Clinical Developments in Antimicrobial Nanomedicine

Chapter 29

Clinical Developments in Antimicrobial Nanomedicine: Toward Novel Solutions Gabriel H. Hawthorne1, Marcelo P. Bernuci1, Mariza Bortolanza2, Ana C. Issy2 and Elaine Del-Bel2 1

University Center of Maringa, Maringa, Brazil; 2University of São Paulo, Ribeirao Preto, Brazil

Chapter Outline 1. Introduction 2. Understanding Clinical Trials 2.1 Phase I 2.2 Phase II 2.3 Phase III 2.4 Phase IV 2.5 Organization and Ethics 3. Overview of Antimicrobial Nanomedicine Clinical Trials 4. Nanoparticles Used 5. Individual Studies Analysis

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5.1 Nanoparticles on Catheters 5.2 Nanoparticle Antibacterial Hand Gel 5.3 Oral Medicine 5.4 Therapeutic HIV Vaccine 5.5 Safety of Silver Nanoparticles 5.6 Antimicrobial Nanomedicines on Hospital Surfaces 6. Final Considerations References Further Reading

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1. INTRODUCTION Antimicrobials are substances used to kill or inhibit the growth of bacteria, fungi, viruses, or other microbes. Despite centuries of searching for antimicrobial substances (Spring, 1975), efficient drugs did not appear until the 19th century, when from extensive research, the domain of antimicrobials was achieved in the form of antibiotic, antifungal, and antiviral drugs. The new antimicrobials were disseminated worldwide and represented a real revolution in medicine, and diseases that caused high mortality could now be cured within weeks. However, in the same century in which this revolution started, there was also the discovery that treatments would need constant updates to cope with the impressive high mutation rates of microbes. Microorganisms have on their side enormous (and still growing) mechanisms of defense, propelled by mutations and an evolutionary history close to 4 billion years (Martinez, 2014). Furthermore, microorganisms have a short life span, and therefore, their reproduction is fast, pulling up their frequency of mutation and improving their capability of adaptation. Also, human agglomeration, increasing in the past decades, poses the ideal scenario for microbial propagation. Successful mutations can generate a novel army of resistant organisms and frustrate therapeutic efforts. Today, it appears that the ability to fight infections is under constant threat. In the past years, researchers have been sounding the alarm that unless we change our conduct regarding treatments, by limiting the use of antibiotics and correctly finishing treatment cycles (Aminov, 2010), the war against microorganisms will lose its equilibrium. Some have been suggesting that we face today a threat that can put the therapeutic range of action close to what it was before the rise of the antibiotics (Qureshi et al., 2015). Indeed, we have being experiencing frequent reports of so-called superbugs, microorganisms resistant to virtually any existent antimicrobial (Chang et al., 2015). Even considered simple bacterial infections are still a challenge despite our entire available antibiotic arsenal. Every day, physicians experience high numbers of treatment failures using modern antimicrobials around the globe.

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One way to deal with the ever-growing antimicrobial resistance is to produce novel antimicrobials, with novel mechanisms of action. However, the development of new antibiotics by the pharmaceutical sector seems to be decreasing (Norrby et al., 2005). The introduction of a new drug is the final step in a chain of several efforts. With regard to this chain we can list some potential reasons for the lack of novelties: (1) Academic research has not produced a great number of antimicrobials, especially antibiotics, with novel principles of action in the past few years, presenting fewer options for clinical studies. Preclinical studies themselves require time and financial incentives, which are limiting factors for the conduction of new studies. (2) Even if a promising research line exists, conducting a clinical trial is an extremely expensive investment and requires years of highly controlled sequential studies. (3) On top of that, infection treatments tend to be short, usually lasting weeks (with notable exceptions, such as HIV treatment); this contrasts with the treatment of a chronic disease, which can ensure financial return to pharmaceutical companies. For these reasons and others, a small number of drugs with completely novel mechanisms have been introduced to the market in recent years (Norrby et al., 2005). At the same time, according to World Health Organization reports (2012), lower respiratory infections, HIV, and diarrheal diseases (which are mainly caused by microorganisms) caused more than 6 million deaths. These infectious etiologies, together, are the third leading cause of death in the world. In low-income countries, lower respiratory infections were the top cause of death in 2012. This information shows the importance of infectious diseases in the global health scenario. More than that, it suggests that the antimicrobials available in our arsenal may not be helpful in many situations. In this context, nanomedicine appears as a new strategy, enabling the development of novel ways to fight infections. Nanomedicine is the application of nanotechnology in the health sciences. The term “nanotechnology” was coined in 1986 (Drexler, 1986), but the first ideas regarding the potential of working with small technologies can be traced back to Feynman, in 1959. Technologies situated on the nanometric scale, often under 100 nm, qualify for the label nanotechnology. However, some organizations such as the European Medicines Agency consider any structure under 1000 nm designed to have specific proprieties as nanotechnology. The first studies applying nanotechnologies to medicine appeared at the end of the 1980s. Being a derivation from nanotechnology, the potential of nanomedicine to increase the fight against infections is incredibly high. If we turn the discussion to the principles of the field, which outline what is ultimately possible, as presented by authors such as Eric Drexler (1986), we will realize that, guided by concepts such as precise atomic manipulation, not only infections but many others conditions could be potentially resolved with few unwanted effects. Although physical, mathematical, and biological principles to achieve these deeds have been assumed possible (Drexler, 2013), these achievements belong to a still unknown future. What is happening today, nevertheless, is still incredible and promissory. The ways in which nanomedicine approaches may help in the war against microorganisms include not only direct antimicrobial effects, but improved delivery of drugs, better bacterial detection, and expansion of vaccination capabilities (Gao et al., 2014). Aided by this technology, antibiotics, surface cleaners, hand washers, vaccines, oral medicine upgrades, and several other options have appeared in the past few years in the antimicrobial field. However, nanomedicine is a very new science. It is true that the majority of approaches, especially the most innovative ones, are theoretical or restricted to animal models. Regarding antimicrobial therapy, nanomedicine is also a science of the present. As novel and interesting studies appear and consolidate themselves, there is a tendency toward the conduction of clinical trials. Nanomedicine approaches have being tested in humans by several clinical trials for more than 20 years (Kattan et al., 1992). A fraction of those trials generate solid results, and with the investments made, new products are developed and end up reaching the market. Many nanomedicine products are available today, sold as pharmaceutical drugs, mainly anticancer drugs (Noorlander et al., 2015). In a quick database search, thousands of nanomedicine studies are found involving cancer therapy and, consequently, dozens of clinical trials regarding this matter. Unfortunately, this scenario of clinical investment does not apply to all the conditions in which nanomedicine is involved. Topics such as neurodegenerative disorders have received less attention, even with plenty of laboratory studies. For instance, by means of a systematic review (Hawthorne et al., 2016) we confirmed that at least a dozen promising nanoparticles exist with potential to improve drugs used in Parkinson’s disease treatment (like levodopa or bromocriptine) in animal models, but no clinical trial involving Parkinson’s disease and nanomedicine could be found in any database (screening deadline was June 19, 2015). In addition, there is a lack of integrative reviews that help in organizing the advances brought by novel laboratory research. It is also possible that a gap exists between basic researchers, physicians, and organizations that provide clinical trial assistance. On top of that, a credible clinical trial is expensive and bureaucratic. Despite these obstacles, a fair number of clinical trials investigate antimicrobial nanomedicine. This fact, at minimum, shows the relevance and potential of the field. As seen in Fig. 29.1, nanomedicine started to impose improvements on antimicrobial therapy around 70 years after the most important advances in antimicrobial medicine took place. The first section of this chapter attempts to review the concept of the clinical trial and the procedures involved in each step of this study type.

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FIGURE 29.1 Diagram showing advances in antimicrobial medicine. Some of the important antimicrobial drugs are represented in the timeline. One of the first advances, the sulfa drugs, was introduced for clinical use in 1936. In 2005 (red box (dark gray in print versions)), the first clinical approach using nanomedicine in antimicrobial therapy appeared: Dermavir, a plasmid DNA nanomedicine for HIV applications. The first report came in 2008 (Lackner et al., 2008) (green box (light gray in print versions)), describing a study in which silver nanoparticles were used to achieve antimicrobial effects in external ventricular catheters. Most of the studies were conducted after 2010, and therefore, the majority of reports came after this date (blue box (gray in print versions)). This is better discussed in Section 3. Based on Lewis, K., 2013. Platforms for antibiotic discovery. Nat. Rev. Drug Discov. 12 (5) 371e387.

2. UNDERSTANDING CLINICAL TRIALS Clinical trials evaluate the use of drugs and medical procedures in humans. These studies are the standard in testing novelties (e.g., a new nanodrug) or new applications to already clinically used approaches (e.g., testing if deep brain stimulation, useful for Parkinson’s disease, is also useful for anxiety disorders), being the foundation for evidence-based medicine. After a hypothesis is generated from studies with animals (for instance, a certain drug may improve a certain condition), clinical experiments are designed and conducted, and the resulting data are analyzed and then reported, enabling peer update. After the newly generated information is deeply analyzed in context, it can guide medical decisions. For instance, trials in which drugs already have clinical utility and are assessed regarding novel applications, such as the role of an antiinflammatory drug in the progression of a neurodegenerative disease, and how influential are these results. Furthermore, trials help in the production of guidelines, updating clinical conduct in a more disseminated level. With little effort, references regarding clinical trials can be found in any credible medical book; authors of those volumes compare and contrast several clinical studies and reviews. Armed with knowledge about clinical trials, researchers can better prepare their studies. Novel fields may encounter a certain resistance to being clinically tested, and thus correct preparation is important. As Noorlander et al. (2015) pointed out, some researchers or organizations even prefer not to associate their studies with the name nanomedicine, indicating the possible existence of a negative bias toward the field. Public perception of the matter may be distorted, sometimes propelled by the media, generating unrealistic fears (Florczyk and Saha, 2007). In this perspective, preparation efforts become crucial, especially in a field in which there is an urgent need for new solutions, like the antimicrobial field. It is not a casualty that the trials already conducted concerning nanomedicine and antimicrobial therapy tend to have a pattern: often conservative, providing information for widely useful interventions (not highly specific ones), and not highly invasive. This is consistent with the initial trials of a novel technology, in which safety must be the first concern and the possibility of application wide, to justify the investments. Clinical trials are divided by phases, which enable us to quickly understand at what stage the research is. The distinctive process of each phase is summarized in Fig. 29.2. The first phase we should understand is a preclinical phase, which is not directly linked to clinical trials, but is necessary to their conduction. These are the studies that, in addition to generating a hypothesis, prepare an intervention to enter into the clinical phase. Essentially, if a hypothesis is generated by in vitro

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FIGURE 29.2 Schematic overview summarizing key information regarding clinical trials. Only around one-quarter of the initial approaches end up successful, according to Watt et al. (2013). The whole process takes several years to be concluded. (A) Preclinical phase uses animals to investigate a novel hypothesis, collecting pharmacological and safety information, which is then presented to a regulatory agency for approval. (B) After the approval, phase I trials focus on safety of the new intervention in a few tens of individuals. (C) If the approach is proven safe, phase II is conducted, enrolling a larger group of individuals, aiming to obtain evidence of the mechanisms of action of the given intervention. (D) Phase III trials have as their main objective proof of the statistical superiority of the approach compared to placebo or other used therapies, and enroll hundreds or thousands of patients. (E) Additional investigations are conducted in phase IV trials, often enrolling subgroups of the population (e.g., elders, children).

evaluations, this hypothesis is generally tested in animal studies. In the preclinical phase, approaches generally need to have a full toxicity and pharmacokinetic assessment (absorption, distribution, metabolism, excretion) in at least two animal species (one of them being a nonrodent). The duration of dose administration to these animals must be at least equal to what is going to be used in humans. Whenever possible, the route of administration of the tested drug should also be the same as designed for humans. The potential impact on women’s fertility and fetal development also needs to be described (Watt et al., 2013). All these measures aim to increase the safety of the intervention, and all the data generated in animal or in vitro studies help to establish initial parameters to study in humans (see Fig. 29.2A). All the information collected in the preclinical phase is submitted to the local regulator agency; in the United States, this agency is the Food and Drug Administration. This proposal must present scientific evidence that the benefit of the approach surpasses the potential risk. The trial is then authorized to move to phase I.

2.1 Phase I The goal of phase I trials is to assess the safety of the new compound in humans. When authors refer to “first in human trials,” they are addressing phase I clinical trials. These trials are conducted with emphasis on collecting pharmacological data, not on therapeutic evaluation. Because a diseased individual may present physiological alterations and is more vulnerable to metabolic variations, phase I trials are conducted in healthy individuals (who may receive a monetary compensation, depending on the country’s legislation); there is also no need for a large group, so study groups tend to have fewer than 30 participants in this phase (Watt et al., 2013) (summarized in Fig. 29.2B). If the approach is too invasive to be conducted in a healthy volunteer (like new chemotherapeutics), disease-suffering individuals can be selected. In terminal diseases such as cancer, patients tend to voluntarily chase and enroll in clinical trials, facilitating the conduction of the

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study. This may, at least partially, explain why cancer clinical trials are the most numerous in the field of nanomedicine (Noorlander et al., 2015). Select the dosage to start the phase I trial is a problem faced by the researchers, because no trial has ever been conducted in humans before (hence the name “first in human” trials). A maximum starting dose can be calculated based on the preclinical research findings. After studies have been conducted in animals, what is sought is called human-equivalent dose. The species of animals from which data were extrapolated, the toxicity and risk inherent to the intervention, and the objective of the trial are the factors to consider in this process of finding the equivalent dose. Researchers start from a maximum dose that does not cause adverse effects and apply a safety factor to correct for imperfections and add safety, often dividing the initial dose by 10. When proved that this initial dose is safe, doses are escalated following conservative patterns to a point at which toxicity starts to appear at low levels in humans, where the maximum tolerated dose is established (Watt et al., 2013). Recently, some have been calling for phase 0 (zero) trials that are done with extremely low doses, usually 1% of the phase I normal values. This enables researchers to assess some pharmacological and metabolic data in a much safer zone. However, as the doses are unrealistic, the quality of this assessment may be prejudiced. Nanotechnologies overall, including antimicrobial nanomedicine approaches, may benefit from phase 0 trials to generate the first amounts of data, as less rigid requirements are necessary (Svendsen et al., 2015).

2.2 Phase II Phase II trials are conducted after the discovery of a safety range of dosing and other pharmacological parameters of the drug or intervention by the first in human trials. The primary objective of phase II trials is to obtain proof of the mechanism of action in humans. Therefore, the population in question usually has the disease of interest. Now, the focus shifts to therapeutic evaluation, although safety is never (or should not be) ignored in any step of any trial. Efficacy measurements are produced by comparing multiple dosing regimens with placebo or no intervention at all and then describing the outcomes. Usually, a medium number of subjects are enrolled, a few hundred individuals (Guptill and Chiswell, 2013) (see Fig. 29.2C). At the end of this phase, the efficacy of the drug or intervention is estimated, the target population is delimited, and the most suitable doses, dosing intervals, and duration of treatment are established. As the drug can also be given together with other medications, interactions between them may be explored. Many trials are discontinued in this phase if promising parameters are not achieved, preventing the loss of the huge investments usually made to conduct phase III trials. If the aim is to use a drug in several diverse populations, it is often necessary to conduct trials in different geographic regions, aiming to cover different genetic variations. Although this may be done as well in phase III or, especially, phase IV, there is great overlap between those phases. In this sense, phase II trials may be conducted in multiple centers internationally, evaluating different populations.

2.3 Phase III If a safe and effective dose is obtained in the phase II studies, several hundred or even thousands of subjects, in multiple study centers, are recruited to a phase III trial. As one may deduce, this is often the most expensive phase of a trial. The aim of phase III is to demonstrate a statistically relevant clinical outcome, compared to actual marketed strategies, placebo, or no therapy at all. Sometimes, to prove the superiority of a new pharmacological strategy compared to a current one, a great number of patients may be needed (Hafley et al., 2013) (Fig. 29.2D), for example, obtaining proof that an antimicrobial nanosilver coating for catheters is superior at reducing infections compared to a commonly used antibiotic coating. Because of the great number of patients involved, occasionally, the whole process of conduction of a superiority trial becomes unviable (Hafley et al., 2013). Another option is to conduct a trial to obtain proof of equality or of noninferiority, which requires fewer participants. This comparison between interventions provides statistical information for regulatory agencies, so the new drug or approach can be approved to reach the market. There are several randomization and statistical techniques that ensure the safety of the data collected, trying to remove biases. Although we may imagine the drug development process as a linear chain, this is not always true. Phase III can occur while phase II trials are also active, for instance, and this may also happen with other phases if safety is preserved and committees are in concordance with it.

2.4 Phase IV Additional studies are usually considered phase IV studies. These studies may be conducted to better evaluate efficacy in subgroups (children, very elderly individuals, patients with comorbid conditions, and others) or to actualize information

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regarding optimal use or risks of a medication (Fig. 29.2E). Phase IV studies may also be conducted to obtain additional data regarding interactions with other medications. Sometimes, regulative organizations may request more extensive studies on a topic that is controversial or in some case in which more information is needed (Hafley et al., 2013).

2.5 Organization and Ethics As seen, not only clinical trials are bureaucratic, but also a considerable time span is needed to conclude all phases. For this reason, frequently, a considerable time is required for the drug or intervention to reach the market. Moreover, there are several costs attached to this process. Costs should be considered regarding the preclinical studies, the development of the hypothesis, the production of the novel product (for instance, production of a nanosilver nanoparticle gel for use in 200 patients), the staff and investment in training professionals, the structure necessary for the accommodation of the patients and professionals during the trial, and finally, the collection and management of data. Financial support is often provided by companies that aim to profit from the possible therapeutic option in the future. Only about a third of phase I trials enter phase III, because phase III trials tend to be at least three times more expensive than phase II trials and up to five times more expensive than phase I trials (Bigelow, 2013). But what kind of organization or group of people conducts clinical trials? Local group efforts can result in small phase I and II trials, but phase III trials necessarily need a larger structure and extensive organization to generate their results. Although financed by pharmaceutical companies, sometimes it is easier and even cheaper for a company developing an idea to hire an organization to conduct these trials. Therefore, a great part of the current clinical research is conducted by clinical research organizations, which offer paid services to interested parties. Conceptually, those institutions can conduct clinical trials faster than companies could by themselves, as they are specialized in this service. In contrast, academic research organizations (like Duke Clinical Research Institute and the Montreal Heart Institute) are nonprofit institutions that conduct clinical trials or help in their conduction. These organizations offer leadership, help with data management, provide statistical analysis, and often conduct entire clinical trials by themselves. The topics investigated by academic research organizations tend to be more relevant to society overall, as they intend to answer strictly medically relevant questions, not profit-driven ones. In the design of the trials, academic research organizations make the effort to guarantee that every outcome, positive or negative, is relevant to change a conduct. These institutes may work in collaboration around the world to conduct large and international studies, producing information about the efficacy of an approach in multiple different environments. In contrast, there is far less collaboration between clinical research organizations (Reist et al., 2013). All the bureaucracy necessary to conduct a trial surely functions as a filter against unethical approaches, but the discrepancy of knowledge between those conducting the trial and those participating in it may facilitate unethical conduct. Regarding ethics evaluation, most research guidelines mandate that a review board or ethics committee assess the principles and objectives of the proposed clinical trial, providing an independent evaluation. This usually comes right after the design of the trial, once preclinical data already exist. This way, a conscientious judgment by the evaluation team may be conducted, analyzing what is the idea and the plan designed to achieve given objective. The clinical trial may then be accepted, altered, or rejected, if considered unethical. Suppose one team wants to conduct a trial based on a new antimicrobial nanoparticle in a country in which the population would probably not get benefits from the outcome, or that a team wants to inoculate dangerous microorganisms into subjects to be able to test the efficacy of their novel drug. As ridiculous as it may appear today, history is filled with analogous episodes. Principles like autonomy, beneficence, and justice always have to be respected. That is why regulatory and ethical organizations need to exist, especially in a universe where several clinical trials are conducted each year. This way, the increasing number of promising approaches obtained by modern science may be tested in a safe and ethical way, with no part being prejudiced (Wong and Schulman, 2013) Clinical trials are indeed a solid evidence-generating tool. However, as seen, many requirements that extend beyond an initial hypothesis and preclinical tests, such as funding, capable professionals, organization, and time, are needed to conduct and conclude such studies. That is why, despite many coordinators making adjustments to simplify some of those steps (in attempt to generate still strong evidence based on the available resources), many appearing promissory approaches may find resistance to being clinically tested. This resistance is especially important when very novel approaches are addressed, like nanomedicine, where little previous evidence is available for analysis by a committee. On the other hand, in the medical arena, it is inevitable to attribute potential to a field based on the outcomes of the clinical trials conducted. After all, a field in which clinical trials are being conducted can be more realistically evaluated.

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3. OVERVIEW OF ANTIMICROBIAL NANOMEDICINE CLINICAL TRIALS We can consider that the clinical testing of antimicrobial nanomedicine has at least 10 years since its very first initiative was taken (Genetic Immunity, 2005). As we know, 10 years in scientific time is a short time span. Furthermore, real impacts on the scientific community came only later, when the reports of these trials were published. The earliest report we could find was available in 2008 (Lackner et al., 2008); thus, the discussion in the field truly started after this date. Because data analysis can be time-consuming, reports generally tend to take a few years to be published after the end of interventions on patients, as seen in Table 29.1. The majority of the clinical reports and clinical initiatives have appeared after 2010. Together, these data are a true indicator of how novel is the movement of clinical testing of antimicrobial nanomedicine hypotheses. As summarized in Table 29.1, at least 14 clinical trials already exist in this field. Regarding localization, trials have been conducted in different parts of the world, including the United States, Brazil, Israel, Italy, Netherlands, Hungary, and Austria. The most-used nanoparticles were silver nanoparticles, but not exclusively. In the next section, we briefly discuss the proposed mechanism of action of various nanoparticles. We noted the existence of a pattern among the clinical trials of the field, which as mentioned, is explained by some constraints on the methodology of clinical trials themselves. The first thing we should consider is that all of them were conservative and safe approaches. This could not be different, as no clinical interventions in the area existed before, and to this point, there are not many clinical data regarding the safety of nanoparticles. That is why three clinical trials, covered in a later section in further detail, were directed toward investigation of the safety of nanoparticles, a necessary first step in any novel field (University of Utah, 2010, 2011; National Institute of Environmental Health Sciences, 2015). The other clinical trials proposed formulations to be applied to the skin, to external surfaces, or in dentistry procedures, and therefore were not very invasive. Therefore, it is clear that safety has been a major preoccupation and has been modeling the field to this point. Regarding investment and return, clinical trials tended to focus on largely applicable innovations. For instance, some of the hypotheses tested were an antimicrobial hand gel, nanoparticles for caries prevention, catheter coating, HIV vaccine, etc. All these innovations are easily reproducible worldwide, with great potential of application anywhere on the planet. This is a desired characteristic not only for this field, but for all clinical trials. Regarding complexity, as interventions are new, all the trials in the field can be considered phase I or phase II trials, testing either safety or efficacy of the mechanism

TABLE 29.1 Basic Information on Clinical Trials Regarding Antimicrobial Nanomedicine Sponsor

Topic of the Trial (Nanoparticle Used)

Conduction (Start Date); Report

Catholic University of Sacred Heart

Central venous access (silver nanoparticles)

2006; Antonelli et al. (2012)

Innsbruck University Medical Center

External ventricular drain (silver nanoparticles)

2006; Lackner et al. (2008)

Madigan Army Medical Center

Hand gel (silver nanoparticles)

2008; Schlicher (2011)

Henry M. Jackson Foundation

Hand gel (silver nanoparticles)

2009; Schlicher (2011)

Genetic Immunity

HIV vaccine (nanoantigens)

2005; Lisziewicz et al. (2012)

National Institute of Allergy and Infectious Diseases

HIV vaccine (nanoantigens)

2006; Rodriguez et al. (2013)

University of Utah

Safety of silver nanoparticles (silver nanoparticles)

2010; Munger et al. (2014)

University of Utah

Safety of silver nanoparticles (silver nanoparticles)

2011; Munger et al. (2014)

National Institute of Environmental Health Sciences

Safety of inhaled silver nanoparticles (silver nanoparticles)

2015; to be published

Hadassah Medical Organization

Resin composite materials (CPQAG nanoparticles)

2006; Beyth et al. (2010)

Hadassah Medical Organization

Root canal treatment (alkylated polyethyleneimine nanoparticles)

2013; to be published

University of Pernambuco

Fluoride formulation (silver nanoparticles)

2012; Santos et al. (2014)

University of Pernambuco

Fluoride formulation (silver nanoparticles)

2014; to be published

Gelderse Vallei Hospital (de Jong, B.)

Surface colonization (titanium dioxide nanoparticles)

2015; to be published

Detailed information on each trial is provided in Section 4. CPQAG, cationic polymers with quaternary ammonium groups.

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in small groups of individuals. To be statistically relevant and able to change conduct, trials need to enroll more patients. Later studies will surely take into consideration the results of these initial trials as a basis for conducting even more important and structured clinical trials.

4. NANOPARTICLES USED Although our focus is on clinical trials, the theorized mechanisms of action of the nanoparticles used in these trials by which they exert their antimicrobial effects are briefly discussed here. Excellent reviews have been conducted on this subject and can complement this topic (Rizzello and Pompa, 2014; Dizaj et al., 2014; Beyth et al., 2015). Silver nanoparticles have been the most-used particles in clinical trials in the field of antimicrobial nanomedicine, as one can infer from Table 29.1, despite our incomplete understanding of the mechanism of action of these particles (Rizzello and Pompa, 2014). Based on several detection techniques, Lee et al. (2014) proposed a complex mechanism that includes accumulation of reactive oxygen species, increased intracellular calcium levels, exposure of phosphatidylserine in the outer membrane, disruption of membrane potential, and DNA degradation. These are common steps in the chain of events involved in apoptosis. It has also been proposed that direct interactions between the silver nanoparticles and cell membranes can occur, causing physical damage (Rizzello and Pompa, 2014). It appears that smaller silver nanoparticles, in the range of 1e10 nm, attach with greater affinity to the surface of the cell membrane because of the comparatively augmented contact surface (Rizzello and Pompa, 2014). It has also been hypothesized that silver ions can intercalate between DNA bases (Rai et al., 2012). Other evidence suggests ribosomes may also be denatured and protein synthesis inhibited (Franci et al., 2015). Therefore, the mechanism of action of silver nanoparticles is probably multifactorial; Fig. 29.3 summarizes some of the discussed points. In addition to size, the shape and concentration are also relevant to the bactericidal effect of silver nanoparticles (Rai et al., 2012). To paint a better picture of their potential effects, in vitro, nanosilver has antibacterial activity on 99% on Escherichia coli cells after 6 h, while norfloxacin, a broad-spectrum antibiotic, has the same effect in 4 h (Lee et al., 2014). Not only bacteria, but fungi, viruses, and algae are also affected by silver nanoparticles.

FIGURE 29.3 Antibacterial effects of silver nanoparticles. The cartoon illustrates some of the supposed mechanisms of action of silver nanoparticles. It is suggested that the particles may impair DNA integrity (1), damage membranes (2), produce reactive oxygen species (3), and interact with organelles (4), triggering the apoptosis chain of events. Therefore, as seen in the box, nanoparticles probably have a multifactorial mechanism of action, benefiting from their augmented contact surface. Based on Rai, M.K., Deshmukh, S.D., Ingle, A.P., Gade, A.K., 2012. Silver nanoparticles: the powerful nanoweapon against multidrug-resistant bacteria. J. Appl. Microbiol. 112 (5), 841e852; Rizzello, L., Pompa, P.P., 2014. Nanosilver-based antibacterial drugs and devices: mechanisms, methodological drawbacks, and guidelines. Chem. Soc. Rev. 43 (5), 1501e1518; Lee, W., Kim, K.J., Lee, D.G., 2014. A novel mechanism for the antibacterial effect of silver nanoparticles on Escherichia coli. Biometals 27 (6), 1191e1201 and Franci, G., Falanga, A., Galdiero, S., Palomba, L., Rai, M., Morelli, G., Galdiero, M., 2015. Silver nanoparticles as potential antibacterial agents. Molecules 20 (5), 8856e8874.

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Three clinical trials, covered in a later section in this review, addressed the safety of silver nanoparticles (University of Utah, 2010, 2011; National Institute of Environmental Health Sciences, 2015). It is logical to assume that such trials would happen, because safety is necessary in any medical intervention. Therefore, if silver nanoparticles are potential new treatments, data regarding their safety needs to exist. These investments are justified because silver is one of the moststudied nanoparticles for antimicrobial effects and also the most-used nanoparticle in clinical trials. Of these safety trials, two had positive outcomes (meaning the particles were safe), and another one is still being conducted. However, silver is not the only metal widely studied for its antimicrobial activities. Iron oxide, titanium oxide, copper oxide, and zinc oxide are examples of other particles that may have action through the generation of reactive oxygen species and other mechanisms (Beyth et al., 2015). For instance, titanium dioxide can generate hydroxy and oxygen radicals in the presence of ultraviolet light, which end up impairing microorganism survival (Dizaj et al., 2014). This nanoparticle has been used in a trial still under conduction that addressed their potential to disinfect hospital surfaces in the intensive care unit (de Jong, 2015). Details will be discussed later. In addition to metal ions, there are also options involving organic nanoparticles. Cationic polymers with quaternary ammonium groups have been used in a clinical trial regarding oral medicine, which we discuss below (Hadassah Medical Organization, 2006b). Although the mechanism of antimicrobial effect is not entirely understood, it is been proposed that those nanoparticles can lyse bacterial cells (Carmona-Ribeiro and de Melo Carrasco, 2013). In oral medicine, this property would partially prevent salivary microorganism growth and impair biofilm formation.

5. INDIVIDUAL STUDIES ANALYSIS In this section we will review the clinical trials of antimicrobial nanomedicine. Comments addressing the repercussions of the studies for the field are made and other important points are considered. In parallel, the methodology, population, and objectives of the studies are exposed, so their outcomes and limitations can be correctly evaluated based on what has already been discussed about clinical trials. Normally, the definition of the population number enrolled in a clinical trial is predetermined based on a statistically significant value, so the study outcome can have statistical credibility. This is necessary, because a small enrollment may generate biased results. This number varies depending on the phase of the trial, the desired objective, and the determined methodology. As discussed, phase I trials, the most frequent in the field, aim primarily at safety, and therefore do not necessarily have to enroll a large group of patients.

5.1 Nanoparticles on Catheters Central venous catheters are used to administer medications and fluids or to collect blood samples. A catheter is positioned inside the jugular, subclavian, or femoral venous site, and then large quantities of medications or fluids can be quickly administered. In the United States alone, more than 5 million central venous catheters are inserted each year (Jasti and Streiff, 2014). Despite high utilization, unfortunately, complications such as infections, pneumothorax, and venous thrombosis are common, especially in patients who need invasive procedures with higher frequency, like cancer patients (Berardi et al., 2015). Regarding the infection problematic, a trial that started in 2006 and ended in 2008 investigated whether central venous catheter (triple-lumen catheters) infections could be reduced by impregnating these catheters with silver nanoparticles (Catholic University of the Sacred Heart, 2006). A later work (Antonelli et al., 2012) presented the results of this trial. The high infection rates associated with central venous catheter use serve as an appropriate illustration of an ideal problematic for the conduction of a clinical trial. The discovery of a new approach that minimized microorganism colonization and infections would significantly change health-care quality for a large number of patients. Furthermore, as central venous catheters are used worldwide, the results could have a great impact on the clinical setting on a global level. Patients were divided into two groups. At the moment of procedure, one group (n ¼ 135) received nanosilver-impregnated central venous catheters, and the other group (n ¼ 137) received conventional central venous catheters. All 272 patients were adults who had similar clinical and laboratory parameters at admission and were randomly selected to receive one of the two options, to reduce bias. All catheters were inserted at subclavian or jugular sites and covered with a dressing. Normal procedures for assessing catheters and replacing them when necessary were conducted, according to local protocols. Whenever the devices started to malfunction, had a suspicion of infection, or were no longer needed, the catheters were retired and then analyzed regarding infections. Based also on blood cultures and on specific criteria, the researchers could classify colonization as probable, definitive, or absent.

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To reduce bias, this study was conducted on patients from five different intensive care units. As regards the initial hypothesis that the silver nanoparticles could reduce infections, the outcomes in the two groups were similar in all centers investigated, as infection rates were close to 31% (about one in every three patients). Therefore, the application of silver nanoparticles on central venous catheters appears to be of little effect on microorganism colonization. The fact that this approach ended up not working as intended illustrates another very important function of clinical trials. As discussed earlier, it is known that silver nanoparticles have an antimicrobial effect. Although promisingappearing approaches are created in vitro inside laboratories and may show interesting parameters in animals, the only way to evaluate the outcome in humans comes from a clinical trial. Therefore, these studies never produce useless results. Clarifying failures, despite ending up as a bad immediate investment for the sponsors, is nevertheless a very important piece of data. When a clinical strategy does not work as expected, this means some approaches should be further studied, improved, or investigated through another angle, or that adjustments to the methodology of the intervention should be made. The failure of an intervention does not necessarily mean the failure of an entire hypothesis; to understand why silver nanoparticles did not work in this scenario, we must evaluate many questions, such as which nanoparticles were used (regarding size and shape, for instance), what was the environment, which bacteria were the most common in those places, etc. Eventually, if after successive studies it has been found that there are critical differences between laboratory results and clinical trials, this may represent the discontinuation of an entire line of research, readjusting resources in a positive way. More than that, every study has conclusions, independent of the outcome. In this study, for instance, the researchers concluded that other preventive measures like hand hygiene, correct asepsis, daily monitoring of catheters, and trained staff are more costly-effective than other interventions for now. In 2006, a different clinical trial addressed the efficacy of nanosilver-impregnated external ventricular drainage catheters. A report was made in 2008 (Lackner et al., 2008). The ventricular drainage procedure is used for management of acute occlusive hydrocephalus and helps in dissipating the obstructed cerebrospinal fluid, thus releasing intracranial pressure. However, as with central venous catheters, complications do exist. Cerebral infection is a serious complication, possibly resulting in encephalitis, ventriculomeningitis, and even death. Catheters may be impregnated with antibiotics that reduce the number of infections, and this can also be done with central venous catheters (Justo and Bookstaver, 2014). As we discussed in the introduction, however, indiscriminate use of antibiotics may lead to bacterial resistance and ultimately greater problems. Therefore, in trying to solve the high infection rates problem, there is a desire for other options. Regarding the population of the trial, there were no significant differences between the control and the intervention group in terms of medical parameters at the beginning of the study. A small population of 19 patients was enrolled in the treatment arm (one had to be excluded owing to the need for catheter replacement), while 20 patients were used as the control group; all participants were adults. In the treatment arm, nanosilver-impregnated catheters were used, while conventional catheters were used in the controls. After cerebrospinal fluid samples were drawn at predetermined days in both groups, at least three times a week, bacterial cultures together with fluid analysis were conducted on these samples. Although colonization of the catheter tip was essentially the same in both groups, five catheter-associated ventriculitis cases were diagnosed in the control group (representing 25%) and none were diagnosed in the silver nanoparticle group. All the catheter infections happened after day 10. The study conclusion was that dressing external ventricular drainage catheters with silver nanoparticles is safe and might be valuable in preventing catheter-associated ventriculitis. However, because the number of enrolled subjects was small, further evaluation by a large randomized trial is necessary to obtain more certain conclusions. As results are obtained, building evidence, larger steps may be taken. Note how the first trials in a novel field begin, generally, with few patients, minimizing exposure to adversities.

5.2 Nanoparticle Antibacterial Hand Gel In these studies, researchers tried to find a better approach than what is currently available for use regarding hand antimicrobial effects. Hand antisepsis is a necessary procedure for all health professionals, sometimes multiple times a day. There is a deep and well-established correlation between hand antisepsis and infection rates. However, compliance with hand disinfection is often low, partially because of unwanted effects such as skin irritation (Kampf and Löffler, 2010). Therefore, we can see how easily hand gels that have a lasting antimicrobial effect can directly affect patient care. Two very similar studies were conducted starting in 2008 (Madigan Army Medical Center, 2008) and 2009 (Henry M. Jackson Foundation for the Advancement of Military Medicine, 2009) in which the antimicrobial effect of a silver nanoparticle gel was assessed. A report of the results was published in 2011 (Schlicher, 2011). In the first study, in addition to the silver nanoparticle solution, an alcohol hand gel was used as control. Apart from the alcohol and nanosilver gels, in the second study they also used a combination gel that contained both alcohol and nanosilver particles in the formulation. The second trial was more complete and took into consideration the results of the

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first one. Therefore, we based our analysis on it. In this study, 53 patients were recruited. After subjects used one of the three gel formulations (alcohol, nanoparticle, or mixed), bacterial counts were obtained after 1 min for evaluation of immediate effect and after 30 min for evaluation of permanent effect. After obtaining bacterial counts, a questionnaire was given, addressing gel acceptability, providing information about the subjective perception of the product by users. Despite the hypothesis, the alcohol-based gel could reduce colony forming units with greater ease in the short period. Regarding persistent efficacy, still no statistically significant difference between the three gel options existed. This finding contradicted a previous finding by the first study, where the nanoparticle gel showed to be better (considering 10 min as the time span for permanent effect). Therefore, according to the new findings brought by the second trial, there were no advantages in using one gel over another when trying to achieve a persistent effect in microorganism count after 30 min. On top of that, in the subjective analysis, users preferred the alcohol-based gel group, followed by the mixed solution, and last of all the silver gel. User confidence in the product, consideration of repeating the use, and recommending the nanogel also followed the same pattern, with the alcohol gel being superior.

5.3 Oral Medicine In dentistry, resin-composite materials are used to substitute hard tissues because of their favorable esthetic properties. However, it is possible that gaps are present around the restoration margins, facilitating the genesis of secondary caries by cariogenic bacteria such as Streptococcus mutans. Therefore, it is desirable that composite materials possess an antibacterial activity. In 2006, a clinical trial was started to assess whether nanoparticles like cationic polymers with quaternary ammonium groups could be of use in this task (Hadassah Medical Organization, 2006b). Previous positive results had been reported in vitro, as expected. However, it was difficult to realistically study this desired antibacterial effect in vitro, as the oral environment is composed of hundreds of different bacterial species, an ambience hard to replicate in a laboratory setting. In situations like these, clinical trials pose as the only viable option for realistic investigation of a hypothesis, and are encouraged as long as the approach is safe enough and approved by committees, as previously discussed. The official report of the trial came in 2010 (Beyth et al., 2010). In this study, an acrylic appliance was intraorally placed in 10 volunteers. In each appliance, two normal resin composites and two resin composites with nanoparticles were inserted. A 4-h period was waited to allow growth of the bacterial biofilm. After removal of the appliance, there were no signs of inflammation (redness, warmth, tenderness, swelling), indicating that the intervention was not an irritant. Volunteers also did not feel discomfort or pain. The biofilms that had formed in the resin composites were analyzed for its thickness and the numbers of dead and live bacteria. In all voluntary samples, there was a statistically relevant reduction of viable bacteria in the biofilm where the organic nanoparticles were used. The study reported that more than half of the entire bacterial population on the surface of the biofilm was dead, even in the most remote parts of the structure. Interestingly, biofilms also tended to be thicker with nanoparticle use. As the authors pointed out, materials like these have the potential to extend the service life of dental restorations. This way, outcomes of a restorative procedure would be better, reducing the need to repeat the intervention and reducing associated costs. This study exemplifies how initial, small clinical trials can provide information regarding the safety of a new intervention. More than that, the intervention provided initial information on the effects of the nanoparticle and its suspected mechanism of action. However, a definite conclusion is not possible based on a such small population. Fluoride preparations are commonly used worldwide to prevent caries, a prevalent condition, especially in undeveloped countries, but require multiple applications per year and may be expensive and not viable in poor regions (Santos et al., 2014). For this reason, other formulations have been studied and developed in the past few years. Silver fluoride approaches appear promising, but when caries lesions already exist, they stain dark, resulting in unpleasant esthetic outcomes. Therefore, nanosilver fluoride, a formulation that contains silver nanoparticles, was developed to try to avoid the black staining. Another possible advantage is that the antibacterial activity of silver nanoparticles increases as particle size decreases (Targino et al., 2014). In 2014, nanosilver fluoride was tested by Santos et al. (2014), and although some criticisms can be directed at their methodology (Burns and Hollands, 2015), mainly involving biases, and lack of additional information, the findings of the study are interesting. Sixty school children from a poor region of Brazil comprised the population of the trial. More than one tooth was treated in each patient; 65 primary teeth were treated with nanosilver fluoride for 2 min (two drops) and 65 teeth from the control group received a drop of water. Treatment was performed only once in 12 months. Final sample size after a year was 51 teeth for the experimental group and 49 teeth for the control, because of not very specified losses, but the results were still statistically significant. Examinations in the children occurred after a week, 5 months, and a year. After 1 year, which is the significant outcome considering the actual clinical use of fluoride, the approach had a success rate of

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66.7%, being more effective in hardening and arresting dentine caries in primary teeth than placebo (success rate of 34.7%). More than that, at no point did the carious lesions stain black. As the authors described, nanosilver compounds tend not to form oxides when contacting oxygen, and that may be the explanation for the lack of staining. What is more, the taste of the nanofluoride is not unpleasant, and the approach is inexpensive. Therefore, silver nanoparticles may be classified as promising components against residual bacteria in tooth cavities and in the restoration margins. Targino et al. also announced in a 2014 publication the conduction of clinical trials in oral medicine. Indeed, in 2014 a trial involving some researchers that participated in the Targino et al. (2014) study was started, addressing S. mutans growth in dental biofilm of children when nanosilver fluoride was used (University of Pernambuco, 2014). These results are expected to be published soon, expanding our understanding of the novel nanotechnology fluoride. This trial can be followed through the identifier NCT01950546 on clinicaltrials.gov. Another trial is being conducted in Israel by Hadassah Medical Organization evaluating the antimicrobial proprieties of alkylated polyethyleneimine nanoparticles in root canal treatment. According to information provided by the authors, the trial is still ongoing as of this writing, although conceived in 2010, involving about 200 patients. Patients older than 18 years who needed root canal treatment were selected. Follow-ups were planned to be at 2 weeks, 3 months, 6 months, and 1 year, with clinical and radiological examinations. The identifier for this study is NCT01167985, where the reader can find further information. Unfortunately, we could not have access to other trials involving antimicrobial oral nanomedicine conducted by the Hadassah Medical Organization. Although we tried to contact the authors and searched for related keywords in databases, our efforts failed. Some of these were intended to begin years ago, and we do not have means to assure that they were truly conducted. These are the referred trials and their respective identifiers on clinicaltrials.gov: “Clinical evaluation of esthetic restorations placed in primary molars with composite resin enriched with insoluble anti bacterial nano particles.” Identifier: NCT00389714. “A clinical study: the effect of addition of insoluble antibacterial nanoparticles (IABN) in resin base provisional cement.” Identifier: NCT00502606. “Antibacterial properties of silicon incorporated with quaternary ammonium polyethylenimine nanoparticles.” Identifier: NCT01007240.

5.4 Therapeutic HIV Vaccine Dermavir is a synthetic DNA plasmid based on nanomedicine against HIV. Essentially, this is a pathogen-like synthetic nanoparticle technology, able to express virus-like particles that end up being captured by dendritic cells. After inoculation, the technology was demonstrated to deliver antigens (15 HIV antigens are expressed by the medicine) to lymph nodes, inducing expansion of the memory T cell population against HIV. Clinical trials were designed to investigate Dermavir’s effects on humans. A phase I trial was conducted in 2005, ending in 2006 (Genetic Immunity, 2005). Nine patients were enrolled and divided into three cohorts. In each of these cohorts, patients received low-dose, medium-dose, or high-dose quantities of the vaccine. A report came in 2012 (Lisziewicz et al., 2012). To minimize biases in the study, all of the subjects had durable suppression of viral load and CD4 count over 300 cells/mm3 before the beginning of the intervention, and antiretroviral therapy was not interrupted in any of the patients. One of the three dose regimens (low, medium, or high) was administered in a 28-day treatment. In addition to the drug being well tolerated, the main finding was a dose-dependent expansion of HIV-specific memory T cells, sometimes as high as 2000-fold, peaking 4 weeks after immunization. Immune reactivity was present even after 1 year of intervention. In contrast, in HIV infection, usually, memory T cells have a life span below 100 days, and then counts start to drop. Is HIV viral load directly related to memory T cell count? To answer the question it may help to know that Rodriguez et al. (2013) cited a clinical intervention that produced a reduction of plasma HIV (HIV-RNA) concentration from baseline compared to placebo when the medium-dose vaccine was used. Unfortunately, no more information was obtained about this study. More trials have been conducted to obtain information on Dermavir. With the goal of investigating toxicity, not clinical outcomes, a trial started in 2006 (National Institute of Allergy and Infectious Diseases, 2006) and was reported in a study published in 2013 (Rodriguez et al., 2013). Twenty-six subjects participated in this study, being again selected by several parameters including CD4 cell count. Because the intervention was well tolerated, the objective of the trial was completed. As additional information, CD4 and CD8 cell counts did not change significantly in any group during the trial, but increase in central memory T cell count was again confirmed; this induced response reached statistical significance at week 17.

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Collectively, these trials suggest that Dermavir, after further testing, could delay initiation or even interrupt the use of antiretroviral drugs in some individuals, if the vaccine can indeed reduce plasma HIV RNA levels. In this scenario, Dermavir would represent a new treatment option for patients infected with HIV. This would be desirable because the HIV epidemic causes millions of fatalities annually, and management of treatment is complicated. In addition, drug resistance is a major problem and some patients have several collateral effects from current HIV treatment. However, as in the case of other approaches, larger and more specific trials are necessary to reach conclusions.

5.5 Safety of Silver Nanoparticles Earlier in the chapter, we discussed silver nanoparticles. Although much has been done to characterize those particles and their potential functions, safety in humans was still an open question. As silver nanoparticles are one of the most-used nanoparticles for antimicrobial purposes, it comes as no surprise that tests regarding the safety of these nanoparticles have been conducted. University of Utah researchers designed clinical trials and studied this matter (University of Utah, 2010, 2011). A report was published in 2013 (Munger et al., 2014). These were possibly the first human studies regarding the safety of any ingested nanoscale product. The main information of these studies are summarized in Fig. 29.4. A total of 60 patients were recruited for this randomized trial (ages 18e80), in which patients would take silver nanoparticles orally. The study was designed to have two dosing phases, so all patients received 10-ppm oral silver and then proceeded to take the 32-ppm nanoparticle formulation; a dose escalation scheme was employed to increase safety for the subjects. The duration of the intervention was

FIGURE 29.4 Safety of silver nanoparticles. The diagram shows the study conducted by University of Utah (2010, 2011) and reported by Munger et al. (2014).

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10e14 days for all patients. To reduce bias, females of child-bearing potential, females breastfeeding, and subjects with history of heavy metal allergy, asthma, chronic obstructive pulmonary disease, renal impairment, or active upper respiratory infection were excluded. The researchers wanted to know if silver nanoparticles had any toxicity, and therefore, blood levels of more than 25 substances were monitored, including enzymatic function. Although detectable silver concentrations could be found in serum, clinically relevant changes did not happen. In addition to blood exams, urine, sputum, metabolism, vital signs, physical integrity, and imaging exams were conducted. No clinically relevant alterations were found. Although the aforementioned clinical studies produced interesting information, more specific data are required. Specific data for the lungs are important because they are the way through nanoparticles on gel and surface formulations would probably enter the body. Researchers from the National Institute of Environmental Health Sciences in the United States are conducting a clinical trial as of this writing to access the safety of silver nanoparticles via inhalation (National Institute of Environmental Health Sciences, 2015). It is going to be a single-center study, in which the primary objective is to learn how nanosilver affects the lung immune system and lung function. Researchers also want to discover if those nanoparticles are absorbed into bloodstream after being inhaled. Nonsmoking adults from 18 to 60 years are being recruited to this task and will be screened before the beginning of the procedures to reduce risk of bias. They estimate they will work with a range of 125 patients. To reach the objectives, the design is to conduct lung function tests before and after the administration of the silver nanoparticles. Urine samples are also going to be taken. Results on this trial will possibly provide crucial information regarding the feasibility of nanosilver formulations in the future. Together, these studies are of pivotal importance, because safety is still one of the biggest concerns in nanomedicine. The fact that silver nanoparticles appear to be safe is an extra incentive for the conduction of gradually more invasive studies, aiming to answer bigger questions. Now, where to move from here? As Munger et al. (2014) suggest, other systems should be evaluated, like the reproductive system and the central nervous system. It may be interesting to evaluate other ways of delivery, like transdermal. What is more, chronic particle exposure needs to be studied, because no study up to this point has addressed this question.

5.6 Antimicrobial Nanomedicines on Hospital Surfaces Hospital-acquired infections (nosocomial infections) are an important cause of disease and mortality, especially in intensive care units. Hospital surfaces are heavily contaminated by bacteria, sometimes resistant types, which can generate conditions like pneumonia, skin infections, catheter infections, and others in hospitalized patients. As antimicrobial resistance increases, these pathogens represent serious risks, especially because they can survive for weeks or even months in a hospital setting. To overcome this problem, systematic cleaning of the environment is the common norm. Sometimes, however, hospital surfaces are not correctly disinfected. On this matter, a trial conducted in 2015 in the Netherlands, precisely addressing environmental cleanliness, already collected information and as of this writing its results are being analyzed (de Jong, 2015). In this trial, a nanotechnology-based self-disinfecting product, MVX, is being evaluated. The product contains titanium dioxide, which generates hydroxy radicals and oxygen radicals in the presence of light. These radicals are responsible for its antimicrobial proprieties, which may last up to 5 years. The titanium dioxide nanoparticles have potential to kill bacteria, viruses, and fungi. Therefore, investigators want to address the microbial colonization of surfaces in the intensive care unit using MVX. The product was applied to surfaces and cultures were obtained at established periods; the expected outcome is a reduction in the formation of microorganism colonies. The results of this trial have the potential to consolidate MVX as an effective product and open doors to reducing infections in a hospital environment. Given the relevance of nosocomial infections, such advancement could have much impact on daily patient care.

6. FINAL CONSIDERATIONS Antimicrobial nanomedicine is a promising field in the nanotechnology scenario. Different clinical approaches have been already conducted, including hand gels or a coating for central venous access, interventions aimed at HIV infections, and new strategies in oral medicine. Antimicrobial nanomedicine has systematically proven to be promising and suitable enough to undergo clinical testing, even with the consideration about how costly and bureaucratic clinical trials tend to be. As the field of nanomedicine and antimicrobial therapy grows, we can expect that more and more approaches will be clinically tested. Although many results have started to be presented very recently, especially from 2010 on, the level of evidence generated by those studies has certainly helped and will further help to catalyze the progress of the field. Currently, there are clinical trials promissory to stimulate large therapeutic testing within the area. Considering the clinical potential of antimicrobial nanomedicine, the conduction of clinical trials is an important step toward, maybe, introducing nanomedicine into the day-to-day practice of medicine.

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REFERENCES Aminov, R.I., 2010. A brief history of the antibiotic era: lessons learned and challenges for the future. Front. Microbiol. 1, 134. Antonelli, M., De Pascale, G., Ranieri, V.M., Pelaia, P., Tufano, R., Piazza, O., Zangrillo, A., Ferrario, A., De Gaetano, A., Guaglianone, E., Donelli, G., 2012. Comparison of triple-lumen central venous catheters impregnated with silver nanoparticles (AgTiveÒ) vs conventional catheters in intensive care unit patients. J. Hosp. Infect. 82 (2), 101e107. Berardi, R., Rinaldi, S., Santini, D., Vincenzi, B., Giampieri, R., Maccaroni, E., Marcucci, F., Francoletti, M., Onofri, A., Lucarelli, A., Pierantoni, C., Tonini, G., Cascinu, S., 2015. Increased rates of local complication of central venous catheters in the targeted anticancer therapy era: a 2-year retrospective analysis. Support Care Cancer 23 (5), 1295e1302. Beyth, N., Yudovin-Farber, I., Perez-Davidi, M., Domb, A.J., Weiss, E.I., 2010. Polyethyleneimine nanoparticles incorporated into resin composite cause cell death and trigger biofilm stress in vivo. Proc. Natl. Acad. Sci. U.S.A. 107 (51), 22038e22043. Beyth, N., Houri-Haddad, Y., Domb, A., Khan, W., Hazan, R., 2015. Alternative antimicrobial approach: nano-antimicrobial materials. Evid. Based Complement. Altern. Med. 2015, 246012. Bigelow, R., 2013. Introduction to clinical experimentation. In: Lopes, R.D., Harrington, R.A. (Eds.), Understanding Clinical Research, first ed. Lange, (Mc Graw Hill Education). Burns, J., Hollands, K., 2015. Nano silver fluoride for preventing caries. Evid. Based Dent. 16 (1), 8e9. Carmona-Ribeiro, A.M., de Melo Carrasco, L.D., 2013. Cationic antimicrobial polymers and their assemblies. Int. J. Mol. Sci. 14 (5), 9906e9946. Catholic University of the Sacred Heart, 2006. Comparison of Central Venous Catheters with Silver Nanoparticles Versus Conventional Catheters. Chang, H.H., Cohen, T., Grad, Y.H., Hanage, W.P., O’Brien, T.F., Lipsitch, M., 2015. Origin and proliferation of multiple-drug resistance in bacterial pathogens. Microbiol. Mol. Biol. Rev. 79 (1), 101e116. Dizaj, S.M., Lotfipour, F., Barzegar-Jalali, M., Zarrintan, M.H., Adibkia, K., 2014. Antimicrobial activity of the metals and metal oxide nanoparticles. Mater. Sci. Eng. C Mater. Biol. Appl. 44, 278e284. Drexler, E., 1986. Engines of Creation: The Coming Era of Nanotechnology. Anchor, New York. Drexler, E., 2013. Radical Abundance: How a Revolution in Nanotechnology Will Change Civilization: Public Affairs. Feynman, R., 1959. RE: Plenty of Room at the Bottom. Florczyk, S.J., Saha, S., 2007. Ethical issues in nanotechnology. J. Long Term Eff. Med. Implants 17 (3), 271e280. Franci, G., Falanga, A., Galdiero, S., Palomba, L., Rai, M., Morelli, G., Galdiero, M., 2015. Silver nanoparticles as potential antibacterial agents. Molecules 20 (5), 8856e8874. Gao, W., Thamphiwatana, S., Angsantikul, P., Zhang, L., 2014. Nanoparticle approaches against bacterial infections. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 6 (6), 532e547. Guptill, J.T., Chiswell, K., 2013. Phase II clinical trials. In: Lopes, R.D., Harrington, R.A. (Eds.), Understanding Clinical Research, first ed. Lange (Mc Graw Hill Education). Hadassah Medical Organization, 2006b. Clinical Study of Antibacterial Nanoparticles Incorporated in Composite Restorations. Hafley, G.E., Leonardi, S., Pieper, K.S., 2013. Phase III and IV clinical trials. In: Lopes, R.D., Harrington, R.A. (Eds.), Understanding Clinical Research, first ed. Lange (McGraw-Hill Education). Hawthorne, G.H., Bernuci, M.P., Bortolanza, M., et al., 2016. Neurotox. Res. 30, 715. Genetic Immunity, 2005. Single DermaVir Immunization in HIV-1 Infected Patients on HAART. Jasti, N., Streiff, M.B., 2014. Prevention and treatment of thrombosis associated with central venous catheters in cancer patients. Expert Rev. Hematol. 7 (5), 599e616. de Jong, B., 2015. Effect of MVX (Titanium Dioxide) on the Microbial Colonization of Surfaces in an Intensive Care Unit. Justo, J.A., Bookstaver, P.B., 2014. Antibiotic lock therapy: review of technique and logistical challenges. Infect. Drug Resist. 7, 343e363. Kampf, G., Löffler, H., 2010. Hand disinfection in hospitals e benefits and risks. J. Dtsch. Dermatol. Ges. 8 (12), 978e983. Kattan, J., Droz, J.P., Couvreur, P., Marino, J.P., Boutan-Laroze, A., Rougier, P., Brault, P., Vranckx, H., Grognet, J.M., Morge, X., et al., 1992. Phase I clinical trial and pharmacokinetic evaluation of doxorubicin carried by polyisohexylcyanoacrylate nanoparticles. Invest. New Drugs 10 (3), 191e199. Lackner, P., Beer, R., Broessner, G., Helbok, R., Galiano, K., Pleifer, C., Pfausler, B., Brenneis, C., Huck, C., Engelhardt, K., Obwegeser, A.A., Schmutzhard, E., 2008. Efficacy of silver nanoparticles-impregnated external ventricular drain catheters in patients with acute occlusive hydrocephalus. Neurocrit. Care 8 (3), 360e365. Lee, W., Kim, K.J., Lee, D.G., 2014. A novel mechanism for the antibacterial effect of silver nanoparticles on Escherichia coli. Biometals 27 (6), 1191e1201. Lewis, K., 2013. Platforms for antibiotic discovery. Nat. Rev. Drug Discov. 12 (5), 371e387. Lisziewicz, J., Bakare, N., Calarota, S.A., Bánhegyi, D., Szlávik, J., Ujhelyi, E., TTke, E.R., Molnár, L., Lisziewicz, Z., Autran, B., Lori, F., 2012. Single DermaVir immunization: dose-dependent expansion of precursor/memory T cells against all HIV antigens in HIV-1 infected individuals. PLoS One 7 (5), e35416. Madigan Army Medical Center, 2008. Efficacy of Silver Nanoparticle Gel Versus a Common Antibacterial Hand Gel. Martinez, J.L., 2014. General principles of antibiotic resistance in bacteria. Drug Discov. Today Technol. 11, 33e39. Munger, M.A., Radwanski, P., Hadlock, G.C., Stoddard, G., Shaaban, A., Falconer, J., Grainger, D.W., Deering-Rice, C.E., 2014. In vivo human timeexposure study of orally dosed commercial silver nanoparticles. Nanomedicine 10 (1), 1e9. National Institute of Allergy and Infectious Diseases & Group, A. C. T, 2006. Safety, Tolerability and Immune Response to LC002 (Dermavir), an Experimental Therapeutic Vaccine, in Adults Receiving HAART.

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National Institute of Enviromental Health Sciences, 2015. Inhaled Nanosilver Study. Noorlander, C.W., Kooi, M.W., Oomen, A.G., Park, M.V., Vandebriel, R.J., Geertsma, R.E., 2015. Horizon scan of nanomedicinal products. Nanomedicine (Lond.) 10 (10), 1599e1608. Norrby, S.R., Nord, C.E., Finch, R., 2005. Lack of development of new antimicrobial drugs: a potential serious threat to public health. Lancet Infect. Dis. 5 (2), 115e119. European Society of Clinical Microbiology and Infectious Diseases. Qureshi, S., Agrawal, C., Madan, M., Pandey, A., Chauhan, H., 2015. Superbugs causing ventilator associated pneumonia in a tertiary care hospital and the return of pre-antibiotic era! Indian J. Med. Microbiol. 33 (2), 286e289. Rai, M.K., Deshmukh, S.D., Ingle, A.P., Gade, A.K., 2012. Silver nanoparticles: the powerful nanoweapon against multidrug-resistant bacteria. J. Appl. Microbiol. 112 (5), 841e852. Reist, C.J., Rorick, T.L., Berdan, L.G., Lopes, R.D., 2013. The role of academic research organizations in clinical research. In: Lopes, R.D., Harrington, R.A. (Eds.), Understanding Clinical Research. Lange (Mc Graw Hill Education). Rizzello, L., Pompa, P.P., 2014. Nanosilver-based antibacterial drugs and devices: mechanisms, methodological drawbacks, and guidelines. Chem. Soc. Rev. 43 (5), 1501e1518. Rodriguez, B., Asmuth, D.M., Matining, R.M., Spritzler, J., Jacobson, J.M., Mailliard, R.B., Li, X.D., Martinez, A.I., Tenorio, A.R., Lori, F., Lisziewicz, J., Yesmin, S., Rinaldo, C.R., Pollard, R.B., 2013. Safety, tolerability, and immunogenicity of repeated doses of dermavir, a candidate therapeutic HIV vaccine, in HIV-infected patients receiving combination antiretroviral therapy: results of the ACTG 5176 trial. J. Acquir. Immune Defic. Syndr. 64 (4), 351e359. Santos, V.E., Vasconcelos Filho, A., Targino, A.G., Flores, M.A., Galembeck, A., Caldas, A.F., Rosenblatt, A., 2014. A new “silver-bullet” to treat caries in childrenenano silver fluoride: a randomised clinical trial. J. Dent. 42 (8), 945e951. Schlicher, M.L., 2011. Efficacy of Silver Nanoparticle Gel on Bacterial Hand Flora: A RCT. Spring, M., 1975. A brief survey of the history of the antimicrobial agents. Bull. N.Y. Acad. Med. 51 (9), 1013e1015. Svendsen, P., El-Galaly, T.C., Dybkær, K., Bøgsted, M., Laursen, M.B., Schmitz, A., Jensen, P., Johnsen, H.E., 2015. The application of human phase 0 microdosing trials e a systematic review and perspectives. Leuk. Lymphoma 1e28. Targino, A.G., Flores, M.A., dos Santos Junior, V.E., de Godoy Bené Bezerra, F., de Luna Freire, H., Galembeck, A., Rosenblatt, A., 2014. An innovative approach to treating dental decay in children. A new anti-caries agent. J. Mater. Sci. Mater. Med. 25 (8), 2041e2047. University of Pernambuco, 2014. Nanosilver Fluoride to Prevent Dental Biofilms Growth. University of Utah, 2010. In Vivo Assessment of Silver Biomaterial Nano-Toxicity. University of Utah, 2011. In-Vivo Assessment of Silver Biomaterial Nano-Toxicity 32 ppm. Watt, K.M., Chiswell, K., Cohen-Wolkowiez, M., 2013. Phase I trials: first in human. In: Lopes, R.D., Harrington, R.A. (Eds.), Understanding Clinical Research, first ed. Lange (Mc Graw Hill Education). Wong, Y.W., Schulman, K.A., 2013. Ethics of clinical research: an overview and emerging issues. In: Lopes, R.D., Harrington, R.A. (Eds.), Understanding Clinical Research, first ed. Lange (Mc Graw Hill Education). World Health Organization, 2012. The Top 10 Causes of Death.

FURTHER READING Hadassah Medical Organization, 2006a. Clinical Evaluation of Esthetic Restorations Placed in Primary Molars with Composite Resin Enriched with Insoluble Anti Bacterial Nano Particles. Hadassah Medical Organization, 2007. A Clinical Study. The Effect of Addition of Insoluble Antibacterial Nanoparticles (IABN) in Resin Base Provisional Cement. Hadassah Medical Organization, 2010. Antibacterial Properties of Silicon Incorporated with Quaternary Ammonium Polyethylenimine Nanoparticles. Hadassah Medical Organization, 2013. A Clinical Study: The Antibacterial Effect of Insoluble Antibacterial Nanoparticles (IABN) Incorporated in Dental Materials for Root Canal Treatment.