nanoscale biomedical implants.

Journal of Controlled Release 189 (2014) 25–45

Contents lists available at ScienceDirect

Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

Review

Recent advances in micro/nanoscale biomedical implants Ammar Arsiwala 1, Preshita Desai 1, Vandana Patravale ⁎ Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Matunga, Mumbai 400 019, India

a r t i c l e

i n f o

Article history: Received 4 March 2014 Accepted 14 June 2014 Available online 21 June 2014 Keywords: Biomedical implants Micro Nano Stents Bone Neural

a b s t r a c t The medical device industry is growing at a very fast pace and has recorded great research activity over the past decade. The interdisciplinary nature of this field has made it possible for researchers to incorporate principles from various allied areas like pharmaceutics, bioengineering, biotechnology, chemistry, electronics, biophysics etc. to develop superior medical solutions, offering better prospects to the patient. Moreover, micro and nanotechnology have made it possible to positively affect at the sub-micron scales, the cellular processes initiated upon implantation. Literature is rife with findings on various implants and this review comprehensively summarizes the recent advances in micro/nanoscale implantable medical devices — particularly cardiovascular implants, neural implants, orthopedic and dental implants and other miscellaneous implants. Over the years, medical implants have metamorphosed from mere support providing devices to smart interventions participating positively in the healing process. We have highlighted the current research in each area emphasizing on the value addition provided by micro/nanoscale features, its course through the past and the future perspectives focusing on the unmet needs. © 2014 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Cardiovascular implants . . . . . . . . . . . . . . . . . . . . 2.1. Stents — really the saviors? . . . . . . . . . . . . . . . 2.2. Nanotechnology to the rescue . . . . . . . . . . . . . . 2.3. Nanocarriers on stents and angioplasty balloons . . . . . . 2.4. Nanostructured surfaces for stents . . . . . . . . . . . . 2.5. Towards market translation — are we successful yet? . . . . 3. Neuronal implants . . . . . . . . . . . . . . . . . . . . . . . 3.1. Cochlear and retinal implants . . . . . . . . . . . . . . 4. Bone and dental implants . . . . . . . . . . . . . . . . . . . . 5. Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . 6. Sterilization of biomedical implants: let's not take it for granted . . 7. Toxicity assessment: micro/nanotechnology may come with a burden 8. Conclusion and future perspectives . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

⁎ Corresponding author at: Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai 400019, Maharashtra, India. Tel.: +91 22 3361 2217; fax: +91 22 3361 1020. E-mail addresses: [email protected], [email protected] (V. Patravale). 1 Both the authors contributed equally.

http://dx.doi.org/10.1016/j.jconrel.2014.06.021 0168-3659/© 2014 Elsevier B.V. All rights reserved.

Biomedical implantable devices are starting to be recognized collectively as medical bionic implants — where “bionic” refers to the study of biological systems in order to develop artificial systems that can replicate their functions. This market is expected to grow at a cumulative annual growth rate (CAGR) of 7.1% from 2012 to 2017 to reach $17.82 billion by 2017 [1]. Quite evidently, there is a spur of research

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A. Arsiwala et al. / Journal of Controlled Release 189 (2014) 25–45

activity in this field amalgamating principles of pharmaceutics, bioengineering, biotechnology, chemistry, electronics, biophysics etc. Broadly, biomedical implants encompass a range of medical solutions for various bodily disorders and include: (i) cardiovascular implantable devices like stents, vascular grafts, heart valves, defibrillators, pacemakers, etc. (ii) neural devices like neuronal implants and prostheses for central nervous system (CNS), peripheral nervous system (PNS), cochlear and retinal applications (iii) orthopedic prostheses like bone grafts, bone plates, fins and fusion devices; orthopedic fixation devices such as interference screws in the ankle, knee, and hand areas, rods and pins for fracture fixation, screws and plates for craniomaxillo facial repair; bone tissue engineering scaffolds for fractures and dental implants which forms the scope of the current article (Fig. 1). However, the current implant technology faces critical challenges pertaining to masking the “foreign-ness” of these biomaterials towards better host acceptability, desired temporal and/or spatial drug release profile and reduced foreign body reactions. Micro/nanointerventions have the potential to revolutionize implant technology by precisely designing and modulating their properties thereby addressing these concerns. Even mimicking the nanostructures in the body becomes possible with nanostructuring; conferring micro/nanoscale implants the unique capacity to positively affect the biomolecular and cellular events. These may be conveniently classified as implants with nanostructured surface topology or as those comprising micro/nanoparticles (Fig. 2). While the former has the potential to manipulate cellular adhesion stimulating selective proliferation of the desired cell types, the latter serves as a platform for discharging therapeutic vehicles programmed to identify, transfect to, and heal the impaired regions “smartly”. The marriage of micro/nanotechnology with biomedical engineering promises a newer generation of implants which has just started to surface the market and will surely translate successfully, pushing out the ones used conventionally. 2. Cardiovascular implants An estimated 82.6 million American adults were found to have one or more types of cardiovascular diseases in 2012 up from 81.1 million in 2010 [2,3]. The figures in other parts of world are even more disturbing [4]. It is safe to surmise that issues pertaining to the cardiovascular system and related deaths would only escalate with time. One of the most researched among these is the coronary artery disease (CAD) which is almost synonymous to stent implantation or percutaneous transluminal coronary angioplasty (PTCA). A stent is a generally tubular device used to support a segment of a blood vessel or any other anatomical lumen so as to preserve or regain its patency [5]. Apart from stents, other commonly encountered cardiovascular implantable devices include artificial vascular grafts, prosthetic heart valves, defibrillators and pacemakers. Understanding interplay between the biomaterial surface and blood-and-adjoining-tissues has and will

Biomedical implants (BMIs): Scope of the review Cardiovascular BMIs

Stents

Central and Peripheral Neural BMIs

Neural Prostheses

Vascular Grafts Heart Valves

Dental BMIs

Nanoparticles

Coated Nanostructures/Meshes/Fibers Topology Modified Bare BMIs

Monolithic

Nanoparticles

Micro/ Nano

Nanostructures/Meshes/Fibers Micro/Nano entities: Potential for BMI Engineering

Liposomes/ Polymeric Polymeric Vesicles Nanosphere Nanocapsule

Carbon Carbon Metallic/ Inorganic NPs Nanotubes Nanofibers

Miscellaneous

Prostheses (Bone grafts, plates, fusion devices etc.)

Retinal Prostheses

Fixation Devices

Cochlear Prostheses

Bone Tissue Engineering Scaffolds

Pacemakers Defibrillators

Orthopedic BMIs

Surface

(Screws, rods, pins etc.)

Fig. 1. Biomedical implants (BMIs): scope of the review.

Sensors for Biomarker Detection, Micro/nano robots

Bare BMI BMI Coating Drug Fig. 2. Schematic overview of micro/nanoengineered biomedical implants (BMIs). Modified from Desai et al. [207].


(a) Balloon angioplasty

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(b) Stent placement

Fig. 3. Schematic of balloon angioplasty and stenting procedures. (a) A schematic of events in balloon angioplasty starting from guiding the balloon to the blocked vessel with the help of a catheter followed by inflation and finally withdrawing the balloon back after deflation. This ultimately results in vessel broadening. (b) The sequence of events in case of stent placement is similar to that in case of balloon angioplasty except that along with the inflation of the balloon the stent expands as well opening up the block lumen and providing a mechanical scaffold thereafter to avoid elastic recoil.

continue to provide a better understanding of novel formulation approaches to eliminate untoward pathological events and improve their clinical acceptability. Before introduction of coronary stents as bare metallic stents (BMS) in the late 1900s, balloon angioplasty was performed to clear blocked vascular vessels. Angioplasty basically employs an initially deflated collapsed balloon, guided by a catheter to the obstructed lumen, then inflated to widen the lumen and finally deflating again to withdraw it back. In case of stenting, a similar procedure is followed except that the balloon in this case dons the stent over it, which also expands during

balloon inflation and resides as a scaffold thereafter while the balloon is retreated (Fig. 3). However, both balloon angioplasty and stent implantation were, and still are not free of complications as they induce trauma to the vessels [6]. The body responds to this by a set of events comprising elastic recoil, vascular smooth muscle cell (VSMC) migration and proliferation, extracellular matrix (ECM) synthesis, vessel wall remodeling, platelet aggregation and hence thrombus formation [7]. Only a day after the injury resulting from stent deployment (or balloon angioplasty), which provides a powerful stimulus to

Healthy vessel Maximum lumen diameter in normal state

Lumendiameter

Immediately post-intervention

Late loss

Post follow up

Acute gain post intervention Net gain

Minimum lumen diameter in diseased state Diseased/blocked vessel pre-procedure

Fig. 4. Schematic of events taking place post stenting and their effect on lumen diameter. The lumen in diseased state reduces in diameter upon deposition of plaque due to atherosclerosis. As a result, a stent is implanted expanding the lumen and regaining the patency to a great extent. However, after a certain period some of the gain in diameter is lost as a result of neointimal hyperplasia (restenosis).

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platelet activation and thrombus formation, vascular smooth muscle cells start proliferating and continue to do so for about two weeks [8]. A thin layer of platelets and fibrin deposits a few minutes after the injury and hours or days later inflammatory cells begin to infiltrate and VSMCs start migrating to the lumen. The VSMCs are found to convert from their normal contractile phenotype to a synthetic phenotype in which hyper-expression leads to an excessive formation of ECM increasing the thickness of the intima [9,10]. The end result of these inter-related activities is the formation of an additional layer in the lumen referred to as ‘neo-intima’ (literally new intimal layer) [11]. If the vessel response to injury is excessive, most or all of the gain in lumen diameter produced by the initial interventional procedure may be lost to the healing process, with return of a severe stenosis (restenosis) and ischemic symptoms (Fig. 4). As a bodily response to the injury an attempt is made to regain the endothelial layer to serve its normal barrier function of re-constructing the injured endothelial layer — rightly referred to as re-endothelialization. This process of endothelialization is critical as a healthy endothelium serves as a natural anticoagulant and as an anti-proliferative surface providing protection against thrombosis, lipid uptake and inflammation by secreting signal molecules like nitric oxide (NO), thrombomodulin, prostacyclin, heparin-like molecules, growth factors, tissue plasminogen activator and tissue factor pathway inhibitor [12]. Moreover, NO is known to modulate blood vessel tone and vascular permeability and inhibits leucocyte adhesion, platelet activation and VSMC hyperplasia [13]. Consequently, it is essential that a stent material/surface enhances cellular endothelial cell growth [14]. Thus in sum, ultimately, upon injuring the arterial wall, a multifactorial process is initiated, leading to neointimal hyperplasia and restenosis [11,15–17]. So, without treatment, a high rate of early stent thrombosis may be expected. For this reason, it is imperative that anti-proliferative drugs accompanied with this intervention may diminish the adverse events. 2.1. Stents — really the saviors? A promising development in stent technology was the arrival of drug eluting stents (DES), whose surface contained a matrix of polymer bearing therapeutics that regulate cell division and prevent thrombosis. Among these agents, the limus series (sirolimus, everolimus, biolimus etc.) and paclitaxel have been dominating since years [18,19]. In several randomized studies the most prevalently implanted stents, the sirolimus-eluting CYPHER® stent (Cordis, Johnson & Johnson Company, USA) and the paclitaxel-eluting TAXUS® stent (Boston Scientific Corp., USA) have shown their potential in reducing the risk of in-stent restenosis [20,21]. Though the introduction of stents in coronary lesions has had a substantial impact on improving early and late outcome compared with coronary balloon angioplasty alone, there are still problems with this technology as mentioned above. While solving the problem of in-stent restenosis (ISR) by way of DES, an even more critical problem of late stent thrombosis (LST) results [22–24]. This problem is partly due to the “foreign-ness” of the stent surface and partly due to incorporation of polymers, which even when biodegradable, cause inflammation and increase susceptibility to thrombosis. Not only that, they have also been found to disrupt the natural healing process of the endothelium [14]. The aim of a cardiovascular intervention in stenotic arteries is to restore blood perfusion in the myocardial tissues without causing acute or late stent thrombosis or restenosis. The first attempts to use biocompatible polymers to reduce in-stent restenosis backfired in the form of inflammatory reactions and subsequent neo-intimal thickening. The polymers alone exerted inflammation, hyperplastic neointima formation, and in-stent stenosis roughly comparable to bare metal stents, but were unable to reduce in-stent stenosis or late loss without drug elution [25–28]. In fact, for this reason, the superiority of drug eluting

stents over bare metallic stents is still controversial [29,30]. However, in spite of these concerns, stents have emerged as one of the most implantable medical devices and have metamorphosed from merely providing mechanical scaffolding to the recoiling lumen to provide a therapy platform assisting the luminal healing process further. 2.2. Nanotechnology to the rescue The marriage of nanotechnology with cardiovascular device design promises selective proliferation of endothelial cells (ECs), while simultaneously suppressing vascular smooth muscle cells (VSMC) — which seems to be the key. Nanomedicine has the potential to revolutionize medicine, and stent technology is no exception. Progress in nanotechnology now makes it possible to precisely design and modulate, at nanoscales, surface properties of materials used for various applications in medicine, offering newer prospects to patients (Fig. 5). Nanostructured stents have proven to be more effective in this scenario compared to nanocarrier based stent coatings as they augment EC proliferation and suppress VSMC proliferation better. Also since the vascular walls possess numerous nanostructured features (e.g., due to the presence of collagen and elastin in vascular endothelial cellular matrix), micro-topography and nano-topography mimic the endothelium structure [14]. However, stent coatings bearing nanoparticles are also gaining impetus. In general, nanoparticles (NPs) associated with stents are found to penetrate the injured epithelium and are taken up by the arterial tissues when delivered locally forming a depot (from where the drug is released). Other important advantages of NPs as drug carriers include sustained release (minimizing chances of local drug toxicity), high tissue uptake (attributed to their sub-cellular size), higher biocompatibility (lesser or no polymer concentration locally) and protection of chemically labile drugs (by providing an inert casing) [31] (Table 1). The nano-related research for other cardiovascular devices runs quite parallelly, as do the problems faced. Acting similar to stents, vascular grafts are also used in cases of blocked coronary arteries by providing an alternative route to blood flow. Issues like restenosis and thrombosis plague here too [32]. A similar situation occurs upon the placement of heart valves for valvular heart disease [33]. As far as the applications of nanotechnology in cardiovascular devices are concerned, the prevalence is more in case of coronary stents. However these may be safely extended to other devices as well. Below we have summarized the recent literature on nano/microscale stent coatings broadly classified as: (i) nanocarriers on stents, of dimensions 1–1000 nm, which are based on encapsulating antirestenotic therapeutics in nanocarriers (majorly of polymeric or ceramic materials), later coated onto stents; and (ii) nanostructured stent coatings, which include surfaces with a nano-topography (usually carved out of the stent material itself or provided as a coating of nanostructures) that may or may not be loaded with anti-restenotic drugs. The content of some papers has been discussed in more detail based on the importance of the information. We have previously summarized the recent patent literature on nanocoatings on implantable medical devices [5] and herein we present the recent research literature on nanoscale technology applications on cardiovascular implant coatings. 2.3. Nanocarriers on stents and angioplasty balloons Labhasetwar and group were probably the first to study the potential of nanoparticles as an effective treatment for restenosis. However, most of his groups' studies were either carried out systemically or in ex-vivo arterial models. Nevertheless, they have been a major driving force in providing a basis for incorporating nanocarriers on stent surfaces for treating restenosis. Song et al. [34] evaluated the uptake of polymeric nanoparticles (NPs) bearing anti-restenotic drugs in an exvivo model utilizing dog carotid arteries. The NP suspension was allowed to stay in the inflated artery for a certain period after which


29

Resolved: • Lumen patency regained • Arterial re-modelling Unresolved: • Neo-intimal hyperplasia • In-stent thrombosis

BMS Adventitia

Resolved: • Lumen patency regained • Arterial re-modelling • Reduced neo-intima

Media Intima Plaque/ Blockage External Elastic Lamina Internal Elastic Lamina

DES

Unresolved: • In-stent thrombosis

NES Resolved: • Lumen patency regained • Arterial re-modelling • Further reduced neo-intima • In-stent thrombosis

Thrombus Fig. 5. Comparative evaluation of various types of stents viz. BMS, DES, and NES. A vascular lumen shown with its different layers in a diseased state (leftmost). Further the three treatment options with their respective merits and demerits are shown for BMS (Bare metallic stents), DES (Drug eluting stents) & NES (Nanoengineered stents).

the arterial uptake was studied. It is reported that when the artery is not flushed, around 26% of the NP had been retained; whereas if the NP contact was followed by flushing/washing with Ringer's solution, the retention dropped to 6%. This result is of importance as the NPs if coated on a stent surface may be simultaneously desorbed or may get washed away due to vascular flow. Another striking conclusion drawn from the study was that arterial uptake is size dependent, with 100 nm particles penetrating better than 266 nm particles [34]. As an extension to this study, the same group evaluated arterial retention of nanoparticles into porcine coronary arteries through an in-vivo model; however, a lesser uptake than that with the ex-vivo model was seen. These results indicated the need for a superior formulation with a higher uptake that would benefit clinically [35].

As a result, they further modified the nanoparticles with dodecylmethylammonium bromide (DMAB) and/or fibrinogen with an intention to enhance retention in the arterial walls. The efficacy was evaluated in an extensive in vivo study on dog carotid arteries with normal blood flow for 30 min after each infusion to match closely the effects of washing away of NPs by vascular blood flow, if any. Multiple infusions given intermittently proved to be more beneficial (in terms of NP retention) compared to a single infusion of an equivalent dose. This implicates that a slow releasing reservoir of drug releasing NPs (coated on a stent) may show success clinically. The NP modification (with DMAB and/or fibrinogen) resulted in a seven-fold higher arterial wall uptake compared to non-modified NPs and also they accumulated predominantly in the media layer of arteries [36] (Fig. 6).

Table 1 Advantages of nano-engineering on biomedical implants. Feature Nanoparticles coated/monolithic biomedical implants Sustained release Sub-cellular size Minimizing or completely evading polymer usage Inert casing for therapeutics Nanostructured biomedical implants Nanotopography

Advantage Extended therapeutic active exposure Minimizing chances of local drug toxicity High tissue uptake Higher biocompatibility and lesser toxicity Protection of chemically labile drugs

Mimics the submicron topography of the internal tissue enhancing biomaterial-blood/CSF/bone tissue compatibility ▪ Cardiovascular •Augments endothelial cell (EC) proliferation and suppresses vascular smooth muscle cell (VSMC) proliferation — enhancing re-endothelialization while discouraging neo-intimal hyperplasia ▪Neural •Augments neurite growth with branching and suppresses astrocyte adhesion and proliferation thus arresting scar tissue formation — enhancing neuronal tissue regeneration while discouraging scar tissue formation •Enhanced electric impulse conductivity in neural prostheses ▪Orthopedic •Augments mesenchymal stem cell proliferation to form osteoblast and suppresses fibroblast formation — enhancing bone regeneration while discouraging scar tissue formation

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Later, they established the potential of nanoparticles to be taken up rapidly by endothelial cells. They were found to preferentially localize in the cytoplasm [37]. In a separate study, they reported a 27-fold higher uptake of smaller sized nanoparticles (mean diameter: 70 ± 2 nm) than the larger sized nanoparticles (mean diameter: 202 ± 9 nm) [38]. Various recent studies on similar lines have evaluated the antirestenotic potential of therapeutic agents encapsulated in nanocarriers and all have revealed an improvement in NP transfection efficiency and thus therapeutic activity. Paclitaxel loaded nanoparticles with a biodegradable matrix of poly(ɛ-caprolactone), poly(ethylene oxide) and C6 ceramide (used as an apoptotic signaling molecule) depicted an effective uptake of NPs in VSMCs with a significant anti-proliferative effect [39]. In a separate study, sirolimus loaded poly(D, L-lactide) nanoparticles (~ 250 nm) were found to be superior to free drug in inhibiting smooth endothelial cell proliferation without affecting the endothelial cell multiplication [40]. The research on applications of nanotechnology for treating atherosclerotic plaques and reviving the patency of blocked arteries began in the form of nano-suspensions bearing anti-restenotic drugs, as summarized above, and evaluating their potential in ex vivo arterial models and in vivo studies. However, earlier there may be a lack of clarity as to how these would be utilized clinically considering the ill effects of systemically administered anti-restenotic drug solutions [41,42]. The first breakthrough was witnessed in the form of nanoparticle coated catheter balloons which when inflated to expand the stent radially would release nanocarriers locally and provide a burst release of the drug for an initial period later followed by slow drug release from the polymeric matrix on DES stent surface. Westedt et al. [43] studied arterial uptake of fluorescent labeled nanoparticles released from a microporous balloon catheter upon different infusion pressures. A typical channel like particle penetration into the arterial layers was witnessed. Also, a higher uptake of the NPs was seen with higher infusion pressures [43]. Quite evidently, the focus then shifted to extending this idea of coating NPs on balloons to coating them on the stent surface itself. Nakano et al. [44] are first to report an active coating of NPs on a metallic stent surface by a cationic electro-deposition coating technique. The nanoparticles were surface modified by chitosan to render them positive (to achieve electrodeposition). This cationicity further helps intracellular uptake because of interaction with negatively charged cell membranes. Also, it accelerates escape of NPs from the perilous endosomal compartments into the cytoplasm from where the drug is released by diffusion and by degradation of the polymeric matrix. The in vitro study showed highly efficient uptake of fluorescein isothiocyanate (FITC)-loaded NPs compared to only FITC solution. Similarly, the in vivo study in porcine models with NP coated stents showed fluorescence in neo-intima and media layers compared to no fluorescence detection in FITC-polymer coated stents [44]. This reconfirmed the hypothesis that it is the nanoparticulate technology which significantly enhances permeation compared to plain drug solutions. Another research group reported a simple novel coating process for nanoparticles on stents termed as ring shaped surface tension (RST) method. It works on the principle of a liquid being held between two very closely spaced surfaces in the form of a meniscus as a result of capillarity. A specially designed ring trails along the immobilized stent surface held along its axis, just like a ring slides over a finger. The nanoparticle suspension was injected between the stent surface and the ring. The deposition occurred at the wedge where the meniscus met the surface when the ring was moved up or down. The advantage of the method is that the drug mass on the stent may be regulated in various ways. The SEM images showed that the nanoparticles deposited uniformly [45]. Lemos et al. in association with Concept Medical Research Private Ltd. and Envision Scientific Private Ltd. (Surat, India) have very recently reported a polymer-free novel phospholipid based sirolimus encapsulated nanocarrier system coated on stand-alone balloon catheters and on stents with precrimped balloons. An additional calcium-phosphorous

based component sparsely located at the NP membrane is said to tune drug release triggered by pH changes. The preclinical studies were however only carried out with the nanosystem coated balloons. A dose dependent study revealed an appropriate nanoparticle dose and structure to achieve the most efficient NP permeation into the media layers. The clinical studies in human volunteers for the proposed novel carrier are on-going to establish its clinical use [46]. It should be noted that 30–40% of critical lesions cannot be stented, largely because they occur at branch sites or in smaller arteries [47]. For this reason, the prospects of systemic nanoparticle formulations should not be belittled as in such situations nanoparticle based suspensions may stand out at their best. Very recently, sirolimus bearing micelles and liposomes were studied by Haeri et al. for their intramural uptake to subside restenosis. Phospholipid based micelles of size 7–20 nm and PEGylated liposomes of size 90 nm were prepared. Previous studies have reported enhanced accumulation of these in the damaged regions because of their biocompatibility, biodegradability, structure, colloidal size, and their lack of immune system activation or suppression [41,48,49]. The in vivo study with a rat carotid arterial model confirmed the superiority of micelles over liposomes, which is attributed to their lower size [42]. Stent coatings with lipophilic drugs are commonly encountered, however in recent research on anti-restenotics, peptides and genes are also finding place [50–52]. Indolfi et al. [53] evaluated a drug eluting stent coat incorporating microspheres bearing model proteins offering a tunable release profile. However, the effectiveness of microparticles in transfecting the high molecular weight therapeutics is still debatable. In fact various studies have proven the superiority of smaller sized particles over larger ones in piercing through the arterial layers [38]. But, being smaller in size, lipophilic anti-restenotic agents can easily be formulated as nanoparticles unlike the bigger sized protein based therapeutics which require larger carriers consequently leading to larger sized particles [53]. There has been limited research on anti-restenotic drugs other than of the limus-family and paclitaxel and even more limited on such drugs that are nano-encapsulated for anti-restenotic purposes. This is probably due to the great clinical success of drugs like sirolimus and paclitaxel with a strong clinical data of over two decades [47]. One attempt to move away from the conventional drug domain is the evaluation of a novel platelet derived growth factor receptor (PDGFR)-β-specific tryphostin, AGL-2043 formulated as nanoparticles. AGL-2043 seems to be relevant as it selectively inhibits SMCs and not vascular endothelial growth factor (VEGF), enabling endothelialisation. The anti-restenotic potential of these nanocarriers upon intraluminal administration was evaluated in balloon injured rat carotid arteries and in stent injured pig coronary arteries. For both in vivo models a size dependent NP uptake was evident (with 90 nm NPs being uptaken significantly more compared to 160 nm NPs). Also the nano-encapsulated drug proved to be more beneficial in terms of its anti-proliferative potential compared to surface adsorbed drug on blank NPs. Like most other studies, flouroscence micrography of the treated arteries showed the NP accumulation in the media layer [54]. Inflammation is very much intertwined with atherosclerosis and restenosis mechanisms, making it important to tap the inflammatory pathways to attenuate restenosis and augment arterial healing. The degree of neointimal growth has been correlated to a greater inflammatory response after stent implantation in animals and humans [55–57]. This develops a basis for anti-inflammatory drugs exerting profound anti-restenotic effects by subsiding inflammation caused by injury. Glucocorticoids in general might inhibit restenosis post stent deployment as they have anti-inflammatory effects on vascular cells without affecting endothelial re-growth of injured vascular tissues [58]. Joner et al. developed cationic lipid functionalized liposomes of prednisolone phosphate (size ~ 100 nm) to achieve site specific targeting. These were systemically administered to animals with stented arteries which revealed a higher concentration of drug as a result of selective


Adventitia

31

Maximum NP accumulation in media

Media Intima Plaque/ Blockage External Elastic Lamina

Marginal NP accumulation in adventitia

Internal Elastic Lamina

Fig. 6. A schematic of the NP permeation through the arterial layers from a nanoparticle eluting stent (NES). As is evident, maximum NP accumulation is detected in the media layer.

uptake, compared to the placebo and positive control. A marked decrease of neo-intimal inflammation was seen in atherosclerotic rabbits 42-days after treatment with this site specific steroid nano-assembly. The novel nano-assembly of this invention may be loaded on a stent further increasing the targeting only to the lesioned layers and also bypassing the need to administer the anti-restenotic formulation systemically [59]. The incorporation of anti-inflammatory actives has also been extended to nanoporous inorganic matrices (e.g. silica) for neural implants to arrest foreign body response as described in the later section. The same group had earlier carried out a similar study where systemic delivery of nano-paclitaxel (bound to albumin) in animals with stented arteries demonstrated a dose dependent reduction of neointimal growth. Paclitaxel delivered locally from a stent surface through a slowly degrading polymeric matrix is accompanied by risks of toxicity due to excessive drug accumulation in arteries [60]. The systemic nanopaclitaxel on the other hand offered a wide therapeutic index. Systemic treatment is said to have the advantages of treatment of multiple stented regions with a single injection as mentioned earlier [60]. However, if rightly engineered, stents bearing nanoparticles may help utilize the advantages of the systemic NP administration along with tackling the disadvantages of conventional drug bearing polymer coats on stents. As seen above, nanocarriers for treating restenosis are being researched extensively with an aim to enhance the uptake. When decorated with substances that enhance the permeation through the injured lesions, these perform better. Thus, future research in the area should address to further minimize the toxic effects of drugs, explore nonpolymeric approaches to deliver nanoparticles and seek gene therapy as an alternative to engineer novel drugs. Also, the current coating technologies used to deposit nanoparticles from their suspensions are slightly elaborate and sophisticated to be applied on a large scale. A development in this area would further boost the translation of this technology to the market.

2.4. Nanostructured surfaces for stents Apart from nanoparticles as anti-restenotic devices, a great amount of research is being carried out on affecting the vascular pharmacology by way of surface topography of cardiovascular interventions. While random surface topographies may promote VSMC proliferation, highly ordered structures can result in VSMC alignment, decreased proliferation and increased differentiation. It is shown that the positive net electrical surface potential on the metallic stent surface triggers the untoward adhesion and activation of platelets [61]. This is generally taken care of by either coating the BMS with a polymer layer — which contributes to thrombosis in turn [62] or by fabricating a nanomesh on the metal surface. These nano-engineered surfaces possess the unique capacity of directly affecting the molecular and cellular events [63].

A very early study suggested the integration of microstructure technology with vascular coronary stents enabling delivery of antirestenotic therapy into coronary arteries by piercing through the plaque [64]. Unlike nanoparticles that are designed to pave their way through the neo-intima into the arterial layers, earlier therapies were unable to transport therapeutics through the compressed plaque layer. As a solution to this, these researchers fabricated microstructures in the form of pyramids and studied drug permeation through these when embedded over a stent. A number of experiments were conducted to determine the optimum microprobe height and applied pressure to successfully transfect the arterial walls and ultimately deliver the drug [64]. The field of microneedle research for topical drug delivery is gaining impetus and we may soon witness stents structured with drug bearing microneedles on their luminal surface. Moreover, these microneedles may be fabricated as depots releasing nanoparticles to take advantage of both strategies. It was probably this study that sowed the seeds of nanostructuring the stent surface. With further insights in the cell proliferation behavior affected by nanostructures, and finding promising results thereof, the focus has shifted. In fact in our view nanostructures etched on stent surfaces seem to be more promising as next generation stent designs compared to nanocarrier based stent coatings. A recent invention by Wang et al. [65] stresses on a polymer free stent coating by opting for a composite coating of carbon nanotubes (CNTs) and magnetic mesoporous silica nanoparticles (MMSNs). As mentioned before, in spite of their commercial prevalence, stent coatings comprising polymers are directly linked to late in-stent thrombosis (LST) because of delayed vessel wall healing [22–24,66]. Even with biodegradable polymers, the side effects of the degradation products need to be solved [67,68]. This is one of the very few inventions that couples the advantages of NPs to effectively deliver the anti-restenotic drug and of nanostructures (CNTs) to affect positively the adherence and proliferation behavior of SMCs and ECs [69–71]. An electrophoretic coating process was used to achieve a crack-free two layered coating: the base coat comprising a buffer CNT layer and the top layer comprising rapamycin loaded MMSNs and CNTs. An in vitro haemolysis assay and platelet adhesion test confirmed the stent's blood compatibility and an in vivo study in rabbits demonstrated early rapid re-endothelialization when compared to a commercial DES. This invention rightly exploits the application of inorganic nanoentities as biomaterials for cardiovascular applications [65]. An alternative to coating nanotubes onto stents, to create a nanotopology, is to carve out the metallic stent surface to create a nanostructure. A vital incentive here is a polymer-free surface to bypass polymer induced thrombosis — a major complication with currently marketed stents. A very recent study demonstrated that thrombosis rates were significantly higher in DES (polymer coated) than BMS (polymer free) [72]. This in itself puts into question the practice of forming a drug-polymer

32


matrix on the stent surface; it does pave a way to effectively provide a long term drug concentration locally but stems an altogether new problem of thrombosis [73]. Though biodegradable polymer coats are better that way compared to non-biodegradable polymers, the problem still persists. Although, various groups are working on strategies to fabricate fully metallic wires with a dense forest of surface nanostructures, we are yet to see this translate to the market. Recent in vitro research findings have hinted that nanosurface texturing has a potential to influence cell behavior like adhesion, proliferation and differentiation [74,75]. Furthermore, the nanostructures enhance the movement of endothelial cells, minimizing late stent thrombosis, particularly relevant for cardiovascular stents [71]. High temperature chemical vapor deposition is a well-known technique for chemically etching surfaces resulting in desired surface properties. Recently, Loya et al. explored radio frequency (RF) plasma for the creation of radially emanating metallic nanopillar structures on stent surfaces creating a dense and porous texture capable of affecting vascular cells [76,77]. The required dimensions and morphology of the nanostructures were achieved by optimizing the RF plasma processing parameters. The in vitro studies with bovine aortic endothelial cells revealed that the nanopillar textured stent alloy surface was covered with a flat cell monolayer 6 times higher in density compared to non-textured smooth alloy surface. Delayed endothelialization is believed to be a major contributor to stent related thrombosis [22,78] . Such metallic nanostructures on stent surfaces may prove to be useful for enhanced and safe stent performance, with a polymer-free approach potentially reducing thrombus rates in patients. The science of nanostructuring metallic surfaces for stent applications may possibly revive BMS's back in vogue [79]. The various physical and chemical methods to pattern nanosurfaces on metallic implants have been summarized in literature [63]. Tsujino et al. have comprehensively summarized the recent advances in nanoand micro-porous stents [80]. Among others, carbon and titania nanotubes have emerged as promising nanostructures because of their mechanical and biological performance. A very important aspect to be considered for engineering stents or for that matter all kinds of prosthetics is its blood compatibility — to be more specific its interaction with platelets [81,82]. Various previous studies have reported stent surfaces fabricated to either repel platelets or prevent their aggregation [65,83]. Platelet aggregation has been directly linked to thrombosis and thrombogenesis originating from platelet response to endothelium damage [62]. Karagkiozaki and group have studied through a series of experiments the thromogenicity potential of titanium and carbon based nanostructures [84–87] and presented strong evidence that surface chemistry, nanotopology and wetting properties of these nano-substrates greatly determine platelet adhesion. Jia et al. studied nanostructured stainless steel stents loaded with paclitaxel that lead to rapid re-endothelialization, promoted vessel healing with less deposition of fibrin and reduced inflammatory responses when compared to a polymer based sirolimus stent and bare metallic stent. On the other hand, the efficacy of the nanostent was not altered [88]. To understand how endothelial cells interact with titanium dioxide (TiO2) nanostructured surfaces, Peng. et al. [89] conducted in vitro studies on titanium nanosurfaces using aortic ECs and VSMCs with a flat surface as a control. The ECs were found to be elongated compared to their counterparts grown on a flat surface. The elongated cells are reported to show increased proliferation, higher ECM production and faster migration speeds [90,91]. On the other hand, VSMCs depicted the exact opposite by preferring to cluster over each other and maintaining minimum contact with the nanotubes compared to the flat control where numerous long processes (to maximize contact with the substrate) were seen. The selective decrease in VSMCs is particularly relevant to stent applications since restenosis is directly related to VSMC

proliferation (Fig. 7). These results have been attributed to the interplay of the nanotopography of the designed structures and the growing cells. Thus, the use of TiO2 nanotubes represents a unique approach where a stent surface coated with or patterned as these would promote reendothelialization but suppress restenosis [89]. This research group also holds various patents in the area of nano-engineered medical device surfaces [5]. Another material explored in the same category is alumina (Al2O3). Wieneke et al. created a nanoporous alumina coating on 316L stents by first coating an aluminum layer by physical vapor deposition and then electrochemically etching it out to create a nanomesh, later loading it with tacrolimus. Animals implanted with these ceramic-coated stents displayed a milder inflammatory infiltration of macrophages and lymphocytes in the neo-intima compared to that in bare metallic stents. Consequently, a 50% reduction in neo-intima thickness was seen compared to bare metallic stents [92]. Aktas et al. conducted a detailed in vitro study to check the role of Al2O3 nanowires on the adhesion and proliferation of ECs and VSMCs. A comparative analysis of low density and high density nanomeshes revealed that the former selectively enhanced the proliferation of endothelial cells in vitro indicating their potential as stent substrate materials. However, the isolated cell material interactions in the absence of cell–cell interactions and blood–material interactions make the results limited and appropriate in vivo experiments need to be conducted to evaluate the potential of these as stent materials [93]. The principle of gene delivery applied to stent coatings is not new. A recent work based on this combined the applications of nanotechnology to fabricate a hybrid reservoir based stent coating. The major motivation behind the discovery is to combat the loss of therapeutic payload by vascular flow during the stents' long transit. Moreover, in case of NP coated stents these ill effects are more pronounced as even minor physical deformations may lead to an instant loss of NPs in the blood stream. A solution to this has been presented in the form of a three component system: multiple angiogenic gene carrying nanoparticles, carbon nanotubes wrapped with polyacrylic acid (PAA) and a fibrin hydrogel [94]. The nanoparticles carry pro-angiogenic genes: vascular endothelial growth factor (VEGF) and angiopoetin-1 (ANG1); the PAA functionalized CNTs are used as an entrapment device to help retain the NPs via ionic bonding; and the fibrin hydrogel to serve as a reservoir for the NP–CNT complex. The double protection system ensured minimum wastage of the therapeutic entities; in fact an increasing safety trend was observed upon increasing the CNT concentration. In an in vivo study, these nanohybrid stents performed significantly better at achieving re-endothelialization and attenuating restenosis compared to the nanohybrid stent without the therapeutic gene that performed better than a BMS [94]. However, a shortcoming of the study was its lack of control on the inflammatory response. Further studies incorporating multiple genes would surely prove to be more beneficial.

(a) Cell behavior with a conventional BMS/DES

Vascular smooth muscle cells (VSMCs) Endothelial cells (ECs)

(b) Cell behavior with NES Fig. 7. Schematic representation of cell interaction with stents. (a) A conventional BMS or DES resulting in smooth muscle cell proliferation on the stent surface ultimately leading to neo-intimal hyperplasia. (b) A nano-engineered stent surface (NES) that selectively enhances EC proliferation (resulting in improved endothelialization) while discouraging VSMC proliferation (inhibiting neo-intima formation).


Nazneen et al. modified stent surfaces with arrays of nano-pits using ion beam current. The diameters and depths of these pits were in the nanometer range. The viability, adhesion and proliferation of ECs were confirmed on the surface of the nanotextured stents [95]. Nazneen et al. have summarized the various physical and chemical surface modification techniques to fabricate nanoscale structures on stents, thereby manipulating the responses of vascular cells, and is suggested for interested readers [96]. Electron beam deposition was utilized by Lu et al. to create submicron rough (S–Ti) and nanometer rough (N–Ti) surfaces on titanium stents. These lead to a higher endothelial cell attachment with a lesser platelet adhesion compared to stents with flat features. As mentioned earlier, the extent of re-endothelialization to a great extent determines the success of stenting. Further these attached endothelial cells on S–Ti and N–Ti surfaces exhibited the highest nitric acid/endothelin-1 ratio, which indicates the best anti-thrombotic endothelial cellular phenotype. Moreover, a technology like this may even bypass the need for pharmaceutical agents and associated polymers to control their release [97]. In sum, the technology to chisel a metallic stent surface, rendering it meshy at the nanoscale, positively alters the vascular cell responses to specifically boost the adhesion and proliferation of the required cell type i.e. endothelial cells leading to re-endothelialization and recede that of the VSMCs that lead to an adverse response. These are anticipated to not only eliminate the risk of stent related thrombosis but also to contribute to the restoration of physiological function of treated vessels. A general summary of the micro/nanoscale features on implants for cardiovascular applications has been presented in Table 2. 2.5. Towards market translation — are we successful yet? There seems to be some delay in translating nanocoated/ nanostructured stents to the market which is probably due to completely bioresorbable stents (BRS) stealing away the limelight. Most colossal players in the stent market are currently focusing on developing BRS as the next generation stents [98]. Microporous stents have still found their place in the market, with a few in pipe-line too; however “nano”-stents currently investigated by industry are very few in number (Table 3). On one hand a buzz is being created about opting for polymer-free approaches which is re-popularizing bare metallic stents, while on the other, fully biodegradable polymeric stents are also emerging as the latest entrants. 3. Neuronal implants As rightly quoted by Paracelsus, what differentiates a drug from a poison is its dose. In case of neural ailments, the therapeutic window is generally narrow resulting in severe side effects and associated toxicity at non-specific neuronal sites. Further, if given systemically, these drugs are required in a higher concentration owing to fencing properties of blood-brain-barrier resulting in peripheral side effects. To address both these issues, site specific drug delivery forms a chosen slant, but obviously involves neural invasion. Additionally, most of the neural treatments desire long term therapy and to avoid recurrent neural invasions, a sustained release dosage form inserted at the focal site in neuronal tissue is recognized as a promising approach. The critical factors to be considered are biocompatibility, stability and host acceptance of the implant. Broadly the construct of implant varies from biodegradable/bioerodible materials (viz. polymers from natural, semisynthetic and synthetic origin, lipids etc.) to non-biodegradable materials (viz. metallic, nonmetallic and inorganic constructs, polymers etc.) and is used based on the desired application and is described in subsequent sections [99–101]. Use of biodegradable polymers as a delivery tool in treatment of neural ailments is emerging over the past few decades. Apart from biocompatibility, possible modulation of spatial as well as temporal drug

33

release profile which these polymers offer attribute to their prevalent use. Literature reports a wide application of polymeric micro/nanoparticles given as intracranial injections for site specific drug delivery. This though effective, presents disadvantages associated with initial drug burst release, high interstitial-pressure induced expulsion from the delivery site etc. Thus, the rationalized use of micro/nanoparticulate compacts in form of discs, wafers, fibers etc. is gaining recognition in recent years as promising delivery tools. They not only offer a high surface to volume aspect ratio but also provide better stability, implantability and control over drug release [99,100,102]. Among a wide spectrum of neural ailments, malignant gliomas are considered to be most nasty to treat due to inaccessibility, postsurgical recurrence and associated complications. The refractory nature of these gliomas is found to be the paramount reason leading to mortality and thus use of post-surgical implants of antineoplastic drugs to arrest tumor recurrence is anticipated to be the most convincing management strategy [102]. In this setting, polymeric implants of antineoplastic drugs are being widely investigated. A microparticulate based polymeric wafer implant Gliadel® (Eisai Inc.) has made a successful entry in market since 1980 to treat otherwise difficult gliomas. Structurally it comprises spray dried microparticles (1–20 μm) of an antineoplastic drug (carmustine) embedded in polyanhydride-based polymer which is further converted to a wafer of diameter 14 mm with 1 mm thickness using compression molding technique. Clinically, a maximum of eight wafers are inserted in the surgical cavity post glioma excision with an aim to arrest gliomal recurrence. The effectiveness of this implant is well corroborated by about two months increase in mean survival time of patients as compared to placebo group. This could be attributed to sustained focal drug delivery by virtue of slow polymer hydrolysis followed by erosion even up to 230 days as reported by clinical studies. Thus, biodegradable polymer based biomedical implants are gaining wide recognition towards successful clinical use [100]. Among the other polymeric implants, poly-(D,L-lactide-co-glycolide) (PLGA) is the choicest biodegradable polymer, as altering the lactic acid to glycolic acid monomeric ratio allows possible modulation and control over the polymer degradation and thus the drug release [102,103]. For instance, Xie et al. reported PLGA fibers of paclitaxel which were further casted into films to achieve sustained delivery of drug for 60 days [102, 104]. Not only sustained drug release but also the geometry of these implants plays a pivotal role in treatment as the recurrence of glioma is generally observed in an approximately 2 cm vicinity of surgical intervention. To understand this effect of geometry, Ranganath et al. [102] developed paclitaxel-PLGA implants using electrospinning in the form of microfiber discs and sheets (3 × 1 mm and 5 × 5 mm respectively) made up of PLGA 85:15 and submicrofiber discs and sheets made up of PLGA 50:50. These systems conferred not only better sustained release over a period of 80 days but also better implantability. From the geometric viewpoint, submicrofiber structures offered comparatively faster drug release, whereas initial burst release was higher with less compact sheets as compared to discs. Further, in vivo investigation revealed almost two fold better tumor inhibition efficacy for all implants as compared to systemically delivered Taxol® [102]. The same group further compared the efficacy of pre-developed submicron fiber discs with that of nanofiber discs and microparticle embedded discs made up of PLGA 50:50 to understand the mere effect of size variation. Post intracranial implantation, the studies revealed a higher drug penetration potential for nanofiber discs compared to the other groups, thus establishing the potential of nanoscale implants [105]. Not only the size but also the relative arrangement of polymer structures plays an imperative role in implant designing. Further, formation of co-axial fibers that ensure drug entrapment in the core of micro/ nanofibers resulting from modified electrospinning technique is reported to overcome the issues related to initial burst effect. Thus along with geometry, the fiber orientation and its synthesis method are also crucial for implant activity [99].

34


Table 2 A summary of recent advances in micro/nanoengineered cardiovascular biomedical implants. Feature/technology Nanocarrier based/bearing stent surfaces Polymeric nanoparticles bearing anti-restenotic agents Modified polymeric nanoparticles with uptake-enhancing agents Polymeric nanoparticles of paclitaxel with a signaling molecule Nanopaclitaxel systemically delivered Sirolimus bearing PDLA NPs Sirolimus bearing liposomes and micelles NPs of AGL-2043 (platelet derived growth factor) in polymeric carriers Cationic lipid functionalized NPs of prednisolone phosphate Nanoparticle loaded microporous balloon catheter Cationic surface charged NPs coated on stents by electrodeposition A novel ring shaped surface tension method for NP coating on stents Polymer free sirolimus bearing NP coated stents and balloons Protein bearing microspheres coated on stents Nanostructured stent surfaces Polymer-free composite coating of CNTs and silica particles (bearing sirolimus) Radially emanating metallic nanopillar structures on stent surfaces by RF plasma etching Carbon and titanium nanostructures on stent coatings

Nanoporous alumina coating on stent surfaces Nanostructured stainless steel stents loaded with paclitaxel Composite stent coating of NPs bearing an angiogenic gene, CNTs and hydrogel to maintain intactness Arrays of nanopits using ion-beam current

Results

References

Arterial retention of NPs confirmed. Smaller sized particles transfect up to 27-fold more [34,35,38] Seven-fold higher arterial uptake of modified NPs with predominant accumulation in media [36] Effective uptake of the NPs in VSMCs with a significant anti-proliferative effect [39] Stented arteries exhibited enhanced healing compared to plain paclitaxel [60] Superior over free drug in inhibiting VSMC proliferation without affecting EC multiplication [40] Enhanced accumulation in damaged regions; higher penetration with micelles compared to [42] liposomes (attributed to smaller size) Smaller NPs transfect better; nanoencapsulated drug performed better than free drug [54] A marked decrease of neo-intimal inflammation Channel like penetration in arterial layers; higher penetration at higher infusion pressures Surface charge enhances uptake and effective NP accumulation in neo-intima and media Uniform particle deposition NP permeation in media layers in pre-clinical study Protein permeation in arterial layers

[59] [43] [44] [45] [46] [53]

Enhanced bio-compatibility and reduced platelet adhesion attributed to the nanostructured coat 6 fold higher adhesion of ECs on nanostructured surfaces compared to flat control

[65]

Improved bio-compatibility and reduced thrombogenicity potential. Elongated ECs with increased proliferation, higher ECM production and faster migration speeds. Suppressed VSMC proliferation (compared to flat controls) Milder inflammatory infiltration of macrophages and lymphocytes in the neo-intima compared to BMS; selective proliferation of ECs compared to VSMCs Less deposition of fibrin, less inflammatory responses when compared to a sirolimus DES and a BMS Better endothelialisation and suppressed restenosis compared to a BMS Enhanced viability, adhesion and proliferation of ECs with nanotextured stents

Combination of polymers in device fabrication is further appreciated as it allows better avenues with respect to drug release modulation. Taking this into consideration, Vogelhuber et al. [106] developed matrices (diameter: 2 mm and thickness: 1.8 mm) embedded with polymeric mircoparticles bearing the antineoplastic drug carmustine (BCNU). The polymer matrix comprised a 1:1 combination of biodegradable anhydride and PLGA 50:50 by compression molding. The studies in human U-87 MG glioblastoma–astrocytoma tumor induced nude mice revealed dose dependent tumor inhibition efficacy with the implants whereas the intraperitoneal injection of BCNU solution was found to be ineffective. The group further established the importance of dual drug therapy by incorporating paclitaxel in a similar set up and showed complete diminution of tumor in majority of animals. Thus combination approaches can be visualized as the future to current implant technology [106,107]. Higher brain tissue penetration of drug post implant insertion is a critical factor that governs therapeutic efficacy. In one such study, the PLGA microparticles with a chemotherapeutic agent, 5-flurouracil are reported to exhibit restricted drug penetration up to 3 mm post parenchymal implantation and thus necessitates multifocal insertion towards better efficacy [108–110]. Thus, extensive research to ensure deep nerve tissue penetration of actives is desired in near future. Till date, nanodrug delivery based intracranial injections/infusions have proven their superiority in treating CNS ailments and have made a successful entry in the market as well. Though this treatment strategy is very well accepted, it must be understood that it requires periodic administration and being invasive it adds to patient incompliance. To overcome this drawback associated with most injections meant for intracranial delivery, scientists are now designing intracranially injectable implants. This interesting category of implants holds an edge over conventional intracranial injections in terms of temporal control over drug release, achieving sustained release profile and reduced frequency of dosing. These are also beneficial over implantable devices in

[77,78] [14,84–87,89,97]

[92,93] [88] [94] [95]

terms of higher safety and relative ease of administration being devoid of any surgical intervention. Literature reports a wide range of such nanoparticulate gels and/or in-situ gelling composites of polymers, lipids, phospholipids etc. One such example of sequential development from intracranial injection/infusion to an implant is DepoCyt®. The delivery system comprises multivesicular liposomes of an antineoplastic drug, cytarabine and is based on a patented technology DepoFoam®. This is generally administered as an intratumoral injection followed by periodic infusion (given via convection assisted external pumps) for treatment of gliomas. Taking lead from this Qi et al. developed a gel-based injectable implant that post autoclaving exhibited about 75% in vitro drug release over a period of about 20 days in contrast to 12 h in case of simple solution. The implant comprises high density phospholipid vesicles of cytarabine (165.6 ± 71.89 nm) prepared using high pressure homogenization technique. The uniqueness of this delivery system is its gel-like consistency resulting from high density vesicles which additionally offers high entrapment efficacy, a sustained release profile and higher stability over conventional liposomes. As cited by authors, this technology could be further extended to a wide arena of therapeutic moieties as these vesicles allow incorporation of hydrophilic as well as lipophilic drugs [111]. Apart from neural carcinomas, epilepsy is another critical neurological ailment characterized by frequent seizures resulting from atypical cortical neuronal cell activity. Clinically, it results in restriction of patients daily activities and associates itself with occupational as well as social discomfiture. Current use of oral anti-epileptic therapy results in systemic and central nervous system (CNS) side effects due nonsite specific drug exposure, which not only requires higher dose but also results in temporal drug resistance. Considering the focal and periodic pattern of seizures, intracranial biomedical implants can truly serve as promising candidates in this regard. In consistency with this rationale, two categories of implants have made their place in current research realm viz. monolithic implants


35

Table 3 Market Status for micro/nanoscale biomedical implants. Product (manufacturer)

Indication

Technology

Current status

FOCUS™ np (Envision Scientific Pvt. Ltd.)

Blocked vascular vessels

Ongoing clinical evaluation

Nano+ [Lepu Medical Technology (Beijing) Co. Ltd.] VESTAsync™ (MIV Therapeutics India Pvt. Ltd.) BioFreedom™ (Biosensors International Ltd.) Yukon® Choice (Translumina) Gliadel® (Eisai Inc.)

Abluminal coating of phospholipid nanocarriers encapsulating sirolimus Polymer-free nanoporous surface bearing sirolimus Microporous hydroxyapatite coating bearing sirolimus Metallic stent surface with micropits bearing biolimus Rough microporous stent surface bearing sirolimus Carmustine containing polymeric microparticulate wafer

Blocked vascular vessels Blocked vascular vessels Blocked vascular vessels Blocked vascular vessels Malignant glioma and recurrent glioblastoma multiforme Motor function impaired Sensor device to be implanted on brain motor cortex patients Retinitis pigmentosa Wireless sensory micro-circuit with video camera and processing unit to replace the function of light-sensing cells Age-related macular Photoelectrode array to be implanted on retinal surface degeneration Dental implant Nanometric calcium phosphate Bone conserving implant Porous plasma spray coated bone conserving implant

BrainGate™ 2 (Cyberkinetics Neurotechnology Systems Inc.) Argus® II (Bionic Eye) (Second Sight Medical Products Inc.) Bio retina (Nano Retina Inc.) NanoTite™ (Biomet 3i Inc.) Comprehensive® Nano (Biomet Inc.)

which are identified as one solid single unit in form of the discs, fibers, rods etc. and neural prosthetics as discussed in a later section. Among wide range of anticonvulsant actives ranging from sodium channel blockers to δ-aminobutyric acid and peroxisome proliferatoractivated-α receptor modulators, adenosine activation therapy is considered very effective in the management of refractory convulsions as it is devoid of sedation and other side effects associated with conventional anti-epileptic drugs [112,113]. Clinically, adenosine-releasing implants or cells are introduced at the seizure focal site as a maintenance therapy. In agreement to this Wilz et al. developed nanocoated 3-D scaffolds of silk polymer entrapping adenoside molecules with an aim to achieve different dosing regimen of active (40, 200 and 1000 ng/day). The successfully developed implants were introduced in the infrahippocampal area of epileptogenic rats. The implants were found to be safe and demonstrated dose dependent protection against the epileptic symptoms and interestingly epileptogenesis was delayed by around one week with the highest dose. Thus nano-based implant module for sustained delivery of actives is a promising avenue towards management of lifelong neural disorders. The same can be extended for more controlled and focal delivery of other anticonvulsive drugs [114]. The research in nanoenabled site specific implants for sustained neuronal delivery is also being extended to neurodegenerative disorders, as these are identified as progressively disabling and relying on long term therapy. These generally result from gradual neuronal death causing reduction in native neurotransmitters and imbalance thereof. The therapy is primarily aimed at symptomatic relief and in majority of cases it either involves direct use of neurotransmitters or inhibitors of neurotransmitter-lytic enzymes. Systemic delivery of these actives associates itself with severe peripheral side effects owing to peripheral neurotransmitter imbalance. In agreement to this, Pillay et al. [115] fabricated nano-enabled scaffold device, quoted by authors as NESDs, for site specific delivery of dopamine towards management of Parkinson's disease. These NESDs comprised negatively charged cellulose acetate phthalate (CAP) nanoparticles of dopamine (size ~ 197 nm) entrapped in alginate crosslinked scaffold and the fabrication method employed a novel technique of biometric simulation using statistical design model of Box– Behnken. The biocompatibility of the developed implants was assured by cell inhibition assays in white blood cells and SK-N-MC human cancer cell lines that showed negligible effect on normal cell growth. Upon implantation in frontal lobe parenchyma of rats, the implants were found to be biocompatible with maintained cerebrospinal fluid (CSF) concentration of dopamine to about 10 μg/mL over a period of 30 days but with an initial lag of about 3 days [115].

Reference [208]

Ongoing clinical evaluation [209] Ongoing clinical evaluation [210] CE approved [206,211] CE approved [212] USFDA and CE approved [213]


[214]

USFDA and CE approved

[215]


[216]

USFDA approved

[217]


[218]

Apart from neurological ailments as discussed above, in recent years, increase in number of nervous system injuries is being reported. This has resulted in more than 2 million patients worldwide with spinal cord injuries, among which around half a million cases are reported in USA alone [102]. Clinically, minor peripheral nervous system (PNS) injuries are considered to be less severe than those of CNS as they possess a regeneration potential owing to the presence of schwann cells. On the contrary, CNS lacks the presence of these cells that play a prime role in axonal growth and the condition is further worsened by intervention of astrocytes and meningeal cells that result in glial scars arresting the tissue regeneration (Fig. 8). These injuries generally associate themselves with sensory and/or motor and/or cognitive impairment which in most cases is irreversible. Apart from direct neural injuries, retinal degeneration, deafness, neurodegenerative disorders, dystonia, epilepsy, bipolar disorders etc. are also recognized by altered neuronal behavior. Restoration of these disorders is of prime concern towards the symptomatic relief. Post nervous injury, re-establishing the neuronal conduction pathways becomes a priority. This bridging is predictably achieved by implantation of nerve fibers from the same host or donor (auto and allografts respectively) but poses problems associated with lack of availability, immunogenicity in case of allografts etc. [116]. Now biomedical engineering is playing a key role in this area at two interfaces viz. stimulation and restoration of neuronal tissue growth and maintenance of normal electric function of nervous system. This area of biomedical application is broadly known as neuroprosthetics and the devices are termed as neural prostheses. Some of the prostheses that have emerged in market are enlisted in Table 3. Conventionally used neuroprostheses comprise the following classes: (i) semiconductor electrodes made up of platinum, tungsten, iridium, gold and their combinations in the form of microwires, (ii) silicon based implants viz. Utah electrode array, Michigan electrode array etc. wherein the conductive silicon microfilament is designed in association with conducting electrodes, and (iii) conducting polymeric implants. The mechanism underlying the electric signal transmission at the microelectrode–tissue interface involves two mechanisms as described below (Fig. 9): (i) Attraction or repulsion of ions on tissue interface by virtue of change in electrode potential which results in capacitative stimulation. (ii) Electric current induced alternate oxidation and reduction of electrodes that result in charge transfer. Of the two mechanisms listed above, charge transfer is predominant of neuronal signal conduction because capacitative stimulation alone

36


Neural Prostheses

(a) Cell behavior with a bare neural prostheses

(b) Cell behavior with micro/ nano engineered neural prostheses

Neuron

Bare neural prostheses

Astrocytes

Micro/ nanoengineered neural prostheses

Fig. 8. Schematic representation of neural cell interaction with neural prostheses. (a) Conventional bare neural prostheses exhibit enhanced astrocyte adhesion leading to scar tissue formation. (b) Micro/nanoengineered neural prostheses augment neurite growth with branching and suppresses astrocyte adhesion and proliferation thus arresting scar tissue formation ultimately enhancing neuronal tissue regeneration.

results in meager electric signals that are inefficient to stimulate neuronal conduction pathways. It must be carefully understood that though charge transfer is a major mechanism of signal conduction, it associates itself with surrounding tissue damage by triggering side reactions like hydrolysis of water in case of insulation failure and electrode deterioration over a period of time [117]. Microstructures though effective present one major concern associated with their effective insulation to avoid direct neuronal tissue contact and entry of extracellular tissue fluid in the implant device. This is generally achieved by coating the implants with biocompatible insulators viz. teflon, polyimide or Parylene C [118]. Though insulated, the system still presents a series of untoward effects like hemorrhage, activation of immune response, surgical interventions at a gap of 3–5 years to replace the subcutaneously implanted battery and marginal conductivity desiring stronger electrical currents which further result in local injury [119]. Nanointerventions are expected to result in very effective neural prostheses as they can circumvent the neuronal immune response offered by astrocytes, arresting the cascade of complications and they additionally stimulate axonal growth towards neuronal synapse refurbishment. To achieve this, scientists have developed various micro/ nano-electroconducting implants, nanoparticulate coated grafts, arrays of nerve growth factors and other neuroregenerative stimulants, polymeric scaffolds with functional electroconducting and neural interfaces etc. [120,121]. In design and synthesis of nanoelectrodes the fabrication technique is the most critical factor and various techniques viz.

Mechanism of Conduction: Capacitative Stimulation and Charge Transfer

Signal Transduction

Neural Prostheses Neuron

Fig. 9. Schematic representation of signal transduction by neural prostheses.

nanodeposition, lithography and pulsed anodization are being explored. Insulation of nanoelectrodes is still desired and nanoporous coating of inorganic materials like aluminium oxide (Al2O3), titanium dioxide (TiO2), silicon etc. are playing an impressive role in this direction. Among these, Al2O3 and silicon are of choice for neural implants [122]; TiO2 a preference for dental and orthopedic implants (as will be discussed in the later section) and TiO2 and Al2O3 preferred for cardiac implants (as discussed earlier). In this context, to achieve better insulation and barrier properties, scientists have reported combination of insulator layers comprising atomic layered coatings of Al2O3 followed by Parylene C [118]. The silicon based implants are also being investigated for nanointerventions at two interfaces either as (i) grafts/nanocoatings for semiconductor nanoelectrodes where the authors have reported nanowire constructs of platinum made by pulsed anodization grafted with polydimethysiloxane for increased conductance and biocompatibility [123] or as (ii) silicon based micro/nanoconductive implants with a varied geometry ranging from wafers to chips to 3-D constructs. To establish the effectiveness of nonporous silicon, Johansson et al. designed a silicon chip having one surface made up of porous silicon while the other having a smooth silicon surface. This chip upon insertion as a bridge to connect 5 mm damage in rat sciatic nerve indicated tight adhesion and extensive nerve tissue generation on porous silicon surface whereas the smooth surface indicated negligible adhesion [124]. To support this further, Moxon et al. compared the in vivo efficacy of ceramic microelectrodes coated with native silicon and nanoporous silicon in rat brain. Studies revealed that porous silicon coating not only resulted in a better implant tissue interface but also exhibited


enhanced neurite growth in comparison to native silicon coated implants [125]. This effectiveness of nanoporous silicon can be further extended as it allows a possible avenue of drug loading and retention with sustained release pattern. In one such study, Sun et al. reported nanoporous silicon construct loaded with hydrophilic drug dexamethasone to achieve stable neural interface. The drug loading was confirmed by reduction in pore size from about 11 nm to 5 nm in case of empty and drug loaded constructs respectively. This not only increased the wettability of the implant ensuring tissue acceptability but also arrested host foreign body response by preventing astrocyte adhesion by virtue of anti-inflammatory and immunosuppressant activity of glucocorticoid drug [126]. Apart from nanomodifications of conventional neural prosthetic strategies, a newer area of nanoprosthetics is emerging in recent years and is broadly identified as carbon nanomaterials in the form of graphene, carbon nanotubes (CNTs), and carbon nanofibers (CNFs) [102]. The biocompatibility of these unmodified carbon nanostructures in vivo has been confirmed by several authors [127–129]. Additionally, these carbon based nanomaterials are gaining wide attention due to their other unique properties like mechanical stability and high electrical conductance with electric current transmittance potential up to 107 A/cm2 [130]. CNTs can be broadly classified either as single-walled or multi-walled that vary in geometry and thermal conductivity (6600 W/mK and 3000 W/mK at room temperature respectively) and are commonly being investigated for deep brain stimulation to restore neuronal activity. The generally employed form in most literature reports is as nanoarrays which not only are superior to conventional metal electrodes but also show a 10 fold escalation in dopamine detection levels as compared to carbon microfibers ascertaining their higher sensitivity in neural signal detection and transduction [131–133]. Nerve tissue regeneration is the second aim in this treatment strategy. CNTs and CNFs are generally employed in the form of 3-D polymeric nanoscaffolds for this application. Literature reports a wide range of polymers viz. silk, poly(methacrylic acid) (PMAA), poly(L-lactic acidco-caprolactone) (PLCL), poly(3,4-ethylenedioxythiophene) (PEDOT) etc. [134–136]. Guang-Zhen Jin et al. [136] prepared PLCL nanofibers using electrospinning followed by multi-walled CNT coating that resulted in a functionalized scaffold with resultant fiber diameter of 1.3– 1.5 μm with rough surface as compared to plain PLCL scaffolds. Upon incubation with rat dorsal root ganglion cells, both the scaffolds, coated and uncoated, indicated neurite outgrowth post 3 days of incubation but a 1.3 fold higher neurite outgrowth was found with coated scaffolds at the end of 9 days, reinforcing the rationale of CNT incorporation. To understand the mechanistic pathway, both the scaffolds were incubated with PC12 neural cell lines. Studies revealed that a marker enzyme, focal adhesion kinase, a factor essential of signal transduction and neurite outgrowth was expressed more in case of CNT coated scaffolds with enhanced levels of calcium ions (Ca+2) predicatively responsible for faster and higher tissue regeneration [136]. The additional benefit that can be gained with these nanoconstructs is direction regulated growth of neural tissue by optimizing the alignment of grafting on the scaffolds. The 3-D geometry of scaffolds thus plays a crucial role towards efficient synaptic reconstruction. In case of these neural reconstructs the alignment of grafted nanostructures on the scaffold plays a crucial role in regulating the direction of neural growth towards successful regeneration of synaptic reconstructs [119]. The application of carbon nanomaterials in conjunction with polymers is further being explored as nanocoatings on conventionally used semiconductor microelectrodes with the purpose of enhancing stability and conductivity and reducing foreign body response with the additional benefit of neurite adhesion followed by tissue regeneration [137,138] (Fig. 8). Herein, the polymer choice becomes a restricting factor as the polymer itself should have electroconduction potential as impedance reduction and enhanced electrochemical properties are the prime requisites for these functional coatings. The widely employed polymers

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for this application include polypyrrole (PPY), PEDOT, polystyrene sulfonate (PSS), poly(ethylene terephthalate) (PET) etc. [139,140]. Recently Zhou et al. [139] developed platinum microelectrodes, coated with multi-walled CNT grafted PEDOT hybrid film using galvanometric and potentiometric deposition method. Among the coated microelectrodes, galvanometrically deposited microelectrodes showed better electrochemical properties which is attributed to retention of regulated nanodeposition; whereas both were found to be superior to plain platinum microelectrodes owing to the higher conduction of nanocoatings. Further, cytocompatibility of these coated systems was confirmed with PC12 neuronal cell cultures that indicated promising neurite growth which is a positive signal towards tissue regeneration. This was corroborated with in vivo experiments wherein these microelectrodes were implanted in cortex region of male adult Sprague–Dawley rats for a period of 6 weeks which exhibited non-significant foreign body response and promising stability and activity. Thus functionalized polymeric nanocoatings can be considered as safer solutions to address issues related to plain semiconductor microelectrodes. In one such other study, graphene oxide grafted PEDOT polymeric films were coated on gold wire with surface roughness of about 41.2 ± 8.6 nm as indicated by atomic force microscopy. Cyclic voltammogram analysis indicated marked increase in charge storage capacity from 8.27 ± 1.32 mC/cm2 to 86.75 ± 7.44 mC/cm2 with conductive film coating emphasizing on the significance of nanofunctionalization towards better neural prostheses [141]. Moreover, PEDOT polymer presents an additional advantage of conjugation with hydrophilic components and these conjugates can be used as nanocoatings to further improve wettability of highly conducting hydrophobic polymeric 3-D scaffolds. For instance, Bolin et al. [140] developed tosylate grafted PEDOT polymeric films to coat highly conductive hydrophobic nanofibrous (diameter ~ 200–400 nm) 3-D scaffolds of PET. Structural analysis of the scaffolds revealed that the 3-D structure was retained with vapor polymerization technique in contrast to the highly compact structure in case of chemical polymerization. This was also reflected in a difference in hydrophilicity indices as suggested by a marked decrease in contact angle of scaffold constructs (from ~147° to ~62° and ~14° in case of plain, chemical polymerization coated and vapor polymerization coated scaffolds respectively). This was further proven to be beneficial towards the 3-D regulated neurite growth as seen with SH-SY5Y neuroblastoma cells. Thus, the studies revealed that the polymerization technique used for coating plays a significant role in determining scaffold properties and should be closely investigated [140]. The next factor considered to be crucial in neural prosthetics is the effective charge on the implant construct and is reported to have a significant effect on neuronal adhesion and conduction. Considering this, charge functionalization of carbon nanostructure implants is now emerging as the next step. To understand this effect, Hu et al. [142] grafted multi-walled CNTs with poly-aminobenzene sulfonic acid and ethylenediamine to impart negative and positive charge on CNTs respectively. Though, the results indicated a similar cellular adhesion for both, cationically charged CNTs led to faster and a more branched neuronal tissue growth [142]. This possibility of grafting molecules on CNTs opens a newer area of research where nerve growth promoters can be functionalized on CNTs to achieve synergistic benefits resulting from higher conductance by CNTs and neurite growth augmentation by therapeutic actives. Literature reports a wide range of active functionalization of CNTs ranging from actives that enhance intracellular Ca+2 levels viz. 4hydroxynonenal (4-HNE) [143] neuronal matrix proteins, nerve growth factors etc. [144,102]. In this context, both physical adsorption and covalent functionalization are reported to retain activity of nerve growth factors. In one such study, Matsumoto et al. covalently conjugated amino functionalized CNTs with neurotrophin (diameter ~ 25 nm, length ≤ 2 μm), a nerve growth factor and confirmed that neurotrophin

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retained its biological activity post covalent linkage and exhibited extensive neural outgrowth upon incubation with dorsal root ganglionic cells [144].

and cochlear biomedical implants has been presented in Table 4 highlighting the technological feature of each. 4. Bone and dental implants

3.1. Cochlear and retinal implants Cochlear and Retinal implants actually form an extension of neural prostheses that are being widely used for hearing and vision improvement respectively. These implants need special consideration as they are meant not only to record and conduct the signal but also to process the same. A cochlear implant is an assembly of devices performing a cascade of activities towards better hearing and comprise: (i) external device with microphone, processor, and transmitter that collects the mechanical sound signal, (ii) receiver–stimulator device that receives the encoded sound signal which is decoded and transmitted ahead, and (iii) microelectrode array based implant (synonymous to aforementioned neural prostheses) which is implanted in the cochlear region that elicits the electric conduction pathway towards hearing. These devices do not merely play a role of signal transmission but also act as smart devices that process sound signals to electric signals that avail hearing. Recent advances in this area run hand in hand with the neural prostheses as described in earlier sections viz. nano-topological modifications, conducting polymer coatings, polymeric nanocarriers of neurotrophins to escalate neural tissue regeneration etc. [145]; whereas some special biotechnological modifications are also being investigated which are aimed towards improving sensitivity and stability of these devices. For instance, Kang et al. [146] demonstrated potential of molluscs-derived polymer polydopamine as a coating polymer on electrode as well as insulator surface with further casting of biomolecules viz. poly-D-lysine serving as a biocompatible implant with enhanced cell adhesion properties [146]. Further, application of these implants is also being extended to nanocarrier based drug and gene delivery to treat inner ear diseases like sensorineural hearing impairment, vertigo etc. [147]. Thus to sum up, it can be seen that cochlear implants have transformed from just a hearing aid to the multifunctional systems providing additional therapeutic benefits. Prostheses used for vision improvement are broadly classified in two classes depending upon the site of implantation viz. retinal prostheses used for retinal and optic nerve stimulation to reverse blindness resulting from retinitis, macular disorders etc. and retino-cortical prostheses used for CNS stimulation towards better vision which is compromised due to optic nerve damage and CNS ailments [148]. These prostheses are very similar to aforementioned neural prostheses with an additional class of diamond based retinal prostheses emerging in recent years. These are currently being investigated under two main projects named DREAMS and MEDINAS [117,149]. They are being designed with an aim to achieve highly sensitive electric stimulation and conduction pathways with enhanced safety profile considering the delicate retinal site. The diamond based implants are made from a novel material named nanocrystalline diamond (NCD) which is proven to be biocompatible with retinal cells [117,149]. Scientists have reported development of 3-D microelectrode arrays using silicon based deposition technique further coated onto polyimide or Parylene C to achieve flexible interface [117]. A further advancement in this area is diamond on diamond based retinal implants towards enhanced safety and sensitivity wherein Hadjinicolaou et al. [150] developed completely diamond based retinal implants made up of array of NCD which were further coated with a monolithic diamond layer. Upon implantation in rat retina explant, it was observed to stimulate retinal ganglionic cells towards signal conduction and processing with extraordinary electrochemical stability. Thus these diamond-based retinal implants can be visualized to make a market entry soon [150]. A summary of micro/nanoengineered neural, retinal

Bone implants primarily refer to orthopedic prostheses like bone grafts, bone plates, fins and fusion devices; orthopedic fixation devices such as interference screws in the ankle, knee, and hand areas, rods and pins for fracture fixation, screws and plates for craniomaxillo facial repair; bone tissue engineering scaffolds as autografts or allografts for fractures and dental implants [5]. Though conventional implants like artificial knee, hip joints, plates etc. are still the most implantable commercially, a great amount of research activity has been initiated in the past decade on employing principles of nanotechnology to bone tissue engineering, which is more relevant to healing fractures. As far as the former are concerned, nanostructured metal implant surfaces or antibacterial metallic NP coated implant surfaces have been explored already. A bone injury creates a gap leading to accumulation of necrotic bone debris. Blood from broken blood vessels and inflammatory cells accompany, ultimately releasing a set of signals [151,152]. A bone injury triggers a set of inflammatory and bone building signals which mitigate the migration of phagocytic cells removing the necrotic tissue and propagate vascularization providing nutrients to the injured cells. This ultimately initiates the healing cascade [152]. To create a new bone (osteogenesis), synergistically acting processes of osteoinduction (a combination of signals and cells getting rid of the necrotic bone and initiating osteogenesis) and osteoconduction (a material acting as a platform for the bone cells to grow onto) are pivotal [153,154]. Osteo-progenitor cells are necessary as they differentiate to osteoblasts (the bone forming cells) replacing the inserted scaffold [155]. Most therapies aim to intensify this positive response and some nanotechnological approaches have been particularly striking. The clinical translation for bone tissue engineering scaffolds still seems like a distant feat despite advances over the past decades. The reason for this could be limitations of current scaffolds including lack of sufficient vascularization, insufficient mechanical strength, incomplete osseointegration of the bioresorbable scaffold (with the living bone) and osteomyelitis (bone related infection) [156]. An obvious treatment module is to incorporate bone morphogenetic proteins and growth factors in the scaffolds. Also since, vascularity of the growing bone is vital, the use of growth factors such as VEGF, platelet derived growth factor (PDGF) and fibroblast growth factor (FGF) has also been reported [157,158]. Though the in vitro and animal studies have been promising, translation to a clinical setting is still unsuccessful [159,160]. There has been a spur in the number of novel scaffolds reported for bone tissue engineering (BTE). Polymers are one of the most widely explored but recently ceramics have emerged as better candidates. The individual limitations of these two are taken care of by a combination approach, rightly terming this category as composites. A criterion for selection of a scaffold material for BTE is biocompatibility and its ability to discourage any adverse physiological reactions [156]. The biodegradability and their ability to support tissue growth and remodeling before being resorbed by the body make polymers appealing [161]. However they have limited capabilities in achieving a strong degree of bone tissue integration and poor mechanical properties. On the other hand, ceramics offer a good biocompatibility and are rampantly gaining their space too but suffer from low fracture toughness (high brittleness) [162]. To make the most of the two by simultaneously balancing out their shortcomings, composites of polymers and/or ceramics and/or metals are being explored at a fast pace. Recently, Fricain et al., fabricated scaffolds of cross-linked pullulan and dextran supplemented with nano-hydroxyapatite (nHA) particles and in vitro studies revealed the expression of early and late bone specific markers with human bone marrow stromal cells in a medium

Table 4 A summary of recent advances in micro/nanoengineered neural, retinal and cochlear biomedical implants. Feature/Technology

Neural/retinal/cochlear implants Al2O3 and Parylene C nanocoated microelectrodes as neural prostheses Silicon coated microelectrodes as neural prostheses Nanotopology engineered silicon chips as neural prostheses Nanoporous silicone implant with dexamethasone as neural prostheses Silicon based 3-D implants with flexible polymer coating for retinal prostheses CNT coated PLCL nanofiber scaffold as neural prostheses Platinum microelectrodes coated with CNTs as neural prostheses CNT embedded conducting polymer PEDOT on gold microelectrode wire as neural prostheses Chemical functionalized (tosylate) PEDOT polymer coat on PET implant as neural prostheses PEDOT-alginate microelectrode coatings for neural prostheses Charge bearing CNTs as neural prostheses Neurotrophin conjugated CNTs as neural prostheses Poly-D-lysine decorated polymeric coatings on microelectrodes as neural prostheses Drug/gene delivery via prostheses for inner ear treatment Diamond based retinal prostheses Chondroitin collagen 3-D scaffolds as neural prostheses

Results

References

Sustained drug delivery over a period of 60 days Findings suggested importance and dependence of drug release on geometry, polymer grade, synthesis technique etc. Demonstrated efficacy of dual drug therapy with in vitro and in vivo studies Demonstrated importance of CNS tissue penetration on glioma management Demonstrated sustained release profile with enhanced stability over conventional liposome Infrahippocampal implantation indicated dose dependent protection with delayed epileptogenesis

[104] [99,102,105] [106] [108–110] [111] [114]

In vivo studies demonstrated maintained levels of dopamine over prolonged time

[115]

Demonstrated better insulation and conduction as compared to bare microelectrodes Enhanced biocompatibility and conductance In vivo studies demonstrated augmented neurite growth Enhanced tissue acceptability and signal conduction Flexible implant retinal interface Enhanced rate and branching of neurons was observed with possible up regulation of neuronal growth markers viz. focal adhesion kinase and Ca+2 Improved electrochemical properties with functional coating. Demonstrated effect of coating technique on signal conduction sensitivity Enhanced signal conduction sensitivity Demonstrated significance of 3-D geometry towards directed neurite growth and differentiation Better mechanical properties Demonstrated better adhesion and neurite growth with cationic charge imparted CNTs Demonstrated extensive neurite growth Demonstrated better cell adhesion Demonstrated wider application of prosthetics towards sensorineural impairments Flexible retinal implant interface triggering ganglionic cell proliferation towards better vision Demonstrated better electrochemical activity, neurite growth and differentiation

[118] [123] [124] [126] [117] [136] [139] [141] [140] [219] [142] [144] [146] [147] [150] [220]


Polymeric/lipid based implants PLGA microfiber casted film of Paclitaxel for glioma treatment Microfiber and sub-microfiber discs of paclitaxel for glioma treatment Polymeric microparticulate matrices of carmustine and paclitaxel for glioma treatment PLGA microparticles of paclitaxel for glioma treatment High density phospholipid vesicles of cytarabine for glioma treatment Nanocoated 3-D scaffolds of adenosine entrapped silk polymer for treatment of refractory epilepsy Alginate scaffolds containing CAP NPs of dopamine for treatment of Parkinson's disease

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deprived of osteoinductive factors. Moreover this composite matrix induced a highly mineralized tissue in small and large animal models (goat and rats respectively), new osteoid tissue formation in the defect region and direct contact of bone tissue regeneration with the scaffold matrix — attributed to the nanoscale HA [163]. Similarly, chitosan/ nanohydroxyapatite/Cu–Zn alloy NP composite scaffolds with enhanced antibacterial activity have been indicated as potential for bone tissue engineering [164]. The same group has also suggested the incorporation of nano-silica in composite scaffolds for this application [165]. The above mentioned NP based technologies for bone implants are non-comprehensive as they have been mentioned for representational purposes only. The scope is unlimited and research ongoing. Like in case of cardiovascular and neural implants, nanotopographically altered device surfaces are making their mark for positively altering the bone regeneration process. These apart from providing a framework to support bone formation and migration, modulate the growth activity at the nano-scale. Because the bone also has a structural hierarchy in the nanometer regime, nanostructured materials are fundamental to orthopedic research [166]. An improved cellular adhesion, cell spreading, growth, migration and differentiation have been achieved as a result of nano-fibrillar structures [167–171]. In addition, nano-topography has also positively affected the activity of various bone-related markers and gene expression of osteoprogenitor-like cells [172–174]. Nanostructured biomaterials emulate the intricate nanofeatures of natural ECM to direct stem cell differentiation to a specific lineage similar to what ECM does. Titanium and its alloys have been widely used in general for orthopedic and dental implants. Nanoarchitectured bone implant surfaces with titania nanotubes were found to provide a controlled, guided and rapid healing to bone cells. The cells cultured on these anodized nanotubular titania surfaces showed higher adhesion, proliferation, alkaline phosphatase (ALP) activity and bone matrix deposition compared to those grown on flat surfaces [175]. Alumina nanofibres created by the same group earlier, with the same technique showed that osteoblasts responded well to the nanostructures by extending their processes into the nanopores [176]. Hybrid micropitted/nanotubular topographies have depicted promotion of stem cell attachment and spread, collagen secretion and ECM mineralization — all indicating osseointegration [177]. Brammer et al. have comprehensively summarized the in vitro studies of osteoblast and osteoprogenitor cells carried out on various titania based nanostructures and we strongly recommend this article for a detailed review on this subject [166]. Osteogenic cells are known to be anchorage dependent and thus enhancement in terms of adhesion and cytoskeleton changes is a prerequisite to differentiation [178,179]. A recent study reported titanium surface features from sub-nano to sub-micron scales and investigated the corresponding effects on mesenchymal stem cell (MSC) response of osteoblast differentiation and osteoblastic phenotype cells. It was concluded that nano-submicron features are more influential in accelerating subsequent osteoblast differentiation [180] developing a strong basis for nanoscale surface engineering for bone implants (Fig. 10). Another major problem that haunts medical devices in general is bacterial infection which is more pronounced in case of bone and dental implants. Osteomyelitis, as it is referred to, leads to inflammation and pus formation adversely affecting blood flow and vascular supply to the healing bone. Among antibacterial agents, silver NPs have served as the gold standard since years, more so for bone and dental implants, majorly because bacteria do not develop resistance to silver, unlike with antibiotics [181,182]. Thus various studies have corroborated the closeness of surface topography of native bone and nanostructured surfaces making their implementation appropriate [183,184]. By stimulating osteoblast cells to deposit the mineralized matrix and further pushing MSCs to differentiate so that they deliver mature bone-building cells, nanostructures

enhance osseointegration [166]. Also NPs of hydroxyapatite incorporated in composite matrices have demonstrated enhanced efficacy for osseointegration as mentioned before. Furthermore, research in this area has taken a step ahead with the introduction of self-assembling scaffolds. Broadly these are identified as synthetic peptides with a unique feature of spontaneously forming nanofibrous architecture. Moreover, their ability to induce bone mineralization and ease of living cell intercalation towards tissue regeneration and compatibility with existing scaffold materials is appealing. PuraMatrix™ is one such self-assembling synthetic peptide that is being extensively explored for its application in dental osseointegration. In this context, Kohgo et al. reported that PuraMatrix™ forms selfassembled nanofibres (when implanted in combination with MSCs and platelet-rich plasma) resulting in almost 2 fold enhancement in boneto-implant contact index signifying its tissue regeneration potential [185]. To amalgamate the efficacy of conventional scaffolds with smart nanofibers, Sargeant et al. incorporated these self-assembling peptides in titanium foam implants. This assembly represented a bioactive matrix capable of initiating calcium phosphate mineralization upon implantation [186]. Though research in the area of self-assembled implants is in its infancy, it is postulated to add an interesting dimension to the entire micro/nanoimplant technology. To summarize, the situation has been positive as far as the number of regulatory approved bone graft solutions available in various forms for bone regeneration is concerned [156]. In due time, micro/nanoscale bone and dental implants will surely push out the conventionally used strategies from the market.

(a) Cell behavior with a bare orthopedic prostheses

(b) Cell behavior with micro/ nano engineered orthopedic prostheses Mesenchymal stem cell

Bare orthopedic prostheses

Osteoblasts

Micro/ nano engineered orthopedic prostheses

Fig. 10. Schematic representation of bone cell interaction with orthopedic prostheses. (a) Conventional bare orthopedic prostheses exhibit lower potential to augment osteoblast growth and adhesion leading to delayed bone regeneration. (b) Micro/nanoengineered neural prostheses augment adhesion and differentiation of mesenchymal stem cells to osteoblasts ultimately enhancing bone regeneration.


5. Miscellaneous It is evident that the three segments discussed above (viz. cardiovascular implants, neural and retinal implants, and bone and dental implants) dominate over the others in terms of utilizing micro/ nanotechnological principles, however, other less but potential areas must not be belittled. Of these two are particularly striking. Functional nanocarriers are known to offer higher sensitivity and selectivity to diagnostics. In this context, Deng et al. have designed a carbon electrode based sensor coated with nafion/multi-walled CNT-gold nanoparticles for quantitative determination of nitrous oxide. Upon implantation in mouse, it was found to be extremely sensitive with a detection limit up to 19 nM and was proven to be biocompatible with tissues like liver, kidneys, heart, spleen etc. [187]. These promising results with nano-sensor implant have opened doors to newer areas of research towards development of smart nanosensors. Another ground breaking advance in the field of biomedical engineering is the advent of micro/nanorobots. These are devices of the micron and nanoscales engineered to perform myriad functions — targeted drug delivery being the highlight. Medical micro/nanorobots may be capable of carrying a cargo accurately to any part of the body through external wireless steering — even to regions like eye and nervous system which are otherwise difficult to reach [188,189]. These are envisioned to perform tasks currently being perceived as difficult or impossible. Nelson et al. have reviewed the potential of microrobots as minimally invasive medical strategies and is strongly recommended for interested readers [190]. Initially inspired by the design of a bacterial flagellum, these researchers fabricated artificial flagella and studied its flow dynamics which was later extrapolated to create entirely autonomous robots [191]. Finding immense application in targeted drug delivery, these may transform into next generation medical therapies, possibly within a decade. 6. Sterilization of biomedical implants: let's not take it for granted Considering the in vivo implantation of biomedical devices, sterilization becomes a critical parameter. The generally employed sterilization techniques for biomedical implants involve steam sterilization, radiation sterilization, ethanol immersion, dry heat sterilization, ethylene oxide sterilization etc. Among all, ethylene oxide based sterilization is most widely employed in case of bionics and tissue implants [192–194]. Sterilization is observed to impact the release kinetics and the physicochemical parameters of the delivery system and thus needs a deeper understanding towards meticulous selection of the suitable method. As cited in an earlier section, Qi et al. have reported phospholipid vesicular gels of cytarabine. Initially, upon autoclaving it resulted in hydroxyl radical (OH) induced drug degradation of cytarabine to about 74% of initial drug content along with undesired phospholipid oxidation. To overcome this, the authors incorporated sodium sulphite, an OH• radical scavenger to stabilize the drug during autoclaving. This resulted in successful sterilization by arresting the drug degradation and lipid oxidation during autoclaving. Further, though the sterilization process of these implants resulted in particle size increment from about 120 nm to 165 nm owing to vesicular fusion and rearrangement; it showed a two-fold improvement in entrapment efficacy and sustained release profile up to 20 days [111]. Further, Godara et al. compared the effect of gamma and steam sterilization on polyetheretherketone polymeric bone implants incorporated with carbon fibers using nanoindentation and nanoscratch techniques. These studies are of prime importance as the autoclave process may alter mechanical and thermoplastic properties of these polymers and thus their behavior in vivo. Though both the techniques did not alter any matrix properties of the implant to a measurable extent, some alterations in surface morphology along with thickening were observed and in comparison were more significant in case of steam

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sterilization. These studies reveal the importance of sterilization method selection [195]. Shifting the focus to inorganic nanocomposites, Zhao et al. investigated the effect of three sterilization techniques viz. ethanol immersion, autoclaving and UV-radiation on titania nanostructures and correlated their impact on cyto-compatibility. Among the three techniques, UV radiation sterilization was observed to be the best. Interestingly, the results implied that UV and ethanol based sterilization techniques induced higher adhesion and cell proliferation and can be attributed to higher surface free energy resulting from sterilization process. This effect could further be used as a boon towards these implants intended to induce tissue regeneration [196]. Thus to conclude, there is no single sterilization technique that can be universally employed for all the implantable devices. Further it being an integral component, meticulous selection is needed so as to retain the physicochemical and pharmacological activity of implants in vivo. 7. Toxicity assessment: micro/nanotechnology may come with a burden As discussed in earlier sections, though marriage of micro/ nanointerventions is revolutionizing the area of biomedical implant research towards better efficacy and performance, one must pay sincere attention at their impact at cellular sites over prolonged use. The very fact that these micro/nanocarriers mimic the biomolecular and cellular structures due to their small size may impart certain toxicities and thus need systematic evaluation. The other concern here is to address to the issues related to host immune response resulting from “foreignness” of these biomedical implants. Though various techniques have been employed to make this implant-tissue interface bio-acceptable by surface functionalization and coating with hydrophilic polymers, it still requires a detailed investigation with respect to their cellular interactions. One such major concern is the extensive use of carbon nanostructures as a discrepancy associated with their safety is already reported [119]. Though there are many reports suggesting in vivo and in vitro safety and biocompatibility of carbon nanostructures there is a segregated thought process based on experimental evidences that suggests possible toxicity which predominantly results from variation in functionality, dose, contact duration, agglomeration etc. [197–203]. In one such study, Wu et al. demonstrated dose dependent impairment of axonal regeneration over a dosing range of 1–10 μg/mL whereas they were reported to be safe when used at a concentration of 0.1 μg/mL [204]. This stresses on the necessity to perform acute as well as chronic toxicity studies since accumulation of these carbon nanostructures at focal sites may lead to delayed toxicity. NanoTiO2 is the other class of nanocomposite which has found wise application in orthopedic and dental implants. There are recent reports pointing towards cardiac toxicity induction by these NPs especially in patients with cardiac ailments. Thus one must also consider overall pathophysiological state of the patient prior to use of these [205]. To sum up, with amplified use of inorganic, carbon based and nonbiodegradable micro/nanoparticles in the field of bionics we must not overlook the battery of toxicological evaluations ascertaining their safety upon long term use to sustain their successful entry in market. 8. Conclusion and future perspectives We have come a long way from making a bold attempt to surgically intervene and intrude the human body with external foreign devices, to attempting to mask this foreign-ness over the years and improving their body acceptability. Over the years, medical implants have metamorphosed from mere support providing devices to smart interventions participating positively in the healing process. A major boost in this pursuit has been provided by micro/nanotechnology in the form of smart-

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vehicles or as judicious topographies enabling improved bloodbiomaterial interface. Though all the major areas (viz. cardiovascular implants, neural and retinal implants, and bone and dental implants) are being researched extensively, even more after the advent of nanotechnology, there is undoubtedly a distinction. Based on the current market status, micro/ nanoscale stents are leading among others in terms of both of approved and under-clinical-trial ones. The hype associated with these may be partly correlated to their huge market share and the lucrative business potential. However, only time will tell if micro/nanotechnology achieves significant clinical superiority over current technologies, or if they are merely oversubscribed. In any case, the selective proliferation of endothelial cells but not smooth muscle cells in case of nanostructured stents or the increased proliferation of osteoblasts from mesenchymal cells for nano-engineered bone implant surfaces have provided a great lead to develop these as next generation implant technologies. Also, nanocarriers have demonstrated their potential in delivering therapeutics locally from the implant surface only to the lesioned sections circumventing chances of systemic toxicities. With some micro/nanofabricated implantable products already in the market, some in the pipeline and ample at the laboratory level, it would be no surprise to see these sidestep current strategies in the near future.

Acknowledgments Authors are thankful to the INSPIRE Program, Department of Science and Technology, Government of India for providing senior research fellowship.

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