Biomedical applications of nano-titania in theranostics and photodynamic therapy

Abstract

Titanium dioxide (TiO₂) is one of the most abundantly used nanomaterials for human life. It is used in sunscreen, photovoltaic devices, biomedical applications and as a food additive and environmental scavenger. Nano-TiO₂ in biomedical applications is well documented. It is used in endoprosthetic implants and early theranostics of neoplastic and non-neoplastic maladies as a photodynamic therapeutic agent and as vehicles in nano-drug delivery systems. Herein, we focus on the recent advancements and applications of nano-TiO₂ in bio-nanotechnology, nanomedicine and photodynamic therapy (PDT).

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F. U. Rehman

Fawad Ur Rehman has been a Ph.D. candidate in the School of Biological Science and Medical Engineering, State Key Lab of Bioelectronics, Southeast University, Nanjing, China since 2013. Before this, he got his Masters degree from The University of Agriculture, Peshawar and his Bachelors degree (DVM) in Veterinary Medicine and Surgery from the University of Veterinary and Animal Sciences, Lahore, Pakistan. Currently, his interest is in bio-imaging and photodynamic therapy with nano-TiO2 and porphyrin derivatives for various disease theranostics, including rheumatism, cancer, and stem cell therapeutics.

C. Zhao

Chunqiu Zhao was born in 1990. He obtained his B.E. degree from Central South University, China. He has been enrolled in the School of Biological Science and Medical Engineering, Southeast University, China for a doctoral degree since 2013. His research interests are the application of nano materials for bio-imaging and photodynamic therapy.

H. Jiang

Dr Hui Jiang is an associate professor of biomedical engineering at Southeast University. He obtained his Ph.D. (Chemistry) from the University of Science and Technology of China in 2005. After a postdoctoral fellowship at Nanjing University, he joined the State Key Laboratory of Bioelectronics, Southeast University in 2007. His research focuses on nano-based electro-chemiluminescent biosensors and the electron transfer behaviors of the micro–nano interface.

X. Wang

Dr Xuemei Wang is currently a full professor of biomedical engineering at Southeast University. She obtained her Ph.D. in Chemistry from Nanjing University, China, in 1994 and became a lecturer at Nanjing University in 1995. She was an Alexander von Humboldt Fellow in 1997 in the Chemistry Department, University of Saarland, Germany, and then she joined the State Key Laboratory of Bioelectronics, Southeast University in 1998. Her research focuses on bioelectronics and biosensors, nanomedicine, bio-imaging and highly sensitive diagnosis strategies for cancers and other related diseases.

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1. Introduction

The role of nanotechnology (NT) in our life is inevitable. Naturally, the existence of bio-nanotechnology (BNT) can be traced back to the initiation of first life on earth. The inter- and intra- cellular transfer of nanosized biomolecules (e.g. low density lipoproteins), neural transmission and memory storage in biological systems are the prototype examples of BNT.^1,2 Similarly, biomolecules like proteins, DNA and RNA are on the nano-scale and are structural analogues to nanomaterials, which introduces the concept of integrating nanomaterials with biomolecules to understand the cellular signal pathways, functional mechanisms and cellular interactions within living cells and organisms.³ In nature, dust particles, smoke and ink are examples of non-biological NT.⁴ BNT existed from the beginning of life and its potential has been explored with the advancement in human civilization by realizing the great impact BNT has on our day to day life. Similarly, enzymes, antigens, antibodies and ligand receptors with 2–20 nm sizes in either direction, are structural analogues to nanomaterials, and thus they provide plenty of scope for BNT biomedical applications.^5,6

The distinctive properties of fabricated inorganic-nanomaterials (INM), including their high reactivity, increased surface area, lower melting point, ductility and charge conductivity, make them suitable candidates for biomedical applications and BNT.^7,8 Since these INM can readily interact with organic biomolecules by exhibiting excellent conductivity and site recognition within cellular gadgetry, they can be reintroduced to cells and biological systems. Mostly, the physico-chemical properties, including the shape,⁹ size¹⁰ and surface chemistry,¹¹ decide the fate of the INM. Surface modification of INM leads to relevant changes in the physico-chemical properties that make them suitable for certain disease treatments, e.g. –COOH functionalized MWCNT nanowhiskers (NW) are more hydrophilic and less toxic than pristine or untreated nanoparticles and whiskers.¹²

Titanium is one of the most abundantly available elements in nature with the most common forms being the metallic state of T, TiCl₄ and titanium dioxide (TiO₂); the latter one exists with well-known crystalline shapes of rutile, brookite, TiO₂-B and the most reactive – anatase.^13–15 It has also been proven that mixed polymorphs of TiO₂ are more efficient for biomedical applications than single crystals, e.g. anatase 80% and rutile 20% are more efficient than anatase 100%,¹³ similarly TiO₂-B1 and anatase have been reported as being more reactive.^16,17 These polymorph additive effects can be attributed to the surface interface among various polymorphs, reducing the recombination of photo-generated electron hole pairs and thus resulting in more ROS generation.¹³

Currently, more than thirteen hundred nanomaterials are globally available,¹⁸ among them TiO₂ is the second most abundantly consumed material in our daily life.¹⁹ Food grade TiO₂ is named as E171 in the European Union.²⁰ In the US, the daily consumer statistics reveal that every child under the age of ten years is exposed to 1–2 mg of TiO₂ per kg_{body weight (bwt)}, whereas the exposure rate for other age groups is 0.2–0.7 mg per kg_bwt.²⁰ TiO₂ was used as a white pigment in ancient times,²¹ and its long term exposure to humans and the environment guarantees its less toxic nature.

Despite the extensive use of TiO₂ in other fields, the history of TiO₂ in the biomedical field is relatively new. The first nano-TiO₂ biomedical application can be traced back to the early nineties of the 20^th century, when Japanese scientist Fujishima and his coworkers reported that TiO₂ had photodynamic therapeutic properties by killing cancer cells (HeLa).²² At the same time another study reported the successful application of nano-TiO₂ in medical implants by coating TiO₂ as a redox agent on their surface.²³ Soon after, these reports on TiO₂ caused momentum in the biomedical applications field and the number of publications and citations exponentially increased by the start of the 21^st century. In this review we will mainly focus on the most recent advancements of nano-TiO₂ and its bio-applications in the biomedical realms.

2. Nanotechnology and bio-application of TiO₂

Nanotechnology (NT) is based on engineering science at the molecular level and is used in biomedicine for therapeutics and diagnostics, which are collectively termed as “Theranostics”.²⁴ Nano-drug delivery systems, bio-imaging, photodynamics, sonodynamics and photothermal therapy are among the few vital applications of BNT. The European Union defines a nanomaterial as a “manufactured, incidental or natural material containing particles as an aggregate, agglomerate or an unbound; where fifty percent exhibit 1–100 nm size in any dimension”.¹⁴ Generally, the size of the nanoparticle (NP) must be in the range of 1–100 nm in any dimension, as described by the National Nanotechnology Initiative; however the term “Nano” describes the size in several hundred nanometers. Their tiny nano-size is advantageous for crossing easily through various cell and tissue barriers with desired effects. NP lodging in desired sites depends upon the unique microenvironment, i.e. temperature, pH, ROS, metabolic rate, osmotic pressure, etc.²⁵ In some cases NPs are conjugated to antibodies or active ligands, like peptides and folic acid, that are highly specific to achieve sufficient accumulation in the desired tissue. This approach is known as an active mechanism,²⁶ similarly most of the neoplastic tissues bear enhanced vascular permeability and retention (EPR) effects that can be termed as a passive mechanism to achieve the desired nano-drug concentration^27,28 (Fig. 1).

Fig. 1 Schematic presentation of enhanced permeability and retention (EPR) effects in tumor tissue. The nanomolecules can easily cross the blood capillary endothelial cells whereas micromolecules can only stay in the tumor tissue due to the larger intercellular space compared to normal tissue.

As one of the most abundantly used nanomaterials, titanium dioxide (TiO₂) has been widely utilized as a food additive and environmental scavenger, as well as in sunscreen, photovoltaic devices, and biomedical treatments. When TiO₂ is irradiated with a certain light band (i.e. UV), it will generate electron–hole pairs that react with the surrounding oxygen to form various reactive oxygen species (ROS), i.e. OH⁻ or O₂⁻ radicals, by redox reactions.²⁹ Waste water purification via TiO₂ by utilizing the natural sun light is the most valuable application in our day to day life.^30,31 In fact, the photo-generated radicals are used as sanitizers for polluted water containing various dyes and phenols, or for other organic pollutants in the environment.^29,32 It has been reported that due to the wide band gap, TiO₂ photo absorption is limited to the UV light spectrum, i.e. 4 percent of the total sun light, however, metal ions and nonmetal doping can extend TiO₂ photo absorption to the visible range.³³ The aforementioned approaches are the key features of nano-TiO₂ for biological applications and modern theranostics. Mostly, the nano-TiO₂ applications depend upon the type of polymorph used (e.g. one dimensional or three dimensional particles), surface modification, size of the particle and the subject system, i.e. biological, photoelectrical, etc.³⁴

2.1 Nano-TiO₂ fabrication

Various techniques have been used so far for the fabrication of nanomaterials, including the sol–gel method, hydrothermal processes, chemical vapor deposition and electrospinning.^35,36 The later method is more favorable for the fabrication of one dimensional TiO₂ nanomaterials, e.g. nanowhiskers, nanofibers, nanobelts, necklace-like nanofibers and nanowires, etc.^30,37 Moreover, the electrochemical anodization technique is considered more favorable for nanotube synthesis, in addition to the chemical template synthesis and alkaline hydrothermal treatment,³⁸ and allows great control over the dimensions during the synthesis.^39,40 The structure of TiO₂ nanotubes is not clearly defined, however, it is assumed that hydrogen titanate is present where the TiO₂ sheets are separated by H⁺ ions.⁴¹

TiO₂ nanowhiskers are better than particles for photocatalytic activity⁴² and charge transportation, which makes them suitable candidates for solar cells and windscreens.⁴³ Moreover, the better charge recombination rate and oxygen affinity of nano-TiO₂ make it a suitable remedy for environmental recycling and an efficient catalyst.⁴⁴

TiO₂ nanowhiskers are also known for their synergistic effect, which is attributed to their photocatalytic activity, that makes TiO₂ an excellent phototherapeutic agent. Moreover, its large surface area and reactivity provide scaffolds for excellent drug delivery systems. Recently, García-Valverde et al. reported the successful coating of carbon on TiO₂ nanotubes for improved application in biomedical realms.⁴⁵ Similarly, the synergistic effect for carrying anticancer drugs like daunorubicin (DNR) to cancer cells (i.e. hepatic carcinoma cells SMMC-7721) and the PD therapeutic effect when irradiated with UV light, mean that TiO₂ nanowhiskers could play important roles in future cancer and other disease theranostics⁴⁶ (Fig. 2).

Fig. 2 Fluorescence microscopic results showing SMMC-7721 cancer cells treated with DNR (a), DNR–TiO₂ nanoparticles (b), and DNR–TiO₂ nanowhiskers (c). (d) represents the possible mechanism for cellular uptake of TiO₂ nanowhiskers in SMMC-7721 cancer cells. Adapted from ref. 46. Copyright 2009 Elsevier Ltd.

2.2 Nano-TiO₂ pharmacodynamics

TiO₂ nanoparticles are commonly used in industry as consumer products and for biomedical applications due to their strong catalytic activity and smaller size, which provides a large surface area per unit mass and a maximum number of electrons on the surface to enhance their reactivity.¹⁴ The safe dose for TiO₂ nanoparticles reported by previous studies is 5 g per kg_bwt in rat models, with no adverse effects and rapid distribution when administered systemically.^47,48 TiO₂ nanoparticles can be administered orally, as an aerosol or by systemic parenterals (intravenous, intramuscular or sub-cutaneous) without any adverse effects with the exception of less to none transdermal penetration when applied topically.⁴⁹ It is evident that the liver, spleen, kidney and lungs are the main organs for TiO₂ nanoparticle deposition, while blood cells, plasma and the brain are least affected.⁵⁰ Nevertheless, recently it has been reported that 5 nm TiO₂ nanoparticles as intraperitoneal injections for 14 days and 80–155 nm particles in the transnasal location can cross the blood–brain barrier^51–54 (Fig. 3).

Fig. 3 Brain tissue histopathology (H&E stain) after 60 days of TiO₂ nanoparticle administration in ICR mice. Here (a) shows normal brain tissue at 100×; (b) shows 5 mg per kg_bwt TiO₂ nanoparticle administration resulting in higher calcium deposition in the brain tissue (400×); (c) shows 10 mg per kg_bwt TiO₂ nanoparticle administration resulting in spongiocyte proliferation indicating pathological lesions (200×); (d) shows 10 mg per kg_bwt TiO₂ nanoparticle administration indicating proliferating ependyma (100×); (e) shows 50 mg per kg_bwt TiO₂ nanoparticle administration indicating calcium deposition (400×) whereas (f) shows the 50 mg per kg_bwt TiO₂ administered group with spongiocyte proliferation. Adapted from ref. 52. Copyright 2010 Elsevier Ltd.

3. Biomedical applications of nano-TiO₂

3.1 Photodynamic treatment

Photodynamic therapy (PDT) is a therapeutic technique with three main components, i.e. a photosensitizer (PS), a light source and an oxygen molecule. A nontoxic photosensitizer (i.e. a macromolecular compound⁵⁵ or nano-sized organic⁵⁶ or inorganic particle⁵⁷) can be introduced into living tissues and cells. These PSs are excited by the absorption of photons from the visible light source and transfer energy to the oxygen molecule to produce singlet oxygen (¹O₂) and reactive oxygen species (ROS).^58,59 PDT has more advantages over the conventional theranostics for cancer, i.e. it has a higher safety record, is minimally invasive, is cost effective, and is highly localized with low to no complications.⁶⁰

PDT is most effective in delicate areas and natural cavities such as the bladder, pulmonary tissues, head and neck, brain, pancreas, prostate, mammary tissue and intraperitoneal cavity, where invasive surgery always carries a great deal of risk.^61–63 PDT is still an ideal candidate and first choice for dermal neoplastic and non-neoplastic maladies. Moreover, the first ever therapeutic effect of PDT was for dermal malignancies.⁶⁴ PDT is more effective for the treatment of tiny and superficial tumors, e.g. on internal body organ linings, above and just below the skin and exposed body cavities (i.e. oral, vagina, rectum, urethra, bladder, etc.). In deep seated tumors, its efficacy is low due to less penetration of the excitation light to generate an ample therapeutic quantity of singlet oxygen (¹O₂) and other reactive oxygen species (ROS). PDT’s potential against dermal and oral microbial infections (e.g. halitosis) has been proven to be more efficient.⁶⁵

The early diagnosis of benign and malignant tumors is still a challenge for modern therapeutics and the only available treatment is invasive surgery, chemotherapy or radiotherapy. Hence, fluorescence imaging provides a suitable alternative for early diagnosis and PDT for cancer^66,67 as well as for other infectious diseases.^68,69 This fluorescence imaging and therapy as a combination can also be termed as “photodynamic therapy” (PDT).⁷⁰ At the beginning of the 20^th century, PDT was rediscovered by Oskar Raaband Hermannvon Tappeiner, although in ancient China, Egypt and the subcontinent PDT was already well established for dermal infections and as a natural sanitizer for households.^65,71 The role of PDT is not only vital in onco-therapy but also in non-neoplastic cardiovascular,^72–74 pulmonary,^75,76 dermal,^77,78 ophthalmic,⁷⁹ oral^59,80 and inflammatory diseases along with rheumatoid arthritis as an autoimmune reaction suppressant.^81–83 Mostly, photosensitizers and drug delivery systems are sensitive to UV light in the range of less than 400 nm wavelength,⁸⁴ however, this range has limited penetration (because of the short wavelength) and can damage the DNA as an adverse effect.^85,86 For deep tissue penetration, the near infrared (NIR) light range (620–850 nm) is more favored because of the less harmful and maximum penetration properties.⁸⁷ The NIR light falls in the phototherapeutic window that ranges from 600 to 1000 nm.⁸⁸

Photosensitive nanocomposites like TiO₂ nanoparticles could be administered systemically to the living tissue and upon attaining a certain concentration in the desired location, visible light or NIR light is passed through the subject tissue. These NPs (PSs) absorb the light in the form of a photon and transfer it to local oxygen containing molecules that release various kinds of ROS and ¹O₂. When light energy is passed through the PS, the ground state oxygen is excited to ¹O₂ and then to the triplet oxygen (³O₂) energy state (Fig. 4).⁸⁶ The half-life of singlet oxygen within the living tissue is estimated to be ∼3 μseconds.^89–91 Moreover, once the PS is photoactivated it produces ROS for 18 hours, which is sufficiently long enough to induce apoptosis within the target cell and adverse effects in the surrounding environment.⁹²

Fig. 4 Schematic presentation of ROS and singlet oxygen generation from TiO₂ when photoactivated with UV range light (A). Fluorescence generated from nano-TiO₂ after photoactivation in fibroblast cells, in vitro (B) and in vivo in a rheumatoid arthritis foot in a murine model (C).

Pristine nano-TiO₂ is susceptible to photoactivation under UV range light (400 nm).⁹³ The WHO approved therapeutic window starts at a relatively higher wavelength (more than 600 nm) due to the fact that proteins, haemoglobin and melanin are also excited under UV range light, which could cause severe adverse effects.⁹⁴ To make pristine TiO₂ amenable for PDT desired effects, it should be either coupled with up-converting surface modifiers or coated with materials of the desired range. Synthetic or naturally occurring agents that upon excitation with certain light produce reactive oxygen species (ROS) are termed as “photosensitizers” (PSs).^95,96 PSs are the main and most important components of PDT.^83,97 Porphyrin derivatives are the most common and prototype PSs^98,99 that are composed of macromolecules with excellent potential for singlet oxygen generation and the least known toxicity.^100,101 Porfimer sodium is an example of an approved PS in the US by the Food and Drug Administration (FDA) for clinical applications, whereas zinc(II) phthalocyanine is an example of a second generation of clinically approved PSs, with high tumor cell selectivity and higher reactivity to generate ⁻O₂.⁶⁰

Most of the PSs are hydrophobic in nature, which impedes their parental administration. This bottleneck develops the concept for hydrophilic nano-PSs, such as micelles, liposomes, low density lipoproteins,^102–105etc. Nevertheless, these PSs’ hypersensitivity reactions and accumulation in the skin and eyes are the leading factors for severe phototoxicity.¹⁰⁶ Therefore, to overcome these snags, the concept of nano-drug delivery systems is promising due to the hydrophilic nature and the safe delivery to the target tissue. Most of the drug delivery systems are nano-silica based, organically modified⁵⁷ or selected metallic and non-metallic PSs, e.g. gold,^107,108 silver,¹⁰⁹ zinc,^110,111 carbon fibers and complexes,^112,113 platinum,^114,115 titanium,¹¹⁶ iron,^117,118 cerium,¹¹⁹ ruthenium,¹²⁰etc., and/or their hybrid clusters.^121–124

Recently NT combined inorganic materials with organic macromolecules as PSs to achieve their synergistic effects in PDT.^125,126 Over the last few decades, the potential of nano-TiO₂ as an inorganic PS for PDT has been extensively explored.¹²⁷ Nano-TiO₂ as a PD-therapeutic agent has become popular for various disease therapies, including for cancer^128,129 and microbial infections,¹³⁰ and in food safety and sanitation.¹³¹ The reason for using TiO₂ as a PS in PDT is that it becomes super hydrophilic when exposed to UV light and this functionality can be reversed, depending upon the light it is exposed to.¹³²

Hou et al. designed NIR light up-converting TiO₂ nanoparticles to explore their potential in PDT for cancer amelioration.¹³³ They used NaYF₄:Yb³⁺,Tm³⁺@NaGdF₄:Yb³⁺ as up-conversion nanoparticles (UNCP) to form UNCP@TiO₂ for the photoactivation of TiO₂ nanoparticles by NIR light. When these UNCP@TiO₂ were introduced into cancer cells in vitro and photoactivated by NIR light, the TiO₂-generated ROS induced apoptosis by decreasing the mitochondrial membrane potential, which releases cytochrome C to activate caspase3 for apoptosis initiation in cancer cells. Furthermore, in vitro, these UNCP@TiO₂ also suppressed tumor growth in murine models (Fig. 5).

Fig. 5 Near infrared light activated TiO₂ nanoparticles damage mitochondria to release the cyto C enzyme for caspase activation to induce apoptosis in target cells during photodynamic therapy. Reprinted with permission from ref. 133. Copyright 2015 American Chemical Society.

Nano-TiO₂ has also been used in combination with other nanomaterials and macromolecules where it exhibits excellent synergistic effects, e.g. Zheng et al. used TiO₂–Fe₃O₄ nano-composites for neoplastic disease theranostics, where nano-TiO₂ is used as a PS for PDT and Fe₃O₄ is used as a contrast agent for Magnetic Resonance Imaging (MRI).¹²⁷ Tokuoka et al. used chlorine e6 in combination with TiO₂ nanoparticles for the murine thymic lymphoma cancer cell line (EL-4). The nano-TiO₂ combined with chlorine e6 had a significant effect on the cell line after photoactivation in the visible range compared with separate treatment with TiO₂ and chlorine e6.¹³⁴In vitro these results could be achieved, but in vivo biomedical applications may not be promising due to the weak electrostatic adsorption force of nano-TiO₂ that systemically interacts with the plasma proteins in circulation. Although this concept is challenging, our group recently successfully delivered tetra sulphonatophenyl porphyrin (TSPP) to inflamed joints by TiO₂ nanowhiskers in photodynamic therapy for rheumatoid arthritis early diagnosis by fluorescence bio-imaging and treatment.¹³⁵

3.2 Sonodynamic therapy with nano-TiO₂

Recently, ultrasound activated sensitizers received attention and gained popularity for the remedy of deep seated tumors and other infectious diseases, due to the deep penetration ability of ultrasound radiation. In 1989, Umemura et al. were the first to investigate the concept of sonodynamic therapy by ultrasonically activating hematoporphyrin, which was a well known photosensitizer back then.¹³⁶

Most of the sonosensitizers used are organic macromolecules (porphyrin derivatives, phthalocyanines, 5-aminolevulinic acid, etc.) that produce ROS upon ultrasonic activation.^137–139 Harada and his co-workers found that TiO₂ nanoparticles activation by ultrasound radiation could be utilized to treat neoplastic melanoma C32 in vitro as well in vivo.¹⁴⁰ They activated nano-TiO₂ by 1 MHz, 1.0 W cm⁻² for two minutes and found a significant decrease in the volume and growth suppression of neoplastic tissue (Fig. 6).

Fig. 6 Sonodynamic therapy with nano-TiO₂ to induce apoptosis in cancer cells. Reprinted with permission from ref. 140. Copyright 2011 Elsevier B.V.

Shen et al. combined Fe₃O₄@TiO₂ (core@shell) nanoparticles and a chemotherapeutic agent, i.e. doxorubicin (DOX), to evaluate its potential for the successful delivery to neoplastic tissue and this is termed “chemo-sonodynamic therapy”.¹⁴¹ They found that TiO₂ irradiation with ultrasound generated the ROS and ensured the safe delivery and retention of the chemotherapeutic agent post intravenous injections in murine models. Shen et al. also used Fe₃O₄@TiO₂ nanoparticles for sonodynamic therapy of cancer cells and found more than 50% reduction in cancer cell viability.¹⁴² The synergistic effect in chemo-sonodynamic therapy of NaYF₄–Fe₃O₄@TiO₂ nanocomposites with a hyaluronic acid coating for cancer was explored by Shen et al.¹⁴³ They reported the successful uptake of DOX loaded nanocomposites by MCF-7 and KB cells in vitro. In xenograft models, the combined synergistic inhibitory effect of chemo-sonodynamic therapy was 88.36% as compared to 38.91% and 28.36% for separate sonodynamic and chemotherapy, respectively.

Yamaguchi et al. reported TiO₂–PEG nanoparticles and explored their sonodynamic and photodynamic therapeutic effects on human glioblastoma cells U251. They activated the TiO₂–PEG sonosensitizers at 5.0 mW cm⁻² and evaluated the effect by MTT assay.¹⁴⁴ Their results demonstrate the promising role of TiO₂–PEG sonosensitizers since they significantly decrease the viability and membrane integrity in the subject tumor cell line.

Ninomiya et al. reported S1/S2 protein mobilized TiO₂ nanoparticles for sonodynamic therapy of HepG2 cancer cells in vitro¹⁴⁵ and xenograft tumor murine models in vivo.¹⁴⁶ Ultrasonic activated TiO₂ nanoparticles activated at 0.5 MHz and 1 MHz for 60 seconds induced apoptosis and reduced viability by 46% in the HepG2 cells. Similarly, when 0.1 mg of TiO₂ nanoparticles were directly injected into tumor tissue, five times in 13 days, the tumor was reduced within 28 days.

¹O₂ singlet oxygen generation by TiO₂ nanoparticles is well known after photoactivation. Recently, Harada et al. also reported ¹O₂ oxygen generation by TiO₂ nanoparticles with core–shell type polyallylamine micelles. When these TiO₂ containing micelles were introduced into HeLa cancer cells, they generated ¹O₂ along with other ROS after sonoactivation.¹⁴⁷ These findings are very important and can revolutionize sonodynamic therapeutics in terms of bio-imaging and theranostics, especially for neoplastic maladies.

3.3 Nano-TiO₂ for medical implants

Medical implants have revolutionized orthopedic surgery, especially endoprosthetic surgeries. However, autoimmune reactions against the orthopaedic implants and cardiovascular stents hamper their liberal application. Various metallic combinations have been used so far, but Ti and its various alloys, especially nano-TiO₂ surface coated implants, are reported to be the most promising for reducing autoimmune reactions. The thin passivation layer of nano-sized TiO₂ minimizes the autoimmune reaction between the underlying bone tissue surfaces and the implant.¹⁴⁸

TiO₂ nanotube implants are used in prosthetic articular surgery of orthopedic patients with tibia-tarsal joints (knee) or acetabulum in hip joint fractures, however, their success rate is not very good due to degradation of the materials or promotion of the generation of chronic inflammation within the implanted tissues.^149,150 Nevertheless, some studies suggest that nano-TiO₂ provides more safe scaffolds for tissue recognition in endo-prosthetic surgery.^151,152 Many studies have reported that TiO₂ nanotubes enhance the bone mineralization, osteoblast adhesion in vitro, and strong bone adhesion in vivo.^153–155

In biomedical applications, especially in orthopaedic plating^156,157 and stenting in the cardiovascular system (CVS), it was found that when the surface was coated with nano-TiO₂ or submicron particles, the macrophage chemotaxis was appreciably reduced and the chances of thrombosis as a post-operative complication were delayed (one of the leading causes of stenosis and complications in CVS stenting).^158–160 Ti-6Al-4V (Ti-Alloy) is the most common alloy used in medical implants¹⁶¹ due to its excellent anti-corrosion properties caused by the spontaneous formation of a 2–3 nm oxide film on the alloy surface.^162–164 Nevertheless, these corrosion properties are limited by native oxides that jeopardize the strength of the titanium alloy.¹⁶⁵ Benea et al. increased the surface thickness of the Ti-Alloy from 23.06 nm to 123.35 nm by using the anodic oxidation technique for depositing TiO₂ nanoparticles, which resulted in safer and better tribo-corrosion than the untreated Ti-Alloy.¹⁶⁶ Jin and his coworkers found that TiO₂ nanotubes (<100 nm diameter) had excellent osteoblast adhesion properties with elongated nuclei and up-regulated alkaline phosphatase enzyme, which suggests excellent biocompatibility and interaction with bone tissue for orthopedic implants.¹⁶⁷

The concept of antibiotic loading on prosthetic implants as a prophylactic therapeutic agent is gaining popularity in biomedical realms. Recently, Chennell et al. used various types of TiO₂ nanofibers, nanotubes and smooth surfaces for loading and eluting cefuroxime as an antibiotic on orthopedic implants.¹⁶⁸ They found the peak drug release at a two minute time interval from the TiO₂ nanotubes with the highest concentration release when compared to other nanostructures. Some studies also revealed successful loading and eluting of gentamycin, a broad spectrum antibiotic, on the surface of TiO₂ prosthetic implants and found an inhibitory effect up to six days for Staphylococcus aureus during the in vitro culture sensitivity trial.¹⁶⁹ Kumeria et al. and Popat et al. loaded gentamycin on the TiO₂ nanotubes array by electrochemically anodizing the compound on the titanium surface to inhibit the Staphylococcus epidermis (Gram positive) bacteria and cause adhesion to osteoblast cells in vitro.^170,171 They found excellent osteoblast adhesion differentiation and proliferation, which should be a key feature for any orthopedic prosthetic implant and should keep check on postoperative bacterial infections. TiO₂ prosthetic implants for hip replacements were coated with hydroxyapatite and poly(cyclodextrin) to load two different antibiotics, i.e. tobramycin and rifampicin, to control postoperative Staphylococcus aureus and Enterobacter cloacae.¹⁷² This dual drug loading synergistic effect on the TiO₂ implants was found to be much more efficient at keeping check on infective post-surgical complications. These findings provide new approaches for prophylaxis of prosthetic surgery post-operative infections, which are common complications, by using TiO₂ as a drug delivery vehicle and a core prosthetic implant.

Zhao et al. used chitosan hydrogel mesoporous silica nanoparticles on the titanium surface as a pH and electro responsive component to release ibuprofen, i.e. a non-steroidal anti-inflammatory drug (NSAID).¹⁷³ This approach is amenable to suppress post-operative inflammation. Moreover, the combined antibiotic and NSAID loading on the same TiO₂ prosthetic implant can efficiently decrease the postoperative complications of infection and inflammation due to prosthetic surgery.

3.4 Nano-TiO₂ for theranostics

The therapeutic effect of TiO₂ is solely dependent upon the generation of ROS, including , O₂˙⁻, OH˙⁻ and ¹O₂, irrespective of its shape or crystalline form. However, the various isomorphs can affect the extent of the ROS generation due to their reactivity variation, i.e. anatase has been reported to be more reactive than rutile. Successful ¹O₂ generation has been reported from nano-TiO₂ with a size of more than 10 nm by NIR phosphorescence, whereas nano-TiO₂ with a size of less than 10 nm is unable to generate ¹O₂ due to the inability of the smaller size particles to retain charge separation.¹⁷⁴ Certain short half-life ROS, i.e.¹O₂ and OH˙, can interfere with intracellular signal pathways and induce local membrane lipid peroxidation (attributed to weaker diffusion), while the relatively long half-life ROS, i.e. H₂O₂, O₂˙⁻ and , can strongly diffuse intercellular with a potential to induce apoptosis by interfering with neighbouring cell signal pathways.¹⁷⁵

In some studies, the potential of nano-TiO₂ as a drug delivery vehicle has also been explored because it is easy to adsorb the therapeutic agent and the presence of an ample number of surface electrons provides scaffolds for covalent or ionic bonding to ensure successful delivery and release in the target tissue via a special microenvironment trigger, i.e. pH, osmotic pressure, blood flow, EPR, etc. The highly reactive uncoordinated surface electrons have a strong affinity to the OH⁻ group in oxygen rich molecules,¹⁷⁶ which can be an excellent approach to target specific cell receptors in drug delivery systems.

Recently, our group used TiO₂ nanowhiskers to mitigate the adverse effects of TSPP on circulatory and excretory systems, both in vitro and in vivo.¹⁷⁷ Porphyrin derivatives’ well-known adverse effects (e.g. neuro-toxicity, photo-toxicity, accumulation in tissue other than the target tissue, etc.) have limited their potential use in biomedical applications. However, TiO₂ nanowhiskers’ porous nature and excellent vehicle properties have reintroduced porphyrin derivatives to biomedical realms, especially for neoplastic malady theranostics.

Veerachandra tested various commercially available combinations of TiO₂ isomorphs on Escherichia Coli samples for food sanitation and found that Degusa (P25) was more efficient than the other combinations. Moreover, they also observed that the germicidal effect and sanitation properties of P25 were increased with an increase in light intensity.¹³¹

Titanate, another form of TiO₂ micro- and nano-particles, has gained popularity in recent years. Monosodium titanate (MST) particles are efficient for biomedical applications, especially as a suppressor of human squamous cell carcinoma by holding 30% mitochondrial activity.¹⁷⁸ However, non-lethal effects of MST were reported for oral bacterial infections,¹⁷⁹ and MST is least effective against murine fibroblasts L929 and human monocytes THP1.^180,181 However, Wataha et al. reported the successful delivery of MST metal ions to monocytes and fibroblast cells, and affected the cellular functionality.¹⁸² Originally MST was designed for the decontamination of nuclear waste materials as it is a good ion exchanger. However, later on it was found that it can be efficiently bound to biologically active metals.¹⁸³ Recently Drury et al. demonstrated that nano-MST is more efficient in biological activities than macro-MST.³⁴

Sandoval et al. used europium-III (Eu³⁺) doped TiO₂ nanoshells coated with polyethyleneimine for in vitro cancer cell (HeLa) photo diagnosis.¹⁸⁴ This system could provide scaffolds for cancer theranostics if the anticancer agent is coupled with the TiO₂ nanoshells.

Nano-TiO₂ as a vehicle in a drug delivery system is more sensitive to pH change. Zhang et al. reported that the fabricated TiO₂ nanocomposites were pH sensitive and therefore they could selectively deliver DNR to the tumor cells. It is well documented that due to a higher metabolic rate, the pH is lower in neoplastic tissue than in normal tissue (7.4).¹⁸⁵

In stem cell biology, nano-TiO₂ is also gaining popularity. Recently Lavenus et al. reported the successful tailoring of nano-TiO₂ on the surface of glass slides with different angles to investigate its effect on human mesenchymal stem cells. It was observed that a subtle deviation in the coating angle of nano-TiO₂ influenced the cell behavior, which was evaluated by cellular pathways viz. differentiation, proliferation, migration and cellular morphology alteration¹⁸⁶ (Fig. 7). Similarly, in neural stem cells, the endocytosis and exocytosis of nano-TiO₂ irrespective of its shape and size were investigated by Wang et al.. They found nano-TiO₂ exocytosis within 72 hours. These findings vouch for the safety of nano-TiO₂ and can be used as a landmark for future stem cell investigations.¹⁸⁷

Fig. 7 Cell morphology of hMSCs on the different substrates. A) Staining of the characteristic cell morphology observed on the surface (actin in green, nucleus in blue, scale bar: 25 μm). B) Projected cell area as a function of the surface deposition angle. C) Distribution of the cell morphology on the different substrates (mean ± SEM, *p < 0.05/***p < 0.001 compared to TCPS). Reprinted with permission from ref. 186. Copyright 2015 Elsevier Ltd.

3.5 The role of TiO₂ in nano-drug delivery systems

A nano-drug delivery system is tailored to be highly specific to achieve the desired concentration and safe drug release in the subject system or tissue. Targeted therapy not only provides scaffolds for safe delivery but also allows combinations of various therapies, e.g. the combination of a photothermal therapeutic agent with a chemotherapeutic agent (doxorubicin, daunorubicin, etc.) or others.⁹⁹ Currently, gold and silver nanoclusters, MWCNTs, platinum, iridium and titanium are in vogue for nano inorganic drug delivery systems (NI-DDSs) with multiple therapies. Among the various NI-DDSs, titanium has been recently reported as a promising sonodynamic, photodynamic therapeutic and delivery agent for cancer and other infectious diseases¹⁴⁶ (Fig. 8).

Fig. 8 Chemotherapeutic agent intake by the drug resistant leukemia cells (K562) in the presence of nano-TiO₂. Reprinted with permission from ref. 29. Copyright 2006 Elsevier Ltd.

Recently, Yin et al. conjugated mesoporous TiO₂ up-converting nanoparticles with hyaluronic acid and doxorubicin for cancer therapy.¹⁸⁸ These up-converting nanoparticles convert the NIR light to UV light for PDT with mesoporous TiO₂ in the tissue and the hyaluronic acid ensures the controlled drug release of the chemotherapeutic agent (doxorubicin) within the neoplastic tissue. Kawashita et al. formed TiO₂ microspheres including SiO₂ nanoparticles and loaded them with Rhodamine B as a model hydrophilic drug. They found rapid elution of Rhodamine B for the first six hours.¹⁸⁹ They exploited TiO₂ (anatase/rutile polymorphs) in drug delivery systems, however, for clinical applications, the burst release of the loaded drug may be a problem that needs to be addressed accordingly.¹⁹⁰ TiO₂ nanoparticles have also been used as a photoprotectant in sunscreens and as a vehicle in drug delivery systems to release terpinen-4-ol during topical applications.

The porous nature of nano-TiO₂ has been widely investigated as a vehicle for drug delivery systems. Recently Wang et al. modified porous TiO₂ nanoparticles with polyethylineimine to deliver an anticancer drug (paclitaxel) to neoplastic tissue by additionally conjugating with folic acid for site specificity.¹⁹¹ The TiO₂ was photoactivated by UV light as a trigger for drug release by producing super oxides and polyethyleneimine was used to prevent premature drug release until its concentration was attained in the desired tumor. The porous TiO₂ nanoparticles not only produced the ROS but also released the chemotherapeutic agent upon photoactivation. This property can be efficiently explored for cancer and other malady treatments.

Yuan et al. developed a protocol to ensure the safe delivery of photoactivatable nanoparticles to the cytoplasm of HeLa cancer cells.¹⁹² They specially designed Fe₃O₄@TiO₂ nanoparticles to attach to the epidermal growth factor receptor (EGFR), which ensured the safe delivery of the nanocomposites to cellular nucleus without interfering and compromising the EGFR and karyopherin-β interaction, a key protein factor for nucleus translocation. After photoactivation, they found more DNA double strand breakage due to the effect of those photosensitizers, which remained in the cytoplasm.

4. Adverse effects of nano-TiO₂

Most nanomaterials that are known to be inert perform totally differently when injected into the body.¹⁹³ Exposure to nanomaterials may be oral, transdermal, or via inhalation, which exert adverse effects on our various body systems⁷ and vital organs, including the respiratory system,^194,195 gastrointestinal tract,^19,196,197 reproductive system,⁷ excretory system (kidney, liver), circulatory and nervous system,¹⁹⁸etc. The length and rigidity of the nanomaterials hamper their clearance from the pulmonary tissue, provoking the release of inflammatory mediators, e.g. multi-walled carbon nanotubes (MWCNT), nano-titania, platinum, etc.^44,199

TiO₂ nanomaterials are one of the most commonly studied nanomaterials for pulmonary toxicity. Nano-TiO₂ was considered as the safest among all the nanomaterials in vogue, until it was reported as a potential carcinogen when murine models were exposed in vivo for prolonged durations²⁰⁰ and then it was ranked as a 2B human potential carcinogen.^201,202 It has been reported that chronic and sub-chronic exposure of nano-TiO₂ leads to inflammation, epithelial hyperplasia and pulmonary carcinoma in murine models.¹⁹³ Moreover, TiO₂ nanoparticles are considered more toxic than nanowhiskers, similarly larger size TiO₂ is reported as being less toxic than smaller sized TiO₂.²⁰³

4.1 Sexual health

The widespread use of TiO₂ nanoparticles has led to severe adverse post-therapeutic complications in animals in general and in advanced primates, including humans in particular.²⁰⁴ Notably, complications have been reported in the reproductive system, such as the decreasing viability and proliferation of laydig cells,^205,206 the decreasing follicle survival rate, less griffin follicles and an altered morphology of the follicle and oocyte in murine models.²⁰⁷ When TiO₂ nanoparticles were injected into pregnant mice, then their offspring became infertile with abnormal development of seminiferous tubules, decreased sertoli cell numbers and increased spermatozoa primary abnormalities.^208,209

TiO₂ nanoparticles can cross the blood–testis barrier and damage the testicular interstitial tissue or sertoli cells by up-regulating caspase 3 to induce apoptosis and down-regulating the anti-oxidative enzymes, i.e. Superoxide Dismutase (SOD), Glutathione Peroxidase (GPX), Catalase (CAT), Glutathione S-Transferase (GST), etc., leading to an increase in ROS production.⁷ Super oxides (O˙⁻) are formed when oxygen acquires an additional electron. These O˙⁻ are scavenged by the SOD, converting them to H₂O₂ and ⁻O₂.^210,211 DNA oxidative damage has also been reported by many studies, suggesting that the smaller sized TiO₂ nanoparticles (<20 nm) can cause damage to the DNA double helix along with micronuclei formation and lipid peroxidation of membranes within the cell either in the presence or absence of photoactivation.^212,213

4.2 Nervous system

In Central Nervous System (CNS) studies, it has been reported that intravenous nano-TiO₂ induces oxidative stress to the brain by increasing the ROS production and decreasing the anti-oxidative enzymes and melatonin level. Moreover, it also activates caspase 3, which initiates apoptosis in the brain tissue.²¹⁴ TiO₂ nanoparticles can translocate in the CNS via olfactory pathways and cause lesions in the brain, including neuronal vacuoles and fatty degeneration of the hippocampus in murine models in vivo.^47,52 In recent studies, Younes et al. also reported that intraperitoneal 20 mg per kg_bwt of TiO₂ nanoparticles on alternate days could alter the neurobehavioral performance and elevate the serum enzymes (ALT, AST, LDH), indicating the additional hepatic malfunction in murine models.⁵⁰

4.3 Musculoskeletal system

In the musculoskeletal system, the role of nano-TiO₂ is very vital because in many orthopedic implants and plates, nano-TiO₂ has been used to suppress the autoimmune reactions and inflammatory response in the bone tissues. Nevertheless, there are some studies suggesting the toxic role of TiO₂ nanoparticles on the osteoblast cells by generating more ROS, hampering the potential use of TiO₂ in prosthetic surgeries. Recently it was reported that nano-TiO₂ damages the double helix DNA structure of bone marrow cells post-oral intake and also promotes the proinflammatory genes in the pro-osteoblast cells (MC3T3-E1 cell line).^17,215 TiO₂ nanoparticles (15 nm) cause impairment of SOD1, SOD2 (Mn SOD) and the sirtuin 3 protein that is present in mitochondria and is responsible for increased catalytic activity and homeostasis in human osteoblast cells (hFOB 1.19). Moreover, the study also revealed ultrastructure alterations in the osteoblast cells^216,217 (Fig. 9).

Fig. 9 TiO₂ nanoparticles in various organelles of hFOB1.19 cells. Reprinted with permission from ref. 217. Copyright 2015 Dovepress.

Cardiac arrhythmias and compromised myocardial function have been reported after administration of TiO₂ nanoparticles to rat models.¹⁹⁸ Earlier, it was also reported that TiO₂ nanoparticles smaller than 100 nm cause endothelial cell leakiness after 60 minutes of exposure, whereas the relatively larger size (more than 600 nm) remained inert.²¹⁸ This approach can be ideal for studies where cancer amelioration is targeted by normalizing the tumor vasculature;²¹⁹ however, the consequences can be more drastic on the surrounding healthy tissue if TiO₂ accumulates in tissue other than the target tissue.

In another study where TiO₂ nanoparticles were used for orthodontic applications, it was found that the nanoparticles accumulated in vacuoles of gingival fibroblast cells without compromising the sensitivity of the human oral squamous cells line (HSC-2) and cytotoxic effects on the surrounding tissue. However, nano-TiO₂ increased the prostaglandin E2 production in synergy to IL-1B as a proinflammatory action of the TiO₂ nanoparticles.²²⁰

5. Conclusions

In summary, from the aforementioned properties of TiO₂ nanoparticles and nanowhiskers, it is evident that TiO₂ can be an excellent photosensitizer and oxidizing agent for biomedical applications and could be a vital part of nano-biotechnology and photodynamic therapy. The relatively lower toxic effects, the existence in various polymorphs, the economical advantages, the high availability and the excellent biocompatibility towards biological systems declare its potential as a vital component in nanomedicine and biomedical realms. The important role of nano-TiO₂ and the relevant nano-biotechnology in early theranostics of neoplastic and non-neoplastic maladies and prosthetic surgeries is exponentially increasing every day. Nevertheless, for the best treatment efficiency, we need to realize the surface functionalization of nano-TiO₂ and bring the current photoactivation energy transfer band (400 nm) to the therapeutic window, which is greater than 600 nm excitation wavelength, as approved by the WHO.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81325011), National High Technology Research & Development Program of China (2015AA020502, 2012AA022703), and the Major Science & Technology Project of Suzhou (ZXY2012028).

Notes and references

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