Titania nanotube arrays for local drug delivery: recent advances and perspectives.

Review

Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by Chulalongkorn University on 01/05/15 For personal use only.

Titania nanotube arrays for local drug delivery: recent advances and perspectives 1.

Introduction

2.

TNTs: synthesis, structure and properties

3.

Biocompatibility of TNTs

4.

TNTs for LDD and DRIs

5.

Perspectives of TNTs for clinical applications

6.

Conclusion

7.

Expert opinion

Dusan Losic†, Moom Sinn Aw, Abel Santos, Karan Gulati & Manpreet Bariana †

The University of Adelaide, School of Chemical Engineering, Adelaide, Australia

Introduction: Titania nanotube (TNTs) arrays engineered by simple and scalable electrochemical anodization process have been extensively explored as a new nanoengineering approach to address the limitations of systemic drug administration. Due to their outstanding properties and excellent biocompatibility, TNTs arrays have been used to develop new drug-releasing implants (DRI) for emerging therapies based on localized drug delivery (DD). This review highlights the concepts of DRI based on TNTs with a focus on recent progress in their development and future perspectives towards advanced medical therapies. Areas covered: Recent progress in new strategies for controlling drug release from TNTs arrays aimed at designing TNTs-based DRI with optimized performances, including extended drug release and zero-order release kinetics and remotely activated release are described. Furthermore, significant progress in biocompatibility studies on TNTs and their outstanding properties to promote hydroxyapatite and bone cells growths and to differentiate stem cells are highlighted. Examples of ex vivo and in vivo studies of drug-loaded TNTs are shown to confirm the practical and potential applicability of TNTs-based DRI for clinical studies. Finally, selected examples of preliminary clinical applications of TNTs for bone therapy and orthopedic implants, cardiovascular stents, dentistry and cancer therapy are presented. Expert opinion: As current studies have demonstrated, TNTs are a remarkable material that could potentially revolutionize localized DD therapies, especially in areas of orthopedics and localized chemotherapy. However, more extensive ex vivo and in vivo studies should be carried out before TNTs-based DRI could become a feasible technology for real-life clinical applications. This will imply the implementation of different approaches to overcome some technical and commercial challenges. Keywords: drug delivery, drug release, drug-releasing implants, electrochemical anodization, localized drug delivery, orthopedic implants, titania nanotube arrays Expert Opin. Drug Deliv. (2015) 12(1):103-127

1.

Introduction

Drug delivery (DD) is referred to approaches, formulations, technologies and systems that enable the introduction and transport of therapeutic compounds in the body to achieve their desired therapeutic effect [1]. In conventional or systemic DD (SDD) administration, drugs are typically delivered by oral (tablets), parenteral (injection), transmucosal and inhalation routes, where drugs are distributed to the whole body and not to the specific site of interest, where the therapy is required [1]. These typical routes of drug administration present many problems and limitations such as poor biodistribution, lack of selectivity, side effects, toxicity, drug solubility

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Electrochemically engineered titania nanotube (TNT) arrays on Ti are increasingly recognized as a promising solution for localized drug delivery (DD) applications. The fabrication technology is low-cost, scalable and can be applied on existing medical implants based on Ti and Ti alloys with different forms (plates, screws, needles, wires, etc.). Outstanding structural properties of TNTs and their excellent biocompatibility proved by cell and in vivo studies make them highly suitable for designing drug-releasing implants. TNTs were tested for the delivery of a broad range of therapeutics, including antibiotics, anti-inflammatory and anticancer drugs, proteins, peptides, genes and growth factors, showing with much flexibility to control their drug release, desired dosage and kinetics including remotely triggered DD. The potential applications of TNTs for localized bone therapy to treat bone inflammations, infections, poor implant osseointegration, cardiovascular stents, dentistry and cancer therapy have been proposed. More in vivo studies are required in the future to translate this technology into the clinical trial stage.

This box summarizes key points contained in the article.

and unfavorable pharmacokinetics [2]. Furthermore, bioactive therapeutics such as proteins, nucleic acids, enzymes and genes tend to destabilize or degrade by enzymes or are eliminated by the reticuloendothelial system (RES) before their reach affected tissues/cells [3]. In addition, not all delivered drugs or drug carriers are capable of circumventing the firstpass metabolism, through the liver, kidney or other barriers including enzymatic, intestinal, capillary endothelial and blood--brain barrier [2]. Another inherent limitation of these therapies is the low efficiency of therapeutics, as only a small percentage (< 1%) of drug molecules is delivered to the site of interest, where the therapy is required and thus higher dosages of drugs are required to achieve an optimized local concentration, which in the case of some toxic therapeutics such as anticancer drugs can generate serious side effects [3]. Furthermore, a large percentage of currently discovered drugs are hydrophobic and water insoluble [4]. This reduces their pharmacological efficiency, and their systemic delivery route is particularly challenging [4]. Therefore, innovative DD approaches are urgently needed in order to address the limitations and disadvantages of traditional SDD [5,6]. These alternative routes of drug administration should provide high effectiveness, efficiency and selectivity with minimized risks and side effects for patients. In that regard, strong interdisciplinary research strategies combining the efforts of engineers, nanoscientists, material scientists, medical experts, biologists and clinicians have recently demonstrated promising results [6]. This combination of knowledge is envisaged to address the inherent limitations of conventional therapies by developing more efficient and 104

rational DD systems, where two concepts based on localized drug delivery (LDDS) and targeting DD systems have emerged as the most promising strategies. Advances in nanoscience and nanoengineering and, in particular, the application of nanotechnology to medicine, referred to as ‘nanomedicine’, have boosted the development of a variety of new nanoscale materials and drug-carriers for DD applications, including nanoparticles, liposomes, polymer micelles, dendrimers, nanogels, carbon nanotubes, fullerenes, graphene, viral vectors and virus-like particles [7,8]. The concept of LDDS has been implemented in different clinical devices capable of providing sustained, controlled, programmable and localized release of drugs at the site of interest in the host body [9]. These approaches have promisingly emerged as potential alternatives to conventional administration of drug for a broad range of clinical treatments. Among the different nanomaterials used to develop LDDS, drug-releasing implants (DRI) based on the generation of nanoengineered surfaces on existing medical implants have attracted great attention [10-13]. Nanoporous and nanotube structures such as nanoporous alumina (NPA) and titania nanotubes (TNTs) arrays produced by electrochemical anodization of aluminium (Al), titanium (Ti) and their alloys are the most explored platforms for developing DRI due to their unique features [13-17]. These nanoporous materials present low-cost fabrication processes, controllable pore and nanotube structures, tailorable surface chemistry, high surface area, high loading capability, chemical stability, mechanical rigidity, versatile geometry, tuneable drug-releasing performance, excellent biocompatibility and more interestingly, can be evenly generated on the surface of existing medical implants [18]. In particular, since the initial report on the fabrication of TNTs in 1999, electrochemically anodized TNTs arrays have attracted a growing interest in biomedical research and have engaged a special niche in LDD technology [19]. In addition to their biomedical applications, as a result of their exceptional structural, optical, catalytic and electrochemical properties, TNTs are used in many research areas, including energy production and storage, photochemical, electrical and environmental applications [18,20,21]. As an example, over the last 3 years, more than 1000 publications related to the fabrication and application of TNTs have been reported [18]. Among them, there are several excellent reviews compiling the most outstanding progresses in the fabrication, characterization and applicability of TNTs, including tissue engineering and DD [18,22-27]. This review is aimed at reporting and compiling the most recent advances on TNTs-based DRI for LDD applications. The fabrication, properties and biocompatibility of this remarkable nanomaterial are briefly introduced and described. Our attention is specially focused on the different drugreleasing concepts based on TNTs and their application for delivering different therapeutics. We discuss in detail the capabilities, advantages and inherent limitations of these systems. Furthermore, we provide comprehensive information

Expert Opin. Drug Deliv. (2015) 12(1)

TNT arrays for local DD

A.

TiO2 (TNT array)

B.

Anode + Ti

Cathode e-

Ti Ti C.


TiO2

100 nm

100 nm

Figure 1. TNTs formation and structure. (A) and (B) Electrochemical cell and anodization process for the formation of titanium oxide (TiO2) TNTs layer on Ti with self-organized and vertically aligned arrays of nanotube structures. (C) Scanning electron microscopy (SEM) images of the top and bottom surface show typical morphology of TNT structures. Reprinted with permission from [25]. Ti: Titanium; TNTs: Titania nanotubes.

about the recent progress in the practical application of TNTs related to bone therapies, stents, dentistry and localized cancer therapy. Finally, we conclude this review with a general overview and a prospective outlook on the future trends, challenges and perspectives in this exciting and promising research field. 2.

TNTs: synthesis, structure and properties

Structure and properties of TNTs TNTs arrays fabricated by electrochemical anodization can be described as a layer of tightly packed, vertically aligned and ordered nanotube structures with hexagonal arrangement, which grow perpendicularly to the Ti surface [28]. Figure 1A and B show illustrations of the chemical cell and the fabrication process of TNTs by electrochemical anodization of Ti. Typical scanning electron microscopy images of prepared TNTs are shown in Figure 1C, with the top and the cross-sectional views showing nanotubes separated into individual entities featuring closed ends at the bottom side [25]. As-prepared TNTs present an amorphous phase structure, although some reports have indicated the presence of nanocrystallites in the nanotube walls, particularly when the anodization process is carried out at high voltages. Typical TNTs arrays are composed of millions of densely packed nanotubes, which can serve as nanoreservoirs with the capacity to accommodate considerable amount of active agents, including insoluble drugs, antibiotics, proteins, genes and drug carriers. The structural features of TNTs include controllable pore diameter (10 -- 300 nm) and thickness (0.5 -- 500 µm), high volume (> 20 m3/g), high surface 2.1

area (180 -- 250 m2/g), high aspect ratio (~ 1000 -- 2000) and nanotube density (~ 1010 nanotubes/cm2) [18]. All these features can be controlled by simple electrochemical fabrication process, which makes TNTs an ideal platform for designing DRI, especially for bone-related therapies. TNTs arrays have been envisaged as functional layers on titanium implants for DD applications due to their outstanding properties, which include non-toxicity, excellent mechanical stability (delamination from the surface), chemical stability (corrosion), outstanding bone integration properties and ideal morphological structures (nanotube array) capable of loading and releasing high amounts of drug molecules. Furthermore, TNTs can be homogeneously grown on the surface of different implants, such as thin needles and wires [29]. These elements could be used as clinical tools for therapies that require a minor surgical invasion for insertion, as compared with current clinically proved DRI (e.g., polymer tablets in brain tumor treatments), which require a large surgical invasion. TNTs synthesis and methods development The fabrication of TNTs on the surface of Ti substrates is a unique electrochemical process termed self-ordering or selforganizing anodization [28]. This process has been used in industry for the decoration and protection of metals for decades. The typical electrochemical setup (Figure 1B) involves two electrodes: titanium (as anode) and an alternate metal (cathode), which are immersed in an appropriate electrolyte and connected to an external voltage (power supply). The initial electrochemical process results into two electrochemical reactions, including the dissolution of metal and the formation of oxide on the Ti surface. When the electrochemical 2.2


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equilibrium between these two reactions is established, the dissolution of the oxide layer is initiated with the formation of self-ordered and vertically aligned porous or tubular oxide nanostructures on the Ti surface (Figure 1C). The dimensions of the resulting nanotube array structures (diameter and length) can be tuned by the anodization parameters such as the type of electrolyte, its temperature and concentration, pH, anodization voltage or current and anodization time [27]. The fabrication of TNTs is very simple, low-cost, scalable and, more importantly, can be applied on Ti and Ti alloys either on two dimensional (2D) or three dimensional (3D) surfaces featuring curved shapes such as plates, screws, meshes, surgical wires and needles, which are clinically used as implants or surgical supports in orthopedics. As the pioneering introduction of the synthesis of TNTs by electrochemical anodization, numerous, studies were focused on achieving higher nanotube growth rates, improved nanotube ordering and controllable dimensions [18,20,21,28]. Different electrochemical protocols were explored in order to improve these technical aspects of TNTs, involving aqueous and organic electrolytes with different chemical compositions and electrochemical conditions. In general, TNTs layers can be formed in aqueous and non-aqueous electrolytes containing small amounts of fluoride ions (hydrofluoric acid [HF] or HF mixtures, NaF or NH4F). Since the pioneering studies, Ti foils were anodized in acidic, neutral and alkaline aqueous electrolytes. The resulting TNTs layers feature short nanotube lengths (from 500 nm to 2 µm) and are described as the first generation of TNTs [19]. However, these TNTs arrays lacked of organized structure and uniformity. These fabrication protocols were improved by Macak et al., who reported on the fabrication of TNTs in a pH-controlled aqueous buffered electrolyte containing fluoride ions, which yielded TNTs with longer lengths (5 -- 7 µm) [30]. This development is considered as the second generation of TNTs and enabled a better understanding of the relationship between the nanotube characteristics (dimensions, ordering and crystallinity) and the electrochemical anodization parameters (electrolyte, pH, temperature, voltage, current and time) [31]. Subsequent studies evolved into the third generation of TNTs, where the anodization process was carried out in non-aqueous polar organic electrolytes (formamide, dimethyl sulfoxide, ethylene glycol, or diethylene glycol) in the presence of fluoride species [32]. These new conditions provided increased growth rates to achieve TNT arrays of longer length (1000 µm). Electrochemical anodization of Ti in organic viscous electrolytes (e.g., ethylene glycol) at an anodization voltage of 80 -- 120 V results in vertically oriented and uniformly close-packed TNTs structures with high growth rate, stable mechanical and optimal geometric characteristics [32,33]. Recently, the fourth generation of non-fluoride-based synthesis of TNTs has been reported, showing further improvement in the growth rate and the quality of prepared TNTs [34,35]. In parallel with these developments focused on TNT fabrications, several new methods 106

for the preparation of TNTs with different geometries were reported. Some examples of these include nanopores, branched, nanolace, bamboo-type, spongy, multi-layered and shaped nanotubes [18,36-38]. The development of novel methods for the preparation of TNTs is still a very dynamic and active research area, and new and more optimized fabrication approaches are expected to be reported in the coming years. The historical progress in the fabrication of TNTs is summarized in Table 1, highlighting the main features of each generation of TNTs. In particular, it is worth emphasizing that TNTs prepared in phosphate solutions exhibit outstanding properties such as high corrosion-resistance in acid and base electrolytes. These properties have been associated with the incorporation of phosphate anions from the electrolyte into the structure of TNTs in the course of the anodization process [39-41]. These TNTs have shown to stimulate the formation of biocompatible hydroxyapatite (HA) in bone cells, which promotes their growth and bone tissue integration, particularly suitable for orthopedic applications. Most recent research involves the preparation of TNTs in new electrolytes, improvement of anodization conditions under ultra violet (UV)-irradiation and ultrasound assistance and extension of nanotube fabrication nanotubes on other materials including Ti alloys and other metals clinically used as medical implants (e.g., TiAl, TiAlV, TiNb, TiAlNb, TiZr, Ta, Hf, W, Zr, stainless steel, etc.) [18,28,42-48]. Although most of the TNTs fabrication technology is focused on the anodization of flat Ti foils, the methodology has been extended to various substrates, including needles, wires and meshes [29]. The use of these various Ti shapes offer more functionalities like 3D release of drug from wire implants, which have been proposed as tiny ‘in-bone’ therapeutic implants. 3.

Biocompatibility of TNTs

Biocompatibility is the ultimate and the most crucial requirement to be fulfilled by any new biomaterial before its use in any clinical application, including DD. Titanium and its alloys, particularly Ti-6Al-4V, have been extensively used in orthopedic and dental implants since 1970. These are the most widespread biocompatible materials used in clinical applications due to their nontoxicity, excellent mechanical and chemical stability and outstanding bone integration properties [41]. The biocompatibility of these Ti-based medical implants is highly influenced by the ultra-thin layer of native oxide formed on the implant surface by natural oxidation of the metal upon exposure to atmospheric moisture. The chemical composition of this native oxide is similar to that of TNTs, which suggests similarity for their biocompatibility behavior. The majority of biocompatibility studies on TNTs were focused on their clinical applications for tissue engineering, vascular implants and stem cells, where the influence of nanometric scale topography and surface modifications (wettability) of



Table 1. Summary on the development of the TNTs fabrication processes. Fabrication of TNTs on Ti Anodization electrolyte

Maximum length

First fabrication of TNTs. Chromic acid electrolyte + HF pH controlled aqueous buffered electrolytes + fluoride


Non-aqueous polar organic electrolytes + fluoride Lactic acid as additive in organic electrolyte + fluoride Other substrate material

Ti-6A1-7Nb/ Ti-6A1-4V TiNb, TiZr and TiTa alloys

Substrate shape

TNT morphologies

Ti-4Zr-22Nb-2Sn Wires Meshes Tubes Nanopores Nanochannels and mesosponge Bamboo-type and hierarchically branched nanotubes Nanolace Double walled

Other enhancements

UV-Vis irradiation Ultrasound irradiation

0.5 µm

Features

Extensive dissolution, non-uniform and dis-organized. Role of Fluoride ions signified. 7 µm Reduced dissolution, Influence of various parameters on nanotube properties 1000 µm Further reduced dissolution. Stable, well-organized TNT structures ~ 20 µm/min Prevention of anodic breakdown at high voltages and very high growth rates Tubes are formed from mixed oxides and can be grown to several hundred nanometers in thickness Randomly organized nanotube bundles composed of mixed oxides TNTs for antibiotic therapy Three dimensional drug release applications Enhanced photo-catalytic activity Influence of electrode orientation defines TNT dimensions Transition of tubes into pores using very low water content in electrolyte Formed in hot glycerol-phosphate electrolyte. Random distribution of pores with very high surface area Fabricated using alternating-voltage anodization. Demonstrates increased light harvesting Fabricated using alternating-voltage anodization Thermal annealing to change the morphology and crystallinity of the TNTs Larger diameters and thicker walls Larger diameters

Ref.

[19]

[30,31]

[32]

[143]

[144] [145] [146] [29] [147] [148] [149] [150] [36,37] [36] [38] [42] [43]

TNTs: Titania nanotubes.

TNTs were explored. Many cell lines have been investigated for their adhesion and functioning, which include osteoblasts, mesenchymal stem cells (MSCS), endothelial cells, vascular smooth muscle cells, chondrocytes, epithelial cells, dermal fibroblasts and epidermal keratinocytes [45-48]. These investigations suggest that TNTs offer a biocompatible and safe environment for cell attachment without any adverse effects and enhance such cell-related features, such as cell differentiation and proliferation. As far as orthopedic applications is concerned, the inspiration for developing implants based on electrochemically prepared TNTs on Ti implants was originated from the human bone structure, which is nanostructured and comprises inorganic particles within the similar size range that TNTs have. In vitro biocompatibility studies Initial studies on the biocompatibility of TNTs were performed by Desai’s group, who demonstrated that the surface of TNT layers presents a favorable nanotopographic surface for bone cells growth and differentiation [45]. These studies clearly evidenced that osteoblast activity can be significantly enhanced using TNTs nanostructures. TNTs surfaces were 3.1

able to promote a higher cell adhesion, proliferation and viability for up to 7 days of culture as compared to plain titanium surfaces [49,50]. Cells cultured on nanotubular surfaces have also demonstrated a higher alkaline phosphatase activity without causing any adverse immune response under in vivo conditions. A significant adhesion/propagation of osteoblasts by the topography of TNTs surfaces, with the filopodia of these cells entering inside of the nanotubes and producing an interlocked cell structure, have been demonstrated by Oh et al. [51-53]. The growth rate of osteoblast cells on TNTs surfaces is considerably accelerated, i.e. more than 300 -- 400% higher than that of bare Ti surfaces [51]. In particular, two research groups found that the adhesion, spreading and differentiation of MSCs are directly related to the diameters of TNTs, which is described as an exceptional discovery in the field of stem cell research [53-55]. It has been shown that TNTs possess the ability to dictate the fate of MSCs and have preferential interface to induce the differentiation of MSCs towards different bone lineages such as osteoblasts, chondrocytes, endothelial cells, fibroblasts or myocytes. Meanwhile, Brammer et al. reported enhanced in vitro


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adhesion of bovine cartilage chondrocyte and found dense extracellular matrix (ECM) fibrils on as-prepared TNTs [56]. TNTs with a diameter of 70 nm showed the highest glycosaminoglycan secretion, aggrecan and collagen type II transcription level [57]. Additionally, an increased growth of dermal fibroblast and decreased epidermal keratinocyte adhesion, proliferation and differentiation in transcutaneous TNTs implants were observed by Smith et al. [58]. Moreover, Feschet-Chassot et al. demonstrated that TNTs have no negative impact on two physiological parameters related to ciliated Protozoan Tetrahymena pyriformis [59]. An alternative cell model was used for in vitro toxicity studies to predict the toxicity of TNTs layer in biological systems and the result showed that TNTs do not affect non-specific esterases activity and population growth rate [59]. It is worthwhile noting that the TNTs topography is not the only influencing factor in terms of biocompatibility and cell adhesion, promotion and differentiation. Some studies have demonstrated that the surface modification of TNTs is another strategy to improve cellular adhesion and promote specific cell proliferation and differentiation. Bauer et al. found considerably enhanced mesenchymal cell attachment to the superhydrophobic TNT surfaces modified by self-assembled monolayers of octadecylphosphonic acid [60]. Furthermore, the surface modification of TNTs with biopolymers such as poly(lactic-co-glycolic acid) (PLGA) and chitosan was found to increase the biocompatibility for human osteoblasts and potentially improved its antibacterial properties [61]. Cell behavior studies on TNTs surfaces The influence of the nanometer scale surface topography and the nanoscale environment on the behavior of cells has received enormous attention in recent years, with many conflicting results in terms of defining cell’s response to different size, shape, charge and surface chemistry of nanostructures. TNTs with well-ordered nanotubular structures and controllable nanotube dimensions have been recognized as a favorable substrate for cell studies towards the development of new platforms for advanced cell culturing with controllable cell behavior and differentiation. By culturing hMSCs on a range of nanotube diameters between 30 and 100 nm, Oh et al. found that cell stretching and the expression of osteogenic differentiation markers achieve their highest level when they grow on 100 nm nanotubes. This was associated with the different response of phenotypic cell lines to TNTs featuring different topographical characteristics [53]. These studies concluded that TNTs featuring nanotubes with larger diameters are more effective for protein adsorption than smaller ones. However, opposite results were reported by Bauer et al. showing the highest adhesion, spreading, growth and differentiation of hMSC stem cells on TNTs with smaller diameters (15 nm) and dramatically decreased cell functions on 70 and 100 nm nanotubes [54,55]. A similar behavior is confirmed on both boneforming cells and bone-resorbing cells [55]. Considering the 3.2

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same size-dependent response to both bone and hMSC cells to TNTs nanotubes, it has been suggested that a surface geometry with a lateral spacing of ~ 15 nm, which corresponds to the dimension of integrin heads, is preferentially recognized by cells [55]. The observed differences can be explained by the use of different methods to prepare TNTs substrates and methods to evaluate the cell behavior. Even though the obtained results are conflicting, all these studies proved that TNTs structures can promote cell differentiation. However, more studies aimed at explaining the mechanism of this process and understanding of controlling parameters are expected in the near future. In vivo biocompatibility studies The first in vivo study using TNTs implants on pigs as the animal model was performed by von Wilmowsky et al., who investigated the effects of TNTs-Ti implants inserted in the skull upon bone formation [62]. Figure 2 shows a bone-implant used by von Wilmowsky et al. for immunohistological analysis. Bone-implant contact and immunohistochemistry analyses were performed at regular time intervals for up to 90 days, showing that the implant interface of TNTs (diameter of 30 nm) was not damaged by mechanical stress and had significantly increased type-I collagen expression as compared to commercially available Ti implants. Moreover, another in vivo study on TNTs implanted in rabbit tibias indicated that TNTs have significantly improved bone bonding strength to the surrounding tissues after 4 weeks of experimental implantation. Their histological analysis confirmed an excellent bone-to-implant interfacial contact, new bone formation and satisfactory levels of calcium and phosphorus content on the nanotube surfaces [63]. Another in vivo biocompatibility study was performed by implanting TNTs surfaces subcutaneously in rats and performing histological analysis during 4 weeks [45]. No chronic inflammation or fibrosis was observed. Increased chondrocyte adhesion on TNTs surfaces compared with bare titanium has also been reported [45]. Furthermore, calcium and phosphorous concentrations were 50% higher on these surfaces, suggesting that matrix deposition was created on the nanotubular surfaces. 3.3

Potential ‘nanotoxicity’ of TNT implants Drug-releasing TNTs-Ti implants are considered as therapeutic medical devices that combine three elements including: therapeutic agent, nanostructured material and an artificial device, which in this case is a titanium implant. These are considered as combination products similar as nicotine patches, antibiotic coatings, drug-eluting pacing lead and drug-eluting stents, classified by FDA as medical devices (Class III), which require specific safety and effectiveness testing [64]. However, these medical devices are susceptible of raising different regulatory challenges, given that their components are normally regulated under different types of authorities worldwide and thus assessed under different criteria (i.e., drug and nanomaterial) [64,65]. It is worth stressing that the differences in regulatory pathways for 3.4




A.

B.

2 mm

2 mm

Figure 2. The first demonstration of implantable drug-releasing titania nanotubes (TNTs) implants. In vivo study of TNTs-Ti implants in the frontal skull of domestic pigs showing regions of interests for the histological and immunohistological evaluations of the bone-implant contact. Adapted with permission from [62].

each of these components can yield discrepancies and heterogeneity in different regulatory aspects of the product (e.g., development and quality control during manufacturing, preclinical testing standards, post-approval modifications, adverse event reporting, etc.). In the case of TNTs, there are some additional safety concerns as the surface of TNTs is composed of nanotube structures, which are defined as nanomaterials and could have potential hazards as a result of nanotubes delamination or degradation after implantation and release of TNTs debris into the host body. This possibility might raise regulatory challenges for clinical approval of TNT implants, given that titania medical implants, drugs and nanomaterials are normally regulated under different types of authorities worldwide and thus assessed under different criteria. The tremendous growth of nanomaterials applied for DD has prompted the investigation of the possible toxicity of TNTs to human health, due to concerns raised by potential delamination and degradation of nanotube layers from TNTs implants. Titania (TiO2) nanoparticles are known to have high bioactivity and, considering their high aspect ratio, it could cause considerable toxicity impact on cells and tissue, including hepatic injury and renal lesions [66]. Similar to other inorganic NPs, TNTs if happened to be present in the systemic circulation, have two potential pathways for clearance, that is, kidneys/urine and bile/feces. Both these routes were proved for ingested TiO2 particles and expected to be the case for TNTs as well [67]. Although in vivo monitoring of TNTs have not been explored and established, NuevoOrdoń˜ez et al. have designed a strategy based on isotope dilution analysis and mass spectrometry, or IDA-ICP-MS, aimed at measuring levels of dissolved titanium in patients with surgical titanium implants. This strategy can assess the

implications of the basal, normal levels of titanium in the bloodstream [68]. They found that control individuals had very low levels of titanium in the blood, whereas titanium concentrations were significantly higher for all the patients with implants showing significant differences for different types of bone fixation devices [68]. The accuracy and precision of this approach could be translated for testing implanted TNTs to monitor their potential degradation over time, which is expected to be explored in future. Biocompatibility: current progress and challenges In summary, these in vitro and in vivo biocompatibility studies have reinforced TNTs arrays as a safe and remarkable biomaterial, which can endow current implantable devices with enhanced biomedical properties and capabilities towards the development of advanced and reliable medical implants. The topography of TNT arrays with relatively large surface area, not only acts as a nanoreservoir for therapeutics and their delivery, but it can also stimulate cellular response, including promotion, adhesion, differentiation and proliferation of bone cells growth. Furthermore, TNTs could increase endothelial cells motility, differentiation and proliferation, while decreasing the proliferation of vascular smooth muscle cells (VSMC) and gene expressions. Other advantages of TNTs layers over plain Ti surfaces are the promotion of HA formation, osseointegration properties and hemocompatibility. Hence, these results suggest that the scope of TNT-based DD systems can be extended to tissue engineering, regenerative medicine and dentistry along with preventive therapy. More in vivo studies, however, need to be carried out in order to address some unanswered questions related to long-term integration of TNTs with bones and other tissues in the host body. 3.5


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TNTs


Ti Antibiotics anti-fungal anti-inflammatory anti-cancer proteins, peptides, hormones antibodies genes, DNA drug nano-carriers nanoparticles

Figure 3. Schematic diagram summarizing the applications of TNTs for localized drug delivery and drug-releasing implants as the most characteristic potential clinical applications where these medical devices can be employed. Ti: Titanium; TNTs: Titania nanotubes.

As far as the potential toxicity of degraded TNT implants is concerned, the available information is still insufficient and thus more exhaustive work must be performed on the safety measures for TNTs as an implantable device. For them to be used safely for clinical applications, a detailed understanding of biocompatibility and toxicity of TNT particles and single nanotubes is needed. At this stage, additional investigations are demanded on these biocompatibility aspects of TNTs through more detailed in vivo and in vitro experiments. These studies will help establishing of future databases containing detailed information on the degree of toxicity on the nanoscale, which will help to clarify the division of toxic effects of nanoscale materials, including TNTs. Furthermore, extensive studies on the interaction between cells/tissues from different organs and parts of the body with TNTs are also required [69]. 4.

TNTs for LDD and DRIs

As we have commented previously, TNTs-based DRI have been explored in several medical fields, such as orthopedic implants, dental implants, vascular (coronary) stents, localized cancer therapy and bone tissue engineering [17,22,25,25,70,71]. In the following sections, we will show examples of numerous therapeutic agents that have been applied in order to implement a broad range of therapies into TNT-based implants. Some examples of these are antibiotics, antifungal, anti-inflammatory and anticancer drugs, bone proteins, peptides, enzymes, vitamins, hormones, 110

genes, antibodies, neurotransmitters, drug nanocarriers and nanoparticles [70-76]. These in vitro studies have proved the capabilities of TNTs to successfully deliver therapeutics for different LDD therapies with desired kinetics, concentration and drugrelease rate, which are critical parameters required for clinical applications. Figure 3 and Table 2 summarizes the key applications of TNTs and the most relevant therapeutic drugs used in combination with TNT-based implants. The specific applications and therapies in which these drug-releasing TNTs-based implants have been used, including bone, cancer, cardiovascular stents and dentistry, are discussed in following sections.

Strategies for controlling drug release kinetics from TNTs

4.1

When TNTs come into contact with the physiological milieu inside the host body, the drug release from nanotubes is governed by a diffusion process, as schematically presented in Figure 4. The release process is established by the mass transfer of drug molecules from the nanotubes, considering that the drug is completely soluble in the physiological solution without precipitation at particular concentrations. This diffusion-controlled process can be described by the Fick’s first law, which is governed by a number of factors such as the molecular size and charge of drugs, the dimensions of nanotubes (diameter and length), their charge and surface chemistry, interfacial interaction between drug molecules and nanotube surface, the dissolution rate of drugs, diffusion coefficient, pH and so on. Over the past 5 years, numerous studies have analyzed the release of different drugs from TNTs layers. Different strategies have been implemented into TNTs-based systems in order to provide a controlled release of drugs. The ultimate aim of these works is the development of DRI with favorable release kinetics, optimized drug release rate and time of release for a broad range of clinical therapies [26]. In particular, the focus of these studies was to eliminate the undesired burst release of drug and to establish strategies capable of providing sustained and longterm release with zero-order kinetics. The zero-order type release is the most desirable release strategy for DRI as it yields the highest dosing stability and resembles the ideal situation, whereby drug is released at a uniform and constant rate, which is independent of time and concentration. However, it is worth stressing that different therapies, disease conditions and parts of the host body require different drug release strategies, which may vary from short to long release, rapid ondemand release or time-programmed release with single or multiple drug loadings. Therefore, TNTs-based drug-releasing systems must be designed with flexible drug release capabilities and optimized parameters in order to fulfill the requirements of different therapies. Recent studies have demonstrated different variety of strategies implemented into TNTs-based systems that can achieve these goals. In that regard, numerous studies from our group and others were focused on exploring different strategies in




Table 2. Summary of the selected therapeutics and key therapies proposed using titania nanotubes. Main Target

Therapeutic loaded

Antibacterial

Vancomycin Penicillin/streptomycin Gentamicin Cefuroxime Silver nanoparticles Zinc ions Antimicrobial peptide hhc-36 (krwwkwwrr) Enrofloxacin Itraconazole Ibuprofen Indometacin Indometacin loaded and chitosan/poly(lactic-co-glycolic acid) coated Dexamethasone Naproxen Gelatin stabilized gold NPs Bone morphogenetic protein-2 (BMP2) Pamidronic acids Pre-synthesized hydroxyapatite Strontium Bisphosphonates (BPs) C-terminal (connective tissue growth factor) fragment Doxorubicin Selenium Paclitaxel Cisplatin Sirolimus Dopamine

Antifungal Anti-inflammatory

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TNTs-based DRI in order to design and advance their drugreleasing performances for specific drugs and therapies [26]. A schematic diagram summarizing these strategies aimed at controlling the release of drugs from TNTs is presented in Figure 4. These include controlled release by: a) structural modifications of TNTs dimensions (diameter and length), b) surface modifications, c) reduction of the pore openings of TNTs by polymer deposition, d) biodegradable polymer coatings, e) polymeric micelles as drug nano-carriers and f) stimulated drug release strategies by external sources (magnetic field, UV light, ultrasound and radiofrequency [RF]). Structural and surface chemistry modifications The simplest strategy for controlling the release of drug from TNTs is to modify their structural features (pore diameters and length) using different anodization conditions (Figure 4A). The diffusion rate of drug molecules from nano-confined structures such as nanotubes, according to Fick’s law, depends on their dimensions [72,74]. As the tubular size in these confined nano-channels can be adjusted precisely at nanoscale, this strategy can be exclusively devised to control the drug release. Studies from several groups confirmed that the drug release is considerably influenced by the TNTs dimensions [26,61,73,77]. These studies have demonstrated that the drug release of several 4.1.1

Additional features In vivo studies Loading using simulated body fluid/osseointegrating Osseointegrating Osseointegrating/in vivo studies Osseointegrating and anti-inflammation/in vivo studies Also targets antibiotic-resistant bacteria

Ref. [101] [102] [49] [151] [103] [152] [104]

Antibacterial and osseointegrating

[153] [74] [154] [61] [61]

Loading using simulated body fluid/osseointegrating

[102,127]

Osseointegrating/mechanical stability studies

Treatment of osteoporosis and Paget’s disease Study on Ti-6Al-4V alloy surface/ In vivo investigations

Antibacterial and osseointegrating Osseointegrating Study on vascular smooth muscle cells Implantable drug delivery system for brain conditions

[109] [112] [155] [156] [111] [157] [158] [82] [83] [132,133] [142] [133] [141]

model drugs e.g., indometacin, paclitaxel dextran and bovine serum albumin (BSA) from TNTs can be extended by reducing their diameters and lengths. This conclusion is supported by the fact that drug molecules are trapped deeper inside the nanotubes and therefore needed a longer time to diffuse out of the TNTs due to capillary force effects. However, it is worthwhile mentioning that the drug loading is also influenced by the TNTs dimensions (i.e., less drug is loaded as the nanotube diameter is reduced), which indicates that this strategy is limited to provide extended release of therapeutics over a period of several months. Another approach was explored by C ¸ alıs¸kan et al. showing that controlling the aspect ratio of the nanotubular space by adjusting the anodization parameters prolonged gentamicin release. The release of the antibacterial agent from TNTs has been demonstrated an effective treatment against Staphylococcus aureus organism [78]. The second approach explored for tuning the drug release characteristics of TNTs was achieved by functionalizing the surface of TNTs. The aim of this approach was to modify the surface chemistry of the nanotubes by imparting hydrophobic and hydrophilic surface properties. This enables a dynamic change of the interaction between drug molecules and inner nanotubes walls, which can alter the drug loading and release kinetics (Figure 4B). This concept was previously


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Figure 4. Strategies for controlling drug release from TNTs. (A) Controlling the nanotube diameters and length; (B) surface chemistry (hydrophobic, hydrophilic, charged); (C) tuning nanotube opening by plasma polymerization; (D) degradation of dip-coated polymer film closing nanotubes (PLGA or chitosan); (E -- F) using drug nano-carriers (micelles) for multidrug delivery; (G -- I) external field triggered drug release using temperature, magnetic field, ultrasound and radiofrequency. Only single nanotube structure is shown to present an array of TNTs. PLGA: Poly(lactic-co-glycolic acid); Ti: Titanium; TNTs: Titania nanotubes.

demonstrated on porous silica particles and was successfully translated into TNTs using several different surface modification methods, including self-assembled monolayers (SAMs) and polymers. The resulting functional monolayers present good stability, and flexibility to use different functional groups, tuneable charge, interfacial and binding properties. TNTs modified with organic silanes (i.e., 3-aminopropyl triethoxysilane, APTES, penta-fluorophenyldimethylchlorosilan, PFPTES) and phosphonic acids (i.e., 2-carboxyethyl-phosphonic acid and 16-phosphono-hexadecanoic acid) were explored to demonstrate the capability of this approach to alter drug loading and release characteristics of TNTs for 112

hydrophobic and hydrophilic drugs [26]. Nonetheless, it was shown that this strategy is still limited to achieving a sustained and extended release of therapeutics from TNTs for a long period of time (> 3 months). Polymer modifications To overcome this problem and to achieve an extended drug release from TNT-based systems with zero-ordered drug release kinetics, our group introduced a new concept using plasma polymer coatings on the top surface of NPA and TNTs [79-81]. This approach makes it achievable to precisely reduce the nanotube opening at the top surface and thus 4.1.2




restrict/extend the release of therapeutics from nanotubes. The proposed advantages of plasma polymer modified TNTs are not only to improve drug-release kinetics but also to improve antibacterial and bone integration properties. This concept is schematically presented in Figure 4C. Our results confirmed that this strategy makes it possible to achieve zero-order release kinetics by significant reduction of the nanotube opening, which can be controlled by the deposited polymer layer [79-81]. This method was successfully proved using more than 10 different drugs with various sizes and properties, including anti-inflammatory drugs (indomethacin), antibiotics (gentamicin and vancomycin), anticancer drugs (doxorubicin and paclitaxel), proteins, BSA, human growth hormone (hGH), bone morphogenetic proteins (BMPs), drug nano-carriers (micelles), antibody rituximab and so forth [76,79-81]. Notice that plasma deposition method is a simple and solution-free process, capable of modifying the top surface of TNTs layers by an unlimited number of polymers [81]. This advantage can be used to impart other desirable properties to TNTs-based implant such as better integration to reduce implant rejection, sensing capabilities, anti-biofouling and antibacterial activity and so on. While plasma technology is widely used in medical practice, the disadvantages of this method are its high cost and potential technical complexity for in situ application caused by the plasma system itself (low pressure chamber required). Recently, atmospheric plasma deposition has been implemented into plasma jet-guns for surface engineering and polymer deposition applications. This new approach could potentially address these problems and enable the design of custom-made coatings on TNTs implants for in situ clinical applications. Dip-coating is another approach explored by our group to modify the top surface of TNTs layers with biopolymers [61,82]. This approach is simpler and cheaper than the polymer plasma coating method and consists of a single step, by which the top surface of drug-loaded TNTs layers is coated with thin polymer films (Figure 4D). In this approach, the thickness and polymer degradability become the factors controlling the drug release rate from TNTs [61]. Two biocompatible and biodegradable polymers (i.e., PLGA and chitosan) were used to prove this concept. These polymers were chosen as a result of their proven antibacterial and osseointegration properties. The obtained results showed that the drug-releasing profile can be tuned by controlling the thickness of the biopolymer film. Furthermore, these biopolymer films significantly suppressed the undesired burst release and extended the overall release for several months with zero-ordered kinetics. Notice that the drug release characteristics of TNTs-based DRI prepared by this approach can be controlled by selecting polymers with different degradation rates and chemical compositions. This design of DRI is particularly attractive for local delivery of antibiotic, which can be extended for up to 6 -- 8 weeks after surgical implantation. Apart from these advantages, TNT layers coated with biopolymer films can enhance the adhesion and proliferation of human osteoblast

cells (HOS) and prevent implants from bacterial infections and biofilm formation due to their outstanding antibacterial properties, especially chitosan films [61]. Therefore, this approach has the flexibility to be applied to Ti implants for specific applications and conditions, from short term drug release scenarios, for example to suppress inflammation, to middle term (1 -- 2 weeks), such as to prevent bacterial infection, and long-term (> 30 days) drug release for other therapies, including improving the osseointegration process, fracture repair or treatment of bone cancer. This concept was explored and extended by other groups as well, which aimed their research at improving cell integration and antibacterial properties of TNTs using different coatings such as HA, calcium phosphate, layer by layer (LbL) organic films, thin metal films (Se), nanoparticles (Ag), hydrogels and so on [83,84]. In summary, the dip-coating method for the preparation of TNTs DRI loaded with desired drugs is a very simple, feasible and low-cost process, which has demonstrated considerable potential to be used in real-life clinical applications. In contrast, while plasma deposition is a relatively more sophisticated approach and warrants more complex protocols, it requires higher facility costs and trained personnel. Advanced TNTs DD systems with multi-drug and stimulated drug release

4.2

The use of conventional SDD therapy based on a single therapeutic agent is not fully satisfactory to treat complex diseases that require more than a type of drug. Some examples of these therapies are cancers, malaria, multiple sclerosis, hypertension, infection, cardiovascular and metabolic diseases [1,70,85]. To meet these criteria, a new concept using polymeric micelles loaded with several drugs (multi-drug nanocarriers) was integrated into TNTs-based DRI by our group [74]. This approach is envisaged to design advanced multi-DRI for combined therapies. The advantages of multi-drug therapy against single-drug therapy have already been proved using traditional medicine and more recently in systemic therapy by multi-drug tablets. Furthermore, additional synergistic effects of different therapeutic substances could offer specific advantages to concurrently treat various diseases, different local tumor and tissues with complex nature, which cannot be treated by means of single-drug strategies due to severe side effects. Multi-DD with micelles as drug carriers from TNTs

4.2.1

The encapsulation of sensitive drugs in nano-carriers such as polymer micelles prior to their loading in TNTs was pioneered by Aw et al., who put forward a prospective solution to simultaneously achieve two goals: i) to protect sensitive drugs and proteins from degradation and ii) to design TNTs-based drug-releasing systems with an extended release capability (Figure 4E) [75]. Four polymeric micelles (i.e., Pluronic F127, d-a-tocopheryl polyethylene glycol 1000 succinate


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[TPGS] and two types of PEGylated phospholipids [DGP 2000 and DGP 5000]) were explored and separately encapsulated with different model drugs and subsequently loaded into TNTs [75,86]. These DRI have shown optimized drug release kinetics (slow release over several weeks) without initial burst release phase. More importantly, further development of this concept was based on the implementation of multi-drug payloads, where polymeric micelles loaded with different drugs were specifically loaded in a layered fashion inside the TNTs. The idea is based on the formation of two or more immiscible layers of drug carriers (regular and inverted polymeric micelles) that have opposite interfacial properties (hydrophobic and hydrophilic), which are used to generate a series of sequential release in a time-controlled fashion [74]. While indomethacin (anti-inflammatory drug) and itraconazole (antifungal drug), both water-insoluble drugs, were encapsulated in regular micelles (TPGS), gentamicin (antibiotic drug) was encapsulated in inverted micelles (DGP 2000). These drug-loaded micelles were loaded in TNT-based implants (Figure 4F). A typical release graph from this multiple-DD system based on TNTs shows a characteristic sequential release, which is divided into two steps over a period of 10 days (5 days each), confirming the independent release of three drugs loaded in two nano-carriers. It was further demonstrated that the sequence or time release of individual layers with loaded drugs can be controlled by changing the ratio and composition of drug-loaded polymeric micelles inside TNTs [74]. Another extended approach generated from this strategy was the development of time-delayed or programmable LDD to provide LDD at lagged times (drug released at a later stage). This strategy is designed for treatments that require the release of drugs at desirable postponed times after surgeries to suppress bacterial infections or to enhance bone integration. Stimulated DD from TNTs with external trigger Most of the previously demonstrated LDD strategies based on TNTs are focused on extended and optimized drug release for long-term therapies, which are inappropriate for critical conditions, where high concentrations of drug are immediately required. Some examples of these adverse conditions are bacterial attack, unexpected onset of inflammation, osteomyelitis or septic arthritis. Therefore, to address these emergency conditions, the ability to control the delivery of therapeutics from externally triggered delivery system is recommended. These adverse conditions can be overcome by implementing externally triggered on-demand control of the delivery of therapeutics into TNTs-based systems. Many investigations were explored to increase the therapeutic efficacy in the required timeframe, which is sometimes narrow and involves high dosage with precise timing, or that is needed only for a short period to treat urgent diseases or unpredictable pathogenic invasion. In that regard, several concepts have been reported, including thermal, magnetic, electromagnetic, radiofrequency and ultrasound actuation. 4.2.2

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The concept of temperature-responsive polymers has been widely explored for designing DD systems based on TNTs with externally stimulated drug release for different nanocarriers (Figure 4G). For instance, Cai et al. explored the viability of controlling drug release from TNTs by using Vitamin B2 as a model drug [73]. In their approach, a coating based on hydrogel was deposited on the top surface of TNTs layers. Hydrogel was used as the temperature-responsive agent on the TNT-based implant. A temperature-responsive polymer (Poly (N-isopropylacrylamide), PNIPAAm) was used to synthesize the hydrogel. PNIPAAm presents reversible volume phase transition properties at its lower critical solution temperature (LCST). When the temperature is lower than the LCST, the composite hydrogel is in a highly water-swollen state. However, at higher temperatures, the hydrogel structure collapses and the drug release from TNTs is triggered. Notice that a common inflammatory reaction generates a temperature higher than 38 C. This principle can serve as a direct temperature controlled stimulus to trigger drug release for a localized therapy [73]. The encapsulation of drug inside nanomagnetic structures has received great attention for designing triggered DD systems because of the exciting possibilities to use these materials for magnetic field triggered drug release. As far as the implementation of this strategy into TNTs-based systems is concerned, Shrestha et al. reported on the development of this concept using TNTs filled with magnetic nanoparticles (MNPs) in order to achieve magnetic- and photocatalyticguided release of drugs [71]. This concept is based on the fact that the UV-induced hole generation in the valence band of the TiO2, leads to chain-scission of a monolayer attached to TiO2, with the cleavage at the anchoring siloxane groups, causing the release of drug molecules. Furthermore, fluorescence microscopy images demonstrated that UV-induced photocatalytic activity with the use of TNTs can be used to kill cancer cells [87]. This is another extraordinary example of TNTs as a therapeutic agent for the treatment of cancer cells, which opens many new opportunities for DD applications in the near future. A different strategy for prompted release of drugs assisted by external magnetic field based on MNPs loaded inside TNTs was proposed by Aw et al. [88]. This system consists of TNTs loaded with drug-encapsulated polymeric micelles at the top acting as drug carriers and MNPs at the bottom of the nanotubes (Figure 4H). The release of drug molecules from this TNTs-based system can be activated by applying an external magnetic field to induce the movement of magnetic particles from the bottom, forcing the release of the drug-loaded micelles. The drug-release characteristic of this system confirms that 100% release of drug nano-carriers were achieved within 1 -- 1.5 h upon the application of the magnetic field. Nevertheless, this system presents some serious limitations such as uncontrolled release, which can be triggered by existing external magnetic fields from the surrounding environment.




To overcome these drawbacks, Aw et al. demonstrated the application of local ultrasonic external field for triggering drug release from TNTs [89]. This drug-releasing system is based on ultrasound-mediated drug and nano-carriers release from TNTs structures, which is achieved by applying oscillating ultrasound waves from a probe close to the location of the TNTs implant (Figure 4I). In vitro studies have shown that drug-micelle release can be arbitrarily completed from 10 min to 2 h, depending on the ultrasound parameters (amplitude, frequency, pressure), which verifies the potential feasibility of this approach for local DD applications. However, some therapies require the use of non-invasive sources of external stimuli. In that regard, radiofrequencyresponsive release becomes an excellent strategy. This concept has been implemented into TNTs-based DRI (Figure 4J). In this system, gold nanoparticles (AuNPs) were used as a stimulant and energy transmitter as result of their capability to be locally heated in a manner similar to the phenomenon observed in RF-induced hyperthermia [90]. After RF exposure and due to the transmission of heat from the AuNPs to the drug molecules, the release of drug is remotely activated from the TNTs-based implants. This system can therefore provide constant release of drugs for 1 -- 3 h, depending on the applied RF energy and the amount of AuNPs loaded into the TNTs. Another approach used to trigger drug releases by an external field was reported by Webster and co-authors, who incorporated drugs into multi-walled carbon nanotubes (MWCNTs) grown out of TNTs, where drug release was controlled from nanotubes through an electrical field [91,92]. Their studies suggested that multiple deliveries in a single TNTs system could be an optimal solution for treating bone diseases and regulating tissue generation at various stages of bone repair. Further advancement of this system is suggested by doping polypyrrole with antibiotics (penicillin and streptomycin) and an anti-inflammatory drug (dexamethasone) and their loading by electrodeposition inside MWCNTs grown on TNT arrays [93,94]. It was observed that approximately 80% of the drug was released when an external voltage was applied, thereby acting like a trigger-enabled drug-eluting bone implant. Although such an ‘on-demand drug-delivery’ technology is in its infancy, it has a huge potential for versatile developments in DD using TNT arrays. Further advances in implant technology would require an in-built sensing functionality with a capability to sense implant conditions, implant environment and trigger drug release when required. Previous studies suggested electrochemical sensing applications to monitor bone formation by fabricating MWCNTs on TNTs-Ti implants based on the ability of MWCNTs to enhance direct electron transfer [94]. This implant was proposed to sense components of osteoblast ECM by detecting their redox reaction profiles. These results suggest that a MWCNTs-modified Ti implant surfaces can serve as electrochemical electrodes to monitor any infection, inflammation that occurred or bone growth from the implant

surface. This technology is anticipated to be tremendously beneficial for implant-related diagnostics in the future. In summary, several exciting and promising concepts for external field triggered drug-release using TNTs were demonstrated in recent years. These elegant and sophisticated approaches have real potential to generate time-programmable, remotely triggered and ‘smart’ DRI. These strategies are also expected to be viable for many applications, including bone therapy and local chemotherapy alone or in combination with systemic chemotherapy. However, these concepts are still at an early stage of development and further studies using ex vivo and in vivo models are required to assess their feasibility for real-life practical applications.

Perspectives of TNTs for clinical applications

5.

TNTs for bone therapy and orthopedic implants Bones play a key role in many critical functions in human physiology. Bone-related diseases and traumatic injuries not only have a direct impact on the quality of life and health of patients but also consume in total 10% of the annual healthcare expenditure [95]. These medical conditions are treated by complicated orthopedic surgical procedures, including total joint replacement, spine fusion and repair of fractures. Typically, orthopedic implants are based on titanium and its alloys [96]. Most of titanium-based orthopedic implants are in the form of screws or plates, the objective of which is to hold the broken fragments together. Bone infections are the most common clinical complications associated with bone implants. This is a serious adverse condition in orthopedics, which requires prolonged hospitalization and complex revision procedures. Furthermore, implant infections can lead to more serious medical complications such as implant failure and rejection and can cause substantial suffering and even death of patients [96]. Traditionally, these clinical complications have been treated by SDD approaches used to combat bone infections or poor implant integration. These, however, are ineffective in reaching the bone site and drug overdose can generate toxicity issues and side effects in patients. Therefore, in that scenario, LDD becomes a potential alternative to overcome these limitations by carrying out direct and local delivery of drugs or proteins from implants. As an example, orthopedic implants coated with drug-releasing layers based on nanoporous materials such as TNTs are considered an excellent alternative approach to address some of these problems. LDDS can be applied in numerous areas, including the suppression of inflammatory reactions, treatment of bone infections, improvement and promotion of bone healing, enhancement of osseointegration and local treatment of primary or secondary bone cancers. 5.1

Bone inflammation When an implant comes into contact with the living tissue, the immune-system of the host body produces an inflammatory 5.1.1


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response, which is initiated during the surgical insertion of the implant. Ainslie et al. investigated the difference in inflammatory responses between TNTs arrays and Ti surfaces (control sample) in order to demonstrate the role of nanostructuring in modulating immune responses [97]. The viability/ morphology of monocytes, inflammatory cytokines and reactive oxygen species production as indication of immune reactions were evaluated. The obtained results showed that TNTs arrays significantly reduced the inflammation response as compared to bare Ti surfaces. In order to improve the antiinflammatory properties of TNTs arrays, Aninwene et al. investigated this subject by loading these nanotubes with anti-inflammatory and immunosuppressant drug dexamethasone, by physical adsorption and deposition from simulated body fluid [98]. Their results showed improved drug-elution characteristics (i.e., release for up to 3 days) along with enhanced osteoblast adhesion. Our group performed several in vitro studies to show the potential applicability of TNTs arrays for loading and releasing water-insoluble indomethacin using planar and wire implants [26,27,61]. Indomethacin is a non-steroidal anti-inflammatory drug, which is commonly used to treat postsurgical bone inflammations and to improve bone healing after implant surgery [99]. Our results demonstrated that a large amount of drug (1 -- 2 mg cm-2) can be loaded inside TNTs arrays and subsequently released in a suitable fashion for therapeutic applications. In addition to that, several concepts for extended, delayed, multi-drug and triggered drug release using indomethacin as a drug model have been presented and discussed in the previous section. These studies provided different proof-of-principle demonstrations for the application of TNTs arrays as DRI, demonstrating that TNTs layers are capable of delivering therapeutics to prevent inflammations in bone-related diseases. Bacterial infection Bacterial infections of bone (osteomyelitis) can lead to devastating medical complications. Typically, bacterial infections occur just after orthopedic implant surgery and must be treated by a prolonged systemic administration of antibiotics [100]. The most common microorganisms linked to implant infections are: S, aureus, S. epidermidis, Enterobacteriaciae and Pseudomonas aeruginosa. To address these infection-related problems associated with orthopedic implants, several approaches have been explored using TNTs arrays loaded with antibiotics, silver NPs (AgNPs) and antimicrobial peptides [49,101-104]. The pioneering study by Popat et al. demonstrated a local antibiotic therapy using TNTs surfaces loaded with gentamicin sulphate (200, 400 and 600 µg) for reducing S. epidermidis adhesion [49]. This strategy significantly reduced the adhesion of bacteria on the surface of TNTs as compared to bare Ti surfaces and unloaded TNTs. Simultaneously, these implants retained their capabilities for promoting osteoblasts adhesion and proliferation, indicating the suitability of this approach to prevent orthopedic implants from bacterial infections and promote 5.1.2

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osseointegration. Furthermore, Zhang et al. demonstrated improved antibacterial activity and biocompatibility of vancomycin-loaded TiO2 nanotubes through a series of in vivo and in vitro experiments [101]. In a similar study, Yao et al. showed an improved method for effective drug loading of penicillin-based antibiotics in TNTs arrays [102]. In this study, TNTs arrays featuring nanotube diameters of 80 nm and lengths of 200 nm were fabricated and loaded with penicillin/streptomycin mixtures, which precipitated with calcium phosphate crystals inside these TNTs arrays. This study demonstrated for the first time that coprecipitated drug coatings on TNTs arrays can delay the release of drug for up to 3 weeks with controllable dosage. In addition, Ercan et al. proved that thermally treated TNTs arrays featuring different nanotube diameters (20, 40, 60 and 80 nm) have varied antibacterial responses for S. epidermidis and S. aureus [105]. These authors found that an annealing treatment (500 C for 2 h) reduced the adhesion of live and dead bacteria for both types of bacteria. Other antibacterial substances that can be loaded inside TNTs are AgNPs. Zhao et al. have reported that the antibacterial effects of TNTs arrays can be achieved by loading them with AgNPs, using a simple process based on AgNO3 immersion and UV irradiation [103]. AgNPs, which possess antibacterial properties, were found to adhere to the inner walls of TNTs arrays. TNTs arrays decorated with AgNPs were capable of destroying all the initial planktonic bacteria present in the culture medium during the first several days and preventing the implant from bacterial adhesion for 30 days. Another approach aimed at preventing the infection of antibiotic-resistant bacteria is based on the use of TNTs arrays loaded with antimicrobial peptides (HHC-36) [104]. These antimicrobial peptides were loaded inside the TNTs arrays and tested against S. aureus. The obtained results showed an ability to kill 99.9% of bacteria and extend the release of these therapeutics over 7 days. Interestingly, the anatase type of TNTs arrays used in this study was found to have a higher release rate as compared to that of amorphous TNTs arrays. This approach demonstrated that TNTs can be used to treat bacterial infections associated with implants in an effective manner, particularly those caused by bacterial resistance from S. aureus, as compared to other therapies based on conventional antibiotic drug administration. Although these studies using TNTs-based implants have shown promising results to solve bacterial infections, they are mainly based on in vitro investigations. Therefore, extensive in vivo studies are required to further validate this technology for real-life clinical applications. Implant osseointegration Another important aspect of orthopedic implants is their osseointegration capability. Poor implant osseointegration due to poor interaction between the implant surface and the biological environment is one of the most common reasons of implant failure. To facilitate a suitable integration of the 5.1.3




implant within the microenvironment surrounding the bone, the implant surface must promote functions of different cell types, including osteoblasts, osteoclasts and stem cells as well as enhance bone healing [106]. To improve osseointegrating properties of Ti implants, several surface roughening approaches have been explored using mechanical methods (machining, grinding and blasting), chemical methods (acid/ alkali etching) and electrochemical anodization [106]. Numerous studies have indicated that nanostructures (nanotube and nanopore morphologies) are more suitable than microstructures for promoting cell adhesion and proliferation functionalities. These studies have indicated that TNTs arrays are excellent interfaces between Ti implants and the biological environment for short- and long-term integrations [107]. One of the key factors to achieve an optimal integration of implants in bones is a fast kinetics of HA formation on the implant surface from the body fluid. As noticed in previous sections, a number of studies have shown that HA formation is accelerated by TNTs arrays as compared to bare Ti and TiO2 surfaces [39-41]. A 3D structure of TNTs was found to be optimal for embedding precursors for HA formation, which promoted HA nucleation and improved osteoblast cells functions. Kunze et al. studied the effects of electrochemically fabricated and annealed TNTs with anatase phase and found that these nanostructures are excellent precursors for the formation of calcium HA [108]. Their results showed that more nuclei were formed on surfaces featuring TNTs than on flat compact titania at the earliest phases of apatite growth, with excellent cell proliferation and osseointegration. Furthermore, in vivo experiments with pigs demonstrated that the surface of TNTs layers can enhance collagen Type 1 and BMP-2 expression [62]. The use of biopolymer coatings such as chitosan and PLGA on TNTs-based implants has been demonstrated as another strategy to increase osteoblast functions such as cell adhesion and proliferation as compared to bare Ti implants and TNTs arrays [61]. Another approach was reported by Neupane et al., who developed a unique method for promoting the growth and attachment of bone cells by loading gelatinstabilized gold NPs (G-AuNPs) into TNTs arrays using lipophilization technique [109]. Osteoblasts attachment and proliferation were significantly enhanced on G-AuNPs/TNTs arrays, producing an interlocked cell structure by the movement of filopodia from growing cells inside the nanotubes. In another example, well-ordered TNTs arrays loaded with strontium (Sr) showed a slow and sustained release of Sr, which led to favorable osteogenic effects [110,111]. Drug-loaded TNTs with a variety of bioactive molecules, such as proteins, enzymes, peptides and calcium phosphate/ HA, have been demonstrated to improve osseointegration. As an example, TNTs were used for the delivery of BMP-2. This system was able to simultaneously promote the proliferation, migration and differentiation of MSCs, opening new opportunities for stem cell engineering and cell-based therapies [112].

Ex vivo and in vivo studies Most of the aforementioned studies on drug release therapies from TNTs were performed through in vitro experiments using PBS solution as eluting medium. This condition, however, is considerably different than that of a real clinical scenario, where real bone tissues and real biological environment are present. Notwithstanding all these promising results obtained from in vitro studies, it is worth concluding that the delivery of drugs to specific skeletal sites in real clinical applications is a technical challenge yet to be addressed. Furthermore, in vivo applications present other challenges such as the use of an appropriate method to accurately monitor the distribution of drug molecules from the implant to the bone structure. To address this problem, our group pioneered a new method for ex vivo study of drug release in bones using The Zetos bone bioreactor. In this system, implantable TNTs-Ti wires loaded with drug are inserted directly into a bovine bone core (Figure 5) [113]. The method is based on the Zetos system developed by D.B. Jones (Germany) and E.L. Smith (USA), which enables the culturing of 3D cancellous bone cores with the ability to apply pre-programmed force compression in real-time, maintain the 3D matrix relationship between the bone cells and the marrow cells and exercise control over the physiochemical bone environment [114,115]. A series of drug concentration profiles in bone were obtained from these images, which showed the capability of this approach to measure the distribution of drug molecules in bones across all directions (x, y and z axes) from the TNTs wire implant. This study also demonstrated that wire or needle-like TNTs implants can be directly inserted inside bones and used as DRI to provide the required drug dosage and release for bone therapy. While ex vivo system bridges the gap between in vitro and in vivo conditions, in vivo examinations are still considered as the real tests for any biomaterial. Therefore, any biomaterial must provide a suitable in vivo performance before it enters the next level of research (i.e., clinical trials). In the case of TNTs fabricated on titanium implants, these have to integrate within the bone tissue and also survive the shearing forces/ stresses experienced during surgical insertion inside the animal model. As described in the previous section, von Wilmowsky et al. studied the implantation of TNTs-Ti implants inside the front skull of domestic pigs. This study reported the positive outcome of bone formation characteristics of TNTs as compared to commercially available pure titanium implants [62]. Similar results were also confirmed by Xiao et al. with TNTs implanted in the osteoporotic jawbone of sheep [116]. In another attempt, Park et al. loaded fibroblast growth factor (FGF) and human fibronectin fragment (hFNIII9-10) fusion protein in TNTs on Ti implants, followed by insertion in rabbit tibia [117]. After 3 months, the animals were sacrificed and bone-implant contact results showed improved osseointegration for protein-loaded TNTs implants [117]. These animal studies provide a valuable information about the performance 5.1.4


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Figure 5. Ex vivo study of transport of drug in bone released form TNTs wire implant. (A) Schematic diagram of drug release from a TNTs; (B) photo of trabecular bone with embedded TNT/Ti wire implant loaded with drug at the centre of the bone core (side view); (C) trabecular bone core with TNT/Ti wire inside the bone chamber connected to perfusion pump which provides culture media to keep bone cells alive; (D) bioluminescent image of the bone core and the embedded implant inside the perfusion chamber to probe the concentration of the model drug released from the implant. Reprinted with permission from [113]. Ti: Titanium; TNTs: Titania nanotubes.

of nanoengineered implants in in vivo environments, and hold much promise for human trials. However, we must stress that these experiments were carried out over a period of 2 -- 3 months, which represented only the initial phase of a typical implant life and thus more studies are required towards investigating longer healing periods and more complex scenarios. TNTs application in dentistry In dentistry, tackling local microbial community is the predominant issue in oral hygiene, and this involves bacterial and planktonic agents that induce bacterial biofilm formation, active fungal, viral infection that elicit inflammatory response from the body and thus inhibit antimicrobial activity. For instance, persistent bacterial infection is a major risk after implantation surgery and sometimes healing is not guaranteed in bones surrounding the implant device. Another example is exhibited by jawbones, which are somewhat thin and delicate inferior maxillary bones. In order to replace a failed implant associated with this particular bone can be difficult, not to mention very expensive -- which costs approximately USD $2000 and $4000 to install a single implant. Other challenges in clinical dentistry are caries control and restoration, tooth remineralization, periodontal disease management and root-canal-related infection [118]. In that regard, TNTs are an alternative technology that can address some of these problems in dentistry as this is an economically affordable technology that can be easily implemented into dental 5.2

118

implants and thus provide the advantages presented throughout the previous sections. Ti and its alloys are routinely employed as dental implants to treat edentulous patients, generally suffering from implant failure due to peri-implantitis and other complications [118,119]. A tight and healthy bone-implant contact and implant-tissue integration by forming a peri-implant soft tissue seal are critical aspects in determining implant stability and long-term bone regeneration, to protect the bone and tissue underneath from bacterial and mechanical damages. The lack of prolonged bonding of the Ti implant to juxtaposed teeth (integration) for clinical implantation can be countered by fabricating biofunctional/bioactive implants via surface modifications, combined ceramic coatings and immobilization of biomolecules [120]. Given that the natural ECM has hierarchical nanostructure, TNTs surfaces are ideal candidates to stimulate the integration of implants due to their nanotopography. Furthermore, the scope of TNTs-based delivery systems can be extended to regenerative medicine and dentistry along with preventive therapy. Figure 6 summarizes a conceptual overview of several aspects relevant to TNTs applications in dentistry, which include studies of osteoblast adhesion to TNTs surfaces and its biocompatibility, HA growth, release of specific drugs and silver nanoparticles and periodontium regeneration by the delivery of multiple growth factors inducing osteogenesis. TNTs can serve as a gene and DD carrier, including growth factors,



B.

C. Apatite

D. Apatite Apatite

10 µm

50 µm

50 nm

A. E.

TNTs

Ti dental implant


200 nm

Figure 6. Schematic diagram summarizing the applications of TNTs in dentistry. (A) Periodontium problem and proposed regeneration by delivery of multiple growth factors inducing osteogenesis, (B) the adhesion of osteoblast cells to TNTs surface, (C) the growth of hydroxyapatite on TNTs. (D) The cross-sectional view of a single TNT revealing the drug sodium naproxen on the inside. (E) Anti-bacterial silver nanoparticles incorporated within TNT. C. Reprinted with permission from [40]. D. Reprinted with permission from [120]. E. Reprinted with permission from [103]. Ti: Titanium; TNTs: Titania nanotubes.

signaling cues and stem cells, which can further enhance osseointegration and bone regeneration relevant to tooth implants. It is known that bioactive molecules, including TGF, platelet-derived growth factor-B (PDGF-B), bone morphogenetic proteins (BMPs), FGFs, Hedgehog and Wnt pathways coordinate the complex molecular and biological orchestration relating to tooth regeneration and local bone formation [120]. Shim et al. demonstrated titanium discs with TNTs layers for controlled delivery of fibroblast growth factor-2-loaded poly-(lactide-co-glycolide) nanoparticles over 2 weeks [120]. Hu et al. loaded TNTs arrays with bone morphogenetic protein 2 (BMP2) and used a coating based on gelatin/ chitosan for controlling the release of BMP2 and stimulating motogenic responses of MSCS and promoting their osteoblastic differentiation [121]. Moreover, Lee et al. examined the feasibility of dental implants based on N-acetyl cysteine-loaded TNTs [122]. This study revealed an increment of newly formed bone volume and bone mineral density in the mandibles of Sprague Dawley rats. Demetrescu et al. showed that TNTs surfaces with a nanotube diameter of < 120 nm exhibits better electrochemical stability in artificial saliva along with improved adhesion and proliferation of human gingival fibroblasts (HGFs) [123]. Additionally, the response of HGFs was recorded by immobilizing silver nanoparticles and FGF-2 onto TNTs arrays to obtain lower cytotoxicity, antibacterial properties, enhancement of HGF cell attachment, proliferation and ECM-related gene expression [124]. This group also investigated the concentration of immobilized FGF-2 and time-dependent response of HGFs to help mimic natural gingival tissueimplants integration [125]. A report by Shokuhfar et al. elucidated the biophysical evaluation of osteoblasts on TNTs grown on the surface of bone screws. This study demonstrated the successful insertion and removal of orthopedic bone screws with TNTs layer without any damage imposed on the

system [126]. TNTs-based delivery systems for antibacterial and anti-inflammatory agents to target implant-associated infections were reported by Zhao et al. using AgNPs, showing preventable post-operation bacterial colonization for 30 days [103]. The intercalation of anti-inflammatory drug, sodium naproxen loaded in liberated TNTs was demonstrated by Shokuhfar et al., who proposed suppression of inflammatory response induced by the release of cytokines caused by innate immunity [127]. Notwithstanding all these advances in the use of TNTs in dentistry-related applications, we must point out that existing LDDS based on nano- and micro-carriers (e.g., agarose beads, collagen sponge, bone cements, alginate gels, PLGA microspheres, hydrogels, inorganic oxide nanoparticles, nanoclays, etc.) provided more practical advantages. Cardiovascular stents Percutaneous coronary intervention (PCI), formerly known as angioplasty with stents, was pioneered by Gru¨ntzig in 1977 and has become one of the most widespread therapeutic techniques performed in medicine [128]. The purpose of coronary stents is to maintain lumen integrity and improve procedural safety and efficacy, eliminating the need for surgical stand-by [129]. Nevertheless, stent-mediated arterial injury elicits neointimal hyperplasia, leading to post-angioplasty restenosis and the need for further revascularization in up to one-third of patients [130]. Thus far, several technological approaches have been explored to overcome these inherent drawbacks associated with cardiovascular stents. To address these problems, the concept of drug-releasing stents was introduced. These systems were traditionally based on stents coated with polymer coatings loaded with antiproliferative agents. These systems showed promising results to reduce the risk of revascularization, as compared to stents based on bare metals [131]. Nonetheless, several studies have 5.3


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questioned the long-term safety of these drug-releasing stents. This led to a reduction in their use and the implementation of an rigorous review of these medical devices by regulatory agencies, which recommended further extension of the dual antiplatelet therapy for at least 1 year post-implantation [132]. In 2007, some studies demonstrated that drug-releasing coronary stents were associated with similar risks of death and myocardial infarction as compared to stents based on uncoated metals, but with an increased risk of stent thrombosis beyond 1 year post-implantation [133,134]. Since then, new technological approaches have been explored to develop improved drug-releasing stents, which are aimed at improving the life expectancy of clinical patients. As a result, different materials have been sought, tested and tried to overcome inherent drawbacks of drug-releasing cardiovascular stents coated with polymers. In that regard, absorbable drug-releasing stents were reported to be an excellent alternative to conventional drug-releasing stents [135]. Another alternative to conventional drug-releasing cardiovascular stents was reported by Wieneke et al., who performed an in vivo study using coronary stents featuring a coating layer of NPA [136]. These cardiovascular stents were produced by electrochemical anodization of stainless steel stents coated with aluminium. The resulting cardiovascular stents featuring nanoporous coatings were infiltrated with tacrolimus and, subsequently, implanted in the common carotid artery of rabbits. The authors verified that the maximum concentration of drug in the bloodstream was reached 1 h after implantation, decreasing gradually during the next 48 h and maintained well within the acceptable therapeutic window. This study demonstrated for the first time the potential applicability of drug-releasing cardiovascular stents based on nanoporous inorganic coatings. Nonetheless, recent in vivo studies in porcine models have demonstrated that the shedding of particle debris released from the nanoporous coatings produces a significant increment of neointimal hyperplasia, as compared to bare stainless steel stents [137]. With regard to this, TNTs could possibly provide better performances than NPA because the surfaces of TNTs promote better interactions with endothelial cells, which lead to the generation of a uniform endothelium on the implant surface, preventing the stent from becoming loose and being dislodged after implantation, and therefore forbidding particle debris release from the implant surface [138]. However, although TNTs layers on Ti implants are well-accepted medical devices for developing DRI, there is concern that Ti does not possess adequate mechanical properties such as sufficient flexibility and expansion capacity to develop cardiovascular stents. Outstanding progress towards the application of TNTs for cardiovascular stents and preventing restenosis problems has been recently reported by Peng et al. suggesting that TNTs are a promising candidate for the next generation of drug-releasing stents and coatings for cardiovascular devices [139]. The confidence is based on the divergent response of endothelial cells and VSMCs to the surface of TNTs arrays. 120

This study also identified certain genes and possible pathways that are correlated with changes in EC and VSMC phenotype, specifically, those that are associated with phenotype changes in response to nanotopography. These results are an important step towards a better understanding of how cells sense and respond to nanotopography and how these interactions can be used to improve the design of medical devices [139]. Nevertheless, we must stress that these studies have been focused on in vitro scenarios, which are far from a real in vivo environment. Therefore, more in vivo studies should be carried out before TNTs-Ti implants become a feasible and reliable alternative to existing drug-releasing cardiovascular stents. Potential application for localized chemotherapy A major problem of current systemic delivery of chemotherapeutics is the limited dosages of toxic anticancer drugs that can be achieved at the cancer site without causing serious adverse complications, such as cardiotoxicity. Surprisingly, the concept of LDD for cancer therapy has not been widely explored yet. So far, there is only one clinically proven system for localized delivery of chemotherapeutics, specifically developed for the treatment of brain tumor (glioblastoma). This implantable system is based on a biodegradable polymer impregnated with the anticancer drug carmustine (Gliadel, Guildford Pharmaceutics, UK) [140]. The Gliadel wafer has a diameter of 2 cm composed of 192.3 mg of polyanhydride copolymer and 7.7 mg of anticancer drug carmustine [140]. An invasive surgical resection is required to place these wafers directly into the tumor cavity in order to deliver the anticancer drug directly to the diseased tissue. The treatment has been accepted in more than 20 counties with more than 50,000 procedures performed to date, showing an increased survival rate of treated patients suffering from glioblastoma. The main disadvantages of the Gliadel wafer are its large size, which requires extensive surgical intervention and prevents its use in many situations, as well as its single drug load, carmustine. To overcome these limitations, our group has recently proposed a new design of DRI for treating cancers such as brain (glioma) and bone (osteosarcoma) locally using TNTs wires loaded with anticancer drugs (doxorubicin, paclitaxel and pro-apoptotic protein Apo2L/TRAIL) [141]. Preliminary results from in vitro (cell) and ex vivo (cancer tissue) studies showed that anticancer drugs were released from the nanotubes and were able to effectively destroy cancer cells. A recent study by Xiao et al. explored the biocompatibility of the TNTs loaded with anticancer drug (cisplatin) for the growth of osteosarcoma (MG-63) cells. Their results showed good biocompatibility, supporting the normal growth and adhesion of MG-63 cells with no need of ECM protein coating [142]. Another appealing approach has been recently demonstrated by Schmuki’s group, which showed that TNTs can be directly used as a photocatalyst for efficient killing of cancer cells (in vitro). Therefore, this system could be potentially 5.4



used for anticancer treatments. They proposed that TNTs can be administrated locally to a tumor, followed by a focused UV-light or X-rays excitation to trigger a photocatalytic reaction on TiO2 and achieve destruction of cancer cells [87]. These preliminary results suggest that the proposed implant technology based on TNTs has the potential to be successfully used for localized cancer therapy, and more studies and more advanced systems are expected in future to explore these concepts.


6.

Conclusion

Recent research studies discussed in this review have demonstrated that TNTs is a promising biomaterial for developing a variety of LDDS capable of overcoming the limitations of systemic drug therapies. The fabrication process of TNTs is based on a well-established and scalable technology using simple and low-cost electrochemical processes. Furthermore, widely available biocompatible materials (Ti and Ti alloys), which have been used for many years as medical implants, can be directly used to produce TNTs-Ti drug-releasing systems. As an example, TNTs layers can be generated on existing medical implants (orthopedic, stents) with no limitation in its shapes and forms, including stents, plates, needles, wires, screws and plates. TNTs were shown to possess a unique set of properties for DD applications, including high surface area, controllable nanotube dimensions, tuneable geometries and surface chemistry, high and versatile drug-loading capacity for several drugs, ability to modulate drug release kinetics and so forth. Moreover, TNTs as biomaterial has shown to have many outstanding properties, including chemical and corrosion resistance, mechanical and thermal stability, rigidity and proved biocompatibility, promoting the growth, adhesion, differentiation and proliferation of bone cells and HA formation, osseointegration and hemocompatibility. Studies mentioned throughout this review confirmed that TNTs can be used for loading and releasing of wide range of therapeutics, with the ability to tune their drug releasing characteristics and provide multi-drug release of different drugs in different fashions (extended, sequential, externally activated, etc.). These approaches, which have been exhaustively presented in this review, are aimed at achieving optimized drug dosage, release rate and time needed for a broad range of specific therapies. Amid these strategies, the design of triggered or ‘ondemand’ drug-release from TNTs using various external signaling sources (thermal, magnetic, electro-magnetic, ultrasonic or mechanical activation) is an outstanding feature offering great perspectives and opportunities for combining TNTs with sensing functions. These could be at the base of a new generation of smart implants and drug-delivery devices. TNTs are shown to be very versatile in terms of their biomedical applications and can be applied as independent DRI or devices. This technology, as this review has demonstrated, is still far from being applicable in real clinical applications even, in vitro, ex vivo and in vivo studies on TNTs have showed very

promising results. Therefore, based on the current progress on this technology, we can conclude that there is considerable potential for TNTs to revolutionize the area of LDD, in particular, for applications related to orthopedics and cancer therapies. Nevertheless, in spite of the success described in the studies mentioned throughout this review, more in-depth research is demanded in order to make DRI feasible for reallife clinical applications. TNTs and LDDS are not going to replace traditional SDD, but it is clear that these systems could provide alternative and synergistic ways to implement more efficient therapies, where the efficacy of SDD is limited, especially in bone therapy, cancer and tissue engineering. 7.

Expert opinion

This review has summarized the recent progress and key findings in the application of electrochemically engineered TNTs arrays for LDD applications. This new and promising material and technology for engineered DD have shown, at this early stage of development, many advantages and potential to be translated into clinical and commercial applications. Key findings to support this statement are based on the outstanding properties of TNTs material. TNTs are highly ordered nanotube structures with precisely controllable dimensions (superior to other competitive materials such as polymers, ceramics). They have morphology that mimics the dimensions of natural bones to enhance their interactions with the surrounding biological environments and HA formation, have excellent mechanical, chemical stability, proved biocompatibility, antibacterial and osseointegration properties. Furthermore, compared with other materials, TNTs offers superior properties in terms of drug loading and release including large loading capacity for a broad range of drugs and drug carriers, including multiple drugs and no limitation for hydrophobic, or hydrophilic, proteins, genes, polymer micelles and nanoparticles, TNTs also possess the flexibility to tailor drug release performance, including dosage, time and kinetics and the implementation of multiple drug release strategies such as extended, delayed, sequential, multi-drug and external field triggered release through external stimuli such as ultrasound, magnetic, RF and X-ray and applicability to different localized therapies (bone, brain cancer, dentistry, cardiovascular). The production of TNTs is based on a simple, scalable, versatile and low-cost fabrication technology applicable on different substrates (Ti, Ti alloys, Al, Al alloys stainless steel, etc.) with different shapes (needles, wires, plates, rods, meshes, etc.) that includes existing titanium implants widely used in medicine. TNTs can be combined or integrated with existing implantable medical devices such as biosensors, microchips and micropumps, to combine sensing and therapeutic function into one device. Nevertheless, some technical challenges must be addressed and further explored before this technology becomes feasible and reliable for real clinical applications. One disadvantage of TNTs is that they are not biodegradable as compared to


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polymer implants. This means that, in some therapies, these implants have to be removed by additional surgical intervention after drugs are released and the treatment finished. However, this may not be necessary if TNTs are formed on the surface of existing implants. In that scenario, TNTs become an outstanding alternative to bare Ti implants given that they enable antibacterial capabilities and osseointegration promotion, which can reduce the risks of implant rejection and enhance bone healing. Another possible disadvantage of TNTs is their long-term stability, and their potential delamination and degradation in the biological environment, which could expose blood with high-aspect ratio and bioactive nanotubes. Even though initial ex vivo and in vivo studies on TNTs implanted in bones confirmed their structural stability without visible damages and delamination, more exhaustive and systematic long-term studies are required to assess these critical aspects of long-term stability. Nevertheless, although still in the early developing stages, numerous in vitro and some ex vivo and in vivo studies have clearly shown the potential of TNTs implants for LDD applications. TNTs have demonstrated their capabilities to address the most critical problems related to orthopedic implants and bone therapies (inflammation, infection and poor osseointegration), dentistry (infections, implants integration), cardiovascular stents (restenosis) and cancer therapy. The obtained results are very promising and strongly suggests the development of TNTs-based DRI towards more exhaustive in vivo studies using animal models and preclinical and clinical studies. To achieve these goals, however, several technical challenges must be addressed, which include further technical development on TNTs fabrication, more studies on toxicity Bibliography

and in vivo studies using animal models. Furthermore, TNTs require more fabrication advances in terms of improvement of their production, scalability and reliability before they start to be produced at industrial scale. Another important challenge to be addressed by this technology is the current public opinion, as the public has serious which have serious reservations about the use of nanomaterials in medicine. As far as this is concerned, more toxicity studies on liberated and fragmented nanotubes are essential in order to eliminate toxicity doubts and gain the acceptance of this new technology when launched into the markets. It is expected that health regulation regarding these medical devices composed of several different components, including nanomaterials and drugs, will be defined in coming years to clarify safety standards and evaluation protocols for these clinical products. Finally, several exciting future developments are expected for the future, which include the integration of TNTs implants with sensors, microchips, development of advanced triggered drug release, the application of TNT implants for combination therapy and potential localized therapy of primary solid cancers or secondary bone cancers.

Declaration of interest The authors gratefully acknowledge financially support provided by the Australian Research Council (ARC) for this work (project grants numbers: DP120101680, FT110100711 and DE14010054). The authors are thankful to the School of Chemical Engineering at the University of Adelaide (UoA) for their support in this research. The authors report no conflicts of interest. The authors are solely responsible for the content and writing of this article.

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Affiliation

Dusan Losic†1, Moom Sinn Aw2, Abel Santos3, Karan Gulati4 & Manpreet Bariana4 † Author for correspondence 1 Resarch Professor, Australian Future Fellow, The University of Adelaide, School of Chemical Engineering, Engineering North Building, Adelaide, SA 5005, Australia Tel: +61 8 8313 4648; Fax: +61 8 8313 4583; E-mail: [email protected] 2 Research Associate, The University of Adelaide, School of Chemical Engineering, Engineering North Building, Adelaide, SA 5005, Australia 3 Research Fellow, ARC DECRA Fellow, The University of Adelaide, School of Chemical Engineering, Engineering North Building, Adelaide, SA 5005, Australia 4 PhD Student, The University of Adelaide, School of Chemical Engineering, Engineering North Building, Adelaide, SA 5005, Australia

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Recent advances on smart TiO2 nanotube platforms for sustainable drug delivery applications.

Titania nanotube arrays as interfaces for neural prostheses.

Recent advances in ocular drug delivery system.

Basics and recent advances in peptide and protein drug delivery.

Bisphosphonates: therapeutics potential and recent advances in drug delivery.

Recent advances of cocktail chemotherapy by combination drug delivery systems.

Nitrogen and transition-metal codoped titania nanotube arrays for visible-light-sensitive photoelectrochemical water oxidation.

Recent advances in targeted nanoparticles drug delivery to melanoma.

Smart electrospun nanofibers for controlled drug release: recent advances and new perspectives.

Hepatitis A. Perspectives and recent advances.

Interfacial Materials for Organic Solar Cells: Recent Advances and Perspectives.

Skin permeabilization for transdermal drug delivery: recent advances and future prospects.

Recent advances in dendrimer-based nanovectors for tumor-targeted drug and gene delivery.

Protamine-oligonucleotide-nanoparticles: Recent advances in drug delivery and drug targeting.

Sensitive and selective electrochemical detection of chromium(VI) based on gold nanoparticle-decorated titania nanotube arrays.

Introduction for the special issue on recent advances in drug delivery across tissue barriers.

Recent Advances in Hydrogel-Based Drug Delivery for Melanoma Cancer Therapy: A Mini Review.

Recent advances in carbon nanotube-based enzymatic fuel cells.

Recent advances in nanoparticle-mediated siRNA delivery.

Atrial remodeling and atrial fibrillation: recent advances and translational perspectives.

Resveratrol: review on therapeutic potential and recent advances in drug delivery.

Recent Advances in Nanomaterials for Gene Delivery-A Review.

Halloysite nanotube-based drug delivery system for treating osteosarcoma.

Disentangling a Holobiont - Recent Advances and Perspectives in Nasonia Wasps.