquatic systems. The fast and efficient degradation of phenol using TiO 2 NPs was studied by Zulfiqar et al. [95]. The authors designed a photocatalytic system based on TiO 2 , which was applied for efficient removal of phenol with 99.48% yield during 540 min irradiation time [95]. Rendel et al. [108] studied the photodegradation kinetics of caffeine under different UV C doses at 254 nm in the presence of hydrogen peroxide and TiO 2 nanopowder. The removal rate of caffeine was higher than 95% for both agents separately [108]. In another study, photocatalytic degradation of atenolol by TiO 2 irradiated with an ultraviolet light-emitting diode was performed, and included a catalyst crystal phase (anatase TiO 2 , rutile TiO 2 , and mixed-phase), catalyst dosage, in the presence of co-existing anions, cations, and pH [96]. Recent approaches towards light-assisted photocatalytic removal of aqueous pharmaceutical pollutants have demonstrated the utility of titania and its derivatives not only with UV, but also with visible light, which was recently reviewed by Majumdar and Pal [109]. Moreover, photocatalytic degradation of pharmaceuticals, such as carbamazepine, diclofenac, and sulfamethoxazole by semiconductor (including titanium dioxide) and carbon materials, was also the subject of interesting research performed by Mestre and Carvalho [110]. In addition, TiO 2 -coated glass slides were applied for the study of a variety of oxidation reactions, including drug candidates and their oxidation products [111]. There are also reports showing that TiO 2 NPs revealed attractive potential as photocatalysts for anti-inflammatory, analgesic drugs [112], cyanide [113], atenolol [114], carbamazepine [115], β-blockers [116], and betamethasone-17 valerate [117]. Moreover, Ruokolainen et al. performed oxidation of tyrosine-phosphopeptides with TiO 2 particles, suggesting their potential as photocatalysts for biomolecules [118].
3 of 32 of metal NPs, including TiO2, depends on many factors, including particle rface coating, size, dose, and exposure route [8,9]. can generally enter the body in three ways: orally, transdermally, or by discussion on the bioavailability of TiO2 from the gastrointestinal tract are here are many indications that titania does not penetrate the gastrointestinal al extent. Animal studies have shown that 24 hours after oral administration e of 100 mg per kilogram of body weight, no significant increase in NP f the tested tissues was detected [10]. Analogous studies using even higher ilar results confirming that orally administered TiO2 does not penetrate the d that penetration is medically insignificant [11]. However, studies using the harmacokinetic modeling technique have indicated that high concentrations to their agglomeration and thus increase their uptake by macrophages. As al. [12], the biodistribution of TiO2 NPs can proceed via two kinetic processes penetrate through the blood vessels to the organs and by phagocytosis of NPs agocyte system. However, it should be emphasized that the pharmacokinetics s administration is different [12]. As in such case, the bioavailability of NPs is ution in the body should be carefully considered. In a study performed by ere administered intravenously with 5 mg TiO2 NPs per kg of body weight 28 days. The animals were healthy and behaved normally throughout the test cal study revealed that TiO2 did not accumulate at detectable levels in blood lymph nodes. However, titania levels were the highest in the liver, while concentrations were observed in the spleen, lungs, and kidneys [13]. rvation concerning TiO2 NPs excretion by the kidneys in rats was made by y noticed that TiO2 is eliminated from the body slowly, which indicates its lation. This issue is not severe considering PDT because the photosensitizer ce or several times during the photodynamic therapy [14]. Besides, the study [15] on rats showed that the TiO2 NPs level in urine was higher than in feces, on as the primary route of TiO2 NP elimination [15].

 To sum up
inetics of TiO2 NPs depends on many factors, including particle type, surface coating, size, dose, and exposure route. t penetrate the gastrointestinal tract at all or to a minimal extent. ical study indicates that after intravenous administration TiO2 NPs accumulate iver, and to some extent in the spleen, lungs and kidneys.
is the primary route of TiO2 NPs elimination. inetics and bioavailability of TiO2 NPs require further and intensive research.

patibility-in vitro and in vivo evaluation of the toxicity of titanium dioxide
ion of titanium dioxide is related to its low toxicity. Many studies with TiO2 of nd microparticle sizes and crystal forms were performed to assess skin, lung, hematotoxicity. Although titania is a quite common ingredient of many especially sunscreens, powders, and eyeshadows, it seems that its size and nd rutile) influence the safety of its usage. vivo studies concerning the skin-related toxicity of TiO2 NPs raised two issues, self and systemic toxicity associated with skin permeation. Wu et al. studied ation of TiO2 NPs in hairless mice and porcine skin after subchronic dermal g to the presented findings, the researchers concluded that the nanosized TiO2 sk to humans after chronic dermal exposure over a relatively long period, tissue distribution. In another study, Crosera et al. studied both TiO2 NPs' ells for 24 h using intact and needle-abraded human skin as well as evaluated keratinocytes. The study demonstrated that the presence of TiO2 NPs was

To sum up
TiO 2 NPs were evaluated for use in pharmacy, especially in pharmaceutical chemistry and technology, as well as medicine, including growing areas related to dentistry and surgery. In dentistry, the photochemical activity of TiO 2 was utilized for the improvement of tooth personal care and teeth whitening. Eggshell-TiO 2 composite was found useful for occluding opened dentine tubules, allowing for efficient dentine occlusion. TiO 2 scaffolds were applied for the preparation of implants for surgery in bone tissue engineering. In pharmaceutical sciences, TiO 2 was applied as a pharmaceutical excipient in the manufacture of tablets, as well as a catalytic system able to eliminate dangerous chemical and pharmaceutical pollutants

Summary
Recently, many studies have been focused on the broad applications of titania in technology and medicine, from dye-sensitized solar cells, photodynamic therapy, to water remediation. It is possible because these nanoparticles reveal excellent photochemical properties and high biocompatibility. Moreover, TiO 2 particles are quite cheap and accessible components. Photosensitizing properties and manufacturing costs of these materials are usually related to the number of active sites on their surface. After irradiation, they can produce reactive oxygen species that spread from the nanoparticles and induce cell death in the neighboring tissues. Therefore, applications of TiO 2 nano-and microparticles in photodynamic therapy are widely explored. Another application of TiO 2 nanoparticles is related to their use as a drug carrier, allowing drugs to reach diseased areas of the body while keeping healthy tissues unharmed. Therefore, much attention must be paid to develop novel formulations allowing to direct the active substances to target cells and minimize the side effects. A plethora of various studies above indicate that an increase of TiO 2 therapeutic efficiency can be reached using targeted drug delivery systems and nanocomposites. It is possible because the surface of TiO 2 NPs can also be labeled with antibodies or markers in order to design drug delivery towards selected, diseased areas.
For more extensive usage of TiO 2 NPs, some of their drawbacks should be overcome. The applications of neat TiO 2 nanoparticles in photodynamic therapy are limited by the necessity to use UV light for their excitation. Fortunately, numerous methods of doping and functionalization of the TiO 2 NPs have been developed. These modifications result in the shift of NPs absorption band towards longer wavelengths, desirable for PDT. Combinations of titania with other nanoparticles such as up-conversion nanoparticles also enable it to bypass the direct use of UV light. Titanium dioxide particles are prone to form agglomerates in physiological pH, hampering their solubility. To prevent this unwelcome issue, the functionalization of TiO 2 with bulky substituents like PEG chains or the use of surfactants was proposed. It could also enable the uniform distribution of the nanoparticles and thus repeatability of dosing accompanied by clinical effect. In the end, the crucial issue concerning the use of TiO 2 in the medical field has to be pointed out. Available data do not present a full profile of the TiO 2 particles' fate in the body as well as their toxicity. Therefore, extensive studies in this area are urgently needed.
The studies discussed in this review indicate that both composites and conjugates of titania with various molecules and biomolecules can significantly improve their broader applications in medicine.