Nanotechnology and Antimicrobials in Veterinary...

Nanotechnology and Antimicrobials in Veterinary Medicine

M. Zampoli Troncarelli1, H. Mello Brandão2, J. Carine Gern2, A. Sá Guimarães2, H. Langoni1

1Universidade Estadual Paulista UNESP (University of Sao Paulo State), Faculdade de Medicina Veterinária e Zootecnia (Faculty of Veterinary Medicine and Animal Science), Departamento de Higiene Veterinária e Saúde Pública (Department of Veterinary Hygiene and Public Health), Distrito de Rubião Jr., s/n, 18618-970, P.O. box 150, Botucatu, São Paulo, Brazil. *[email protected]

2Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Gado de Leite (Embrapa Dairy Cattle), Laboratório de Nanotecnologia Aplicada à Saúde e Produção Animal (Laboratory of Nanotechnology Applied to Animal Health and Production), Juiz de Fora, Minas Gerais, Brazil

Nanotechnology is defined as a scientific knowledge application for matter’s manipulation and control in nanometric scale (1-100 nm). Nanostructures may present new physical and chemical characteristics, demonstrating high reactivity and solubility levels, and better stability than the original actives. Nanotechnology allows the development of new products and also the possibility of reworking conventional substances viewing better efficacy results. This technique is commonly used in Human Medicine; especially involving nanocarriers for quick and specific drug delivery into cells. In Veterinary Medicine, the use of nanoparticles composed by antimicrobials actives are being intensively tested, especially in livestock animals. Nanostructured antimicrobials composed by synthetic or natural actives have shown excellent results against multi-resistant microorganisms such are normally hard to eliminate by using conventional treatments. Pet care nanoproducts, as shampoos composed of silver nanoparticles, are already approved for use in dogs and cats. These products are indicated to act in skin and coat, and also cleaning and softening. Other available nanoproducts are deodorants, surface disinfectants for pets’ environment and owners’ furniture. On the other hand, the growing use of nanomaterials also reveals a need for risk assessment regarding the environment and even food. Viewing to better comprehend the nanoparticles interactions, important research groups and government organizations in several countries have contributed with important safety and efficacy studies regarding the use of nanoparticles in animals and evaluating its impact on animal health, Public Health and environment.

Keywords antimicrobials; nanoparticles; animals

1. Introduction

Despite the great progress with antimicrobials development since the discovery of antimicrobial drugs in 1960’s [1], many infectious diseases, especially intracellular infections, remain difficult to treat. During the infection course, a fusion of phagosomes with early or late endosomes and/or lysosomes occurs where microbes are destined for elimination and clearance. However, in chronic infection cases, the system breaks down and the microbe can survive in these compartments from months to years. Furthermore, many antimicrobials are difficult to transport through cell membranes and have low activity inside the cells, thereby imposing negligible inhibitory or bactericidal effects on the intracellular bacteria [2]. Another relevant point is that the antimicrobial toxicity to healthy tissues poses a significant limitation to their use. Even though the therapeutic efficacy of these drugs has been well established, inefficient delivery could result in inadequate therapeutic index and local and systemic side effects. Thus, the search for means to best deliver antimicrobial drugs directly to organelles that harbor infection is critically important [2; 3 apud 4-7]. Nanostructured biomaterials, nanoparticles in particular, have unique physicochemical properties such as ultra-small and controllable size, large surface area to mass ratio, high reactivity, and functionalizable structure. These properties can be applied to facilitate the administration of antimicrobial drugs, thereby overcoming some of the limitations in traditional antimicrobial therapeutics. In recent years, encapsulation of antimicrobial drugs in nanoparticle systems has emerged as an innovative and promising alternative that enhances therapeutic effectiveness and minimizes undesirable side effects of the drugs [2]. Examples of potential applications of nanotechnology in animal agriculture and Veterinary Medicine include disease diagnosis and treatment delivery systems, new tools for molecular and cellular breeding, the security of animal food products, modification of animal waste, pathogen detection, and many more. Considering the relevance of this theme in the scientific actual context, the objective of the present review is to discuss about Nanotechnology definition, general particularities and applications, and also the advances in Veterinary Medicine obtained by the use of nanoproducts, especially antimicrobials composed of conventional and natural bases.

Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)

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2. Nanotechnology

2.1. Definition

The term ‘nanotechnology’ was first applied in 1974 [8] and was used to describe production technology at ultrafine dimensions, hence the use of the Greek word ‘nano’ - meaning dwarf [9]. According to the published document of International Organization for Standardization, ISO/DTS 80004-1, nanotechnology is defined as a scientific knowledge application for matter’s manipulation and control in nanometric scale [10,11]. The most widely use definition of nanotechnology is provided by the United States Government's National Nanotechnology Initiative. According to the researchers, nanotechnology is defined as: "Research and technology development at the atomic, molecular and macromolecular levels at the scale of approximately 1 - 100 nanometer range, to provide a fundamental understanding of phenomena and materials at the nanoscale and to create and use structures, devices and systems that have novel properties and functions because of their small and/or intermediate size" [12 apud 13]. On the other hand, the National Institute of Intellectual Property in Brazil indicates that nanotechnology cannot be defined at once, due its interface with Chemical, Physics, Biology and Engineer areas [14]. The generic term of 'nano-object' as defined by the EU Commission recommendation on a code of conduct for responsible nanosciences and nanotechnologies research will include all nanomaterials, nanostructured materials, nanoparticles and their aggregation at the nanoscale, nano-systems, and nanoproducts [15].

2.2. The “nano world”

Nanotechnology involves the management of some molecular phenomena and properties that are size and structure-related, in a different way from the natural process that usually involves individual atoms and/or aggregated molecules [3]. Nanomaterials have been defined as having one or more dimensions measuring 100nm or less, or having at least one dimension at this scale which affects the materials’ behavior and properties. To put the nanoscale into context: a strand of DNA is 2.5 nm wide, a protein molecule is 5 nm, a red blood cell 7,000 nm and a human hair is 80,000 nm wide. If one imagines that a nanometre is represented by a person, a red blood cell would be 7 kilometres long! It is like to say that if a nanoparticle was a soccer ball, Earth’s diameter should be one meter. Then, 1 nm (one nanometer) corresponds to 10-9 meters. [16]. Despites the different possibilities of Nanotechnology’s definitions, there is a common sense among scientists that this technology is associated to the matter’s characteristics, involving its origin and/or change (increase/decrease) of its properties, due a scale reduction of at least one of its dimensions [11]. In fact, while the substance has its size reduced, it may present new physical and chemical characteristics, and also demonstrate higher reactivity and solubility. When the active ingredient is nanostructured, the stability of this substance increases, due a protection against oxidant agents, enzymes and other components [11,17]. Nanoparticles can be more chemically reactive and more bioactive than larger particles. Because of their very small size, nanoparticles also have much greater biological access, so they are more likely than larger particles to enter cells, tissues and organs.

2.3. Chemical composition of nanoparticles

A range of nanoparticular systems, which include functionalized fullerenes and carbon nanotubes, liposomes, iron oxide nanoparticles, polymeric micelles, dendrimers, nanoshells, polymeric nanospheres, nanobins, quantum dots, and polymer-coated nanocrystals, among others (Tables 1 and 2), are being applied to improve human disease outcomes [3 apud 18-21]. Antimicrobials are being placed into polymer-coated crystalline nanoparticles, homogenized particulate suspensions, cholesterol-conjugated amphiphilic peptide self-assembled particles, composite hydrogel/glass particles, liposomes, PLGA, cationic, and pDNAcoated gold nanoparticles. Liposome-based therapeutics has been approved by the FDA for indications including fungal infections, for example, liposomal amphotericin B [22]. These nanostructures have shown excellent results, especially in life-threatening diseases such as staph infections and tuberculosis [2]. The bioavailability, solubility, half-life, therapeutic index and immunogenicity of a nanostructured drug are directly related to its physical and chemical characteristics, like composition, shape, surface and size (Figure 1). Searching for strategies of efficacy and safety optimization of nanoparticles for therapeutic use, scientists have been done intensively and detailed studies [22]. All these efforts are directed to obtain accurate medicines with an excellent biodistribution, a specific and direct action in target cells/microorganisms, and minimal or, if possible, null toxicity for the patient.


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Table 1 Nanosystems and its characteristics.

Chemical composition / Structure Characteristics Functions

Functionalized Fullerenes (Buckeyballs) and Carbon Nanotubes

Lack of water solubility Vehicles for nanodrugs delivery; Contrast agents; Photothermal cancer ablation

Lipossomes

Vesicles composed of a lipid bilayer surrounding a hollow core They can be composed of natural phospholipids or other surfactants

Drugs or other molecules can be loaded for delivery to tumors or other disease sites; Liposomes can carry both hydrophobic and hydrophilic drugs and molecules to a target site

Polymeric Micelles

Made with amphiphilic polymers such as the block copolymers poly(ethyleneglycol)-b-poly(e-caprolactone)(PEG-b-PCL),22 poly(styrene) or PLGA

Commonly used for targeted drug delivery; Deliver poorly water soluble drugs such as paclitaxel and amphotericin B

Polymeric Nanospheres

Uniform spherical structures less than a micron in size made from nonbiodegradable or biodegradable polymers

Effective agents for transdermal drug delivery Diagnose human epidermal growth factor receptor 2 (HER2)-positive and integrin-positive cancer cells in vitro

Dendrimers

Large, complex molecules with a well-defined branched chemical structure; Monodisperse, highly symmetric, highly branched, and generally spherical

Allow carriage of drugs or molecules for imaging; Dendrimer-based conventional nanocomposites are been studied as possible antimicrobial agents against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli

Polymer-Coated Nanocrystals

Prevents aggregation and helps in establishing a stable nanosupension

Macrophage- based delivery to sites of HIV infection and sequestration

Nanoshells

Spherical particles consisting of a dielectric core surrounded by a thin metallic shell, most commonly gold

Biomedical imaging and cancer treatment

SPIO Nanoparticles

A core of magnetite or maghemite with a coating of polysaccharides, polymers, or monomers

Iron oxide particles in the range of 1–100 nm possess superparamagnetic properties that make them attractive for biomedical imaging, diagnosis, and therapeutics

Quantum Dots

Semiconductors with spatially confined excitons that afford them unique optical and electrical properties

Their distinct fluorescence spectra make them valuable tools for biomedical imaging

Adapted from McMillan et al., 2011 [3].


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Table 2 Classification of nanoparticle delivery systems.

Nanostructure Derivates

Encapsulated nanoparticles

Lipid-bases capsules => nanoliposomes => archaeosomes => nanocochleates => micelles Polymer-based capsules => nanospheres => nanocapsules => polymersomes => micelles

Polymer-based capsules Natural polymers (e.g. albumin, collagen, algirates, gelatine, chitosan => nanospheres Synthetic polymers (e.g. PLA, PLGA, PCL)

Adapted from Bouuwmeester et al., 2007 [64].

Fig. 1 Architeture of nanoparticles. A: Micelle; B: Liposome; C: Phospholipids chain. Adapted from: Vandamme, T., 2013 “Perspectives d’application thérapeutique des nanotechnologies en médecine vétérinaire” Université de Strasbourg, Faculté de Pharmacie, http://www.academie-veterinaire-defrance.org/fileadmin/user_upload/pdf/pdf_2013/Vandamme.pdf Incorporation of certain functionalities can modulate the responsiveness (assembly/disassembly) of the nanoparticles in biological environments under different pH, enzymatic, oxidative, and reductive conditions, etc. [3; 23].The small size of nanoparticles makes them similar to physiological molecules, and might allow them to utilize the same mechanisms of entry across internal barriers. On this basis, extensive research regarding the possible use of appropriately designed nanoparticles for the diagnosis and treatment of diseases in organs protected by the internal barriers has been developed during the last years [24]. Nanostructures can be equipped with smart components (Figure 2) to allow their delivery beyond certain biological barriers, such as skin, eye, brain, placenta, mucus, blood, extracellular matrix, cellular and subcellular organelles. “Smart’ delivery systems can have multifunctional characteristics to successful targeting and they may also be time-controlled; spatially targeted; self-regulated; remotely regulated; pre-programmed [25]. Monofunctional nanoparticles provide a single function – a liposome can transport drugs but does not have the inherent property to distinguish between healthy and unhealthy cells or tissues – multifunctional nanoparticles combine different functionalities in a single stable construct. For example, a core particle could be linked to a specific targeting function that recognizes the unique surface signatures of their target cells. Simultaneously, the same particle can be modified with an imaging agent to monitor the drug transport process, a function to evaluate the therapeutic efficacy of a drug, a specific cellular penetration moiety and a therapeutic agent [26].


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Fig. 2 Smart drug delivery system. Source: Rajasokkapan S., 2013 [41]. “Aplications of Nanotechnology in Veterinary Science” http://www.slideshare.net/sokkappan/nanotechnology-in-veterinay-science

2.4. Specific properties in nanoscale

In practice, the physical, chemical and biological properties of a substance are determined by the association of physico-chemical phenomena that act on the matter, e.g. gravitational force, inertia, friction, Brownian motion, electrostatic interactions, electrical repulsion, etc. [27]. One conventional substance in metric scale suffers the action of all these phenomena, in different levels. However, once a substance has its size reduced; the action of these forces changes. In addition, their surface area is drastically increased. Around 100 nm, the atoms are more stable in relation to their metric form, requiring therefore a smaller amount of energy to separate them [19]. Then, a size-reduced substance can acquire new physical and chemical characteristics, with greater reactivity and solubility, according to the different environment conditions. When inside a nanoparticle, the active ingredient has increased its stability, remaining protected from oxidizing agents, enzymes or chemical interaction with other molecules [11]. This phenomenon becomes particularly relevant in case of products developed for the treatment of bovine mastitis, since inflammatory processes in the mammary gland is a series of physical and chemical changes that usually interferes with the action of conventional antimicrobial actives. Using milk as an example to the understanding of nanoscale phenomena, it is known that many of its components are naturally at the nanometer scale, e.g. casein, which generally has hydrodynamic radius less than 400nm. At this size, the intensity of the Brownian motion of casein overcomes gravity, preventing its precipitation. However, when the acidification of milk occurs, casein’s micelle is destabilized, and form aggregates. In this condition, the particles have their hydrodynamic radius increased and tend to precipitate, especially because the gravity force overcomes the Brownian motion [11].

2.5. Practical applications

When considering its use in commercial products, nanotechnology is being used in electronic components, packaging for various product categories - highlighting the food - drugs, manufacturing nanosensors, fertilizers and pesticides, special fabrics and a multitude of other applications [17]. Considering the Pharmacology area, it is important to reinforce that nanotechnology allows the development of new products and also the possibility to rework conventional substances in order to obtain better efficacy results [5]. By loading drugs into nanoparticles through physical encapsulation, adsorption, or chemical conjugation, the pharmacokinetics and therapeutic index of the drugs can be significantly improved in contrast to the free drug counterparts. Drug-loaded nanoparticles can enter host cells through endocytosis and then release drug payloads to treat microbes-induced intracellular infections [2]. Nanoparticle-based drug delivery provides many advantages, such as enhancing drug-therapeutic efficiency and pharmacological characteristics. The utility of nanoparticles in improving pharmacokinetics, reducing unwanted side effects, and improving delivery to disease sites has been demonstrated for a number of nanodrug delivery systems [3


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apud 21]. For example, nanoparticles improve the solubility of poorly water-soluble drugs, modify pharmacokinetics, increase drug half-life by reducing immunogenicity, increase specificity towards the target cell or tissue (therefore reducing side effects), improve bioavailability, diminish drug metabolism and enable a more controllable release of therapeutic compounds and the delivery of two or more drugs simultaneously for combination therapy [26 apud 28-29]. Generally, the practical consequences of a pharmaceutical nanostructured substance are: a) Providing a rational use of the active ingredient, considering that both the number of doses and the concentration of the drug may be reduced during the treatment; b) “Renewing” of old pharmaceutical bases which were continued used; c) Prolonging the systemic circulation lifetime of drug [30-32]; d) Releasing drugs at a sustained and controlled manner, preferentially delivering drugs to the tissues and cells of interest [30-32]; e) Delivering multiple therapeutic agents to the same cells for combination therapy [30-32]; f) Providing new perspectives of administration routes for medicines and vaccines; g) Reducing stress for drug administration; h) Reducing toxicity and collateral effects of conventional pharmaceutical actives; i) Providing the use of new molecules and actives in animal therapeutic; j) Producing low (or none) residues in animal products, resulting in no withdrawal needed [2]; k) …and other perspectives

3. Nanotechnology applications in Medicine and Pharmacology

3.1. Human Medicine

Nanomedicine research has focused, in large measure, on cancer and infectious diseases. Of the nanomedical products developed, reformulated pharmaceuticals remain in majority [3 apud 65]. Nanoparticles can be built from various materials (e.g. polymers, lipids, metals) and can host a wide range of active components, including chemotherapeutics, anti-inflammatory drugs [33], contrast agents for imaging and diagnosis [3], proteins, nucleic acids [23], and antimicrobials. Nanostructured antimicrobials are being approved for human treatment for a broad range of microbial infections including tuberculosis [3] The use of nanocarriers is one of the most important aspects of nanomedicine, which in recent years has been marked by a considerable increase in the number of studies which describe improvements in traditional pharmacological bases used, especially in human therapeutics [5,69,70].

3.1.1. Chemotherapy nanomedicines

The development of more effective drugs for the treatment of chronic diseases must be pointed out. In general, a chemotherapeutic agent when is encapsulated in the nanoparticle may cause less toxicity and cellular resistance to the drug compared to traditional chemotherapy medicines. As example, doxorubicin and paclitaxel are very effective in the treatment of ovarian carcinomas when used together, but they present high toxicity. A study using structured lipid nanoparticles of both actives against multidrug-resistant cancer cell lines demonstrated that the nanoparticles were highly effective in controlling tumor cell growth and caused less toxicity in cells [34].


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3.1.2. Anti-inflammatory nanoparticles

Indomethacin is a non-steroidal anti-inflammatory routinely used in human medicine, recommended for application every eight hours. In vitro assays indicated that when incorporated in nanoparticles of poly-n-vinylpyrrolidone, the sustained release indomethacin may allow administration of the drug every twelve hours. This result was obtained due to the effect of promoting sustained release of the drug by the nanocarrier [35].

3.1.3. Nanoantimicrobials

The use of nanotechnology in active antimicrobials has also shown satisfactory results. Penicillin is the first and most ancient antimicrobial agent from the group of β-lactams, which has been used for decades in the treatment of bacterial infections. However, indiscriminate use resulted in the selection methicillin resistant Staphylococcus aureus (MRSA) strains. A study allowed the covalent attachment of a chain of penicillin polyacrylate nanoparticle structure with approximately 100nm. The researchers compared the in vitro activity of nanostructured molecule with a conventional formulation. In MRSA strains, nanostructured formulation is more effective, which was attributed to a probable active protection against action of bacterial β-lactamases and / or protection against penicillin binding proteins [36]. Gaseous nitric oxide (NO) is considered an effective antimicrobial agent. MRSA wound infections are treated with NO-releasing nanoparticle technology [37 apud 38]. An in vivo wound model shows that the NO-releasing nanoparticles display antimicrobial activity against MRSA. Furthermore, NO releasing nanoparticle treatment shows decreased supportive inflammation, minimal bacterial burden, and less collagen degradation cause the speed of infected wound closure increase.

4. Antimicrobials nanoparticles in Veterinary Medicine

Veterinary health care is a highly visible and growing concern not only for pet owners, but also for governments. With an increasingly aging pet population, along with higher costs for medications and veterinary care, the need for new solutions is urgent [13]. In agriculture today, if an animal becomes infected with a disease, it can be days, weeks, or months before some whole organism symptoms appear and the disease is detected; at this point, the infection may be widespread and entire herds/fields might need to be destroyed. Nanotechnology operates at the same scale as a virus or disease-infecting particle, and thus holds the potential for very early detection and eradication [25]. The field of veterinarian sciences stands to gain with nanotechnology as much as human medicine does: diagnostic tools (nanoprobes) that can be used in vitro and on living animals, targeted delivery of medications, therapeutic nanomaterials, vaccine antigen vectors, in vivo imagery, or traceability of products of animal origin. An important increase of scientific researches for nanostructured products development in the last years has been verified in Veterinary Medicine, especially using antimicrobials actives. Conventional synthetic and natural antimicrobial substances are being tested, and have shown excellent results against multi-resistant microorganisms and bacteria strains that are normally hard to eliminate by using the conventional treatment, like Brucella, Mycobacterium bovis, Staphylococcus aureus, Salmonella, Ehrlichia, Anaplasma; Rhodococcus equi, etc. [3,8].

4.1. In vitro studies – conventional antimicrobials

Nanostructured streptomycin and doxycycline were tested against Brucella melitensis strains, and the efficacy results of nanoparticles were better than the conventional antimicrobials [39]. This specific pathogen usually stays inside animal’s macrophages, and its pharmacological control is very hard. In this in vitro study, both antimicrobial actives were encapsulated in PEO-b-PAA nanocomplexes (anfihilic polimers), allowing the nanoparticles to reach the interior of murine macrophages. When tested in vivo (in infected murines), the nanostructured formulation determined 79% reduction of the number of colony-forming unities (CFUs) compared to the control group, and also with a better efficacy compared to the conventional formulation (P<0,05). Escherichia coli and Salmonella typhi bacteria are two common pollutants and they are developing resistance to some of the most used bactericide. Therefore new biocide materials are being tested. Thus, gold nanoparticles are proposed to inhibit the growth of these two microorganisms. Gold nanoparticles dispersed on zeolites eliminate Escherichia coli and Salmonella typhi at short times. The biocidal properties of gold nanoparticles are influenced by the type of support which, indeed, drives key parameters as the size and roughness of nanoparticles. The most active materials were pointed out Au-faujasite. These materials contained particles sized 5 nm at surface and eliminate 90–95% of Escherichia coli and Salmonella typhi colonies [40]. In a study done in USA, a penicillin antibiotic was covalently conjugated in emulsified polyacrylate nanoparticles onto the polymeric framework. These nanoparticles were prepared in water by emulsion polymerization of an acrylated penicillin analogue pre-dissolved in a 7:3 (w:w) mixture of butyl acrylate and styrene in the presence of sodium dodecyl sulfate (surfactant) and potassium persulfate (radical initiator). Dynamic light scattering analysis and atomic force microscopy images show that the emulsions contain nanoparticles of approximately 40 nm in diameter. The


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nanoparticles have equipotent in vitro antibacterial properties against methicillin-susceptible and methicillin-resistant forms of Staphylococcus aureus and indefinite stability towards β-lactamase [38].

4.2. In vitro studies with Ag nanoparticles

The antimicrobial effects of silver (Ag) ion or salts are well known, and also the silver nanoparticles show efficient antimicrobial property compared to other salts. The Ag nanostructures are most effective on E. coli, S. aureus, Klebsiella and Pseudomonas. These nanoparticles preferably attack the respiratory chain and cell division, finally leading to cell death. The STEM (Scanning Transmission Electron Microscopy) confirms the presence of silver in the cell membrane and inside bacteria [41]. Stable Ag nanoparticles were prepared and their shape and size distribution characterized by particle characterizer and transmission electron microscopic study. The antimicrobial activity of Ag nanoparticles was investigated against yeast, Escherichia coli, and Staphylococcus aureus. In these tests, Muller Hinton agar plates were used and Ag nanoparticles of various concentrations were supplemented in liquid systems. As results, yeast and E.coli were inhibited at the low concentration of Ag nanoparticles, whereas the growth-inhibitory effects on S. aureus were mild. The free-radical generation effect of Ag nanoparticles on microbial growth inhibition was investigated by electronspin resonance spectroscopy. These results suggest that Ag nanoparticles can be used as effective growth inhibitors in various microorganisms, making them applicable to diverse medical devices and antimicrobial control systems [42]. However, it is important to reinforce that the Ag nanoparticles may be toxic for some animal species, as demonstrated in a study using a zebrafish model [43]. Continuous toxicity studies must be done in order to clarify the impact of nanoparticles residues in environment, flora and fauna.

4.3. Antimicrobial efficacy of solid lipid nanoparticles

A study was conducted in China, viewing to enhance of tilmicosin antibacterial activity by solid lipid nanoparticles (SLN) [44]. Tilmicosin-loaded hydrogenated castor oil (HCO)-SLN was prepared using a hot homogenization and ultrasonication method. The physicochemical characteristics of SLN were investigated by scanning electron microscopy (SEM) and photon correlation spectroscopy (PCS).The antibacterial activity of tilmicosin- SLN against Staphylococcus aureus was evaluated by growth inhibition and colony-counting method. A therapeutic study of tilmicosin- SLN was conducted by subcutaneous injection in a mouse mastitis modelinfected with S. aureus by teat canal infusion. Therapeutic efficacy was assessed by physical appearance of the mammary gland and measurement of colony-forming units (CFU) pergland.The results showed that the diameter, polydispersivity index, Zeta potential, encapsulation efficiency and loading capacity of the nanoparticles were 343±26nm, 0.33±0.08, 7.9±0.4mV, 60.4±3.3% and 1.2±0.47%, respectively. Tilmicosin-SLN showed a sustained-release effect and sustained and enhanced antibacterial activity in vitro. SLN significantly enhanced the therapeutic efficacy of tilmicosin determined by lower CFU counts and a decreased degree of inflammation. These results demonstrated that the HCO-SLN is an effective carrier to enhance the antibacterial activity of tilmicosin.

4.4. In vitro studies of nanoparticles composed by natural antimicrobial actives

The antimicrobial activity of propolis against a wide range of bacteria, fungi, yeasts and viruses has been investigated since the late 1940s and it showed variable activity against different microorganisms. The alcoholic extracts of propolis inhibited the growth of various bacteria, including strains of streptococci and Bacillus. The inhibition of bacterial RNA-polymerase by the components of propolis is probably due to the loss of their ability to bind to DNA [45] . The bactericide effect of propolis nanoparticle was tested in vitro against Staphylococcus aureus (ATCC 23273) cultivated in Mueller Hinton broth, at EMBRAPA Dairy Cattle (Brazil). Nanoparticles with 380.4 nm medium diameter needed a minimum concentration of 512 µg/mL propolis, while low nanoparticles (250 nm) needed only 256 µg/mL propolis concentration [46]. It is important to reinforce that the minimum inhibitory concentration (MIC) is directly dependent of propolis source.

5. Use of antimicrobial nanoparticles in Livestock animals

The main safety and efficacy studies of nanoparticles in Veterinary Medicine are being done in livestock animals. Examples of potential applications of nanotechnology in the science and engineering of agriculture and food systems include disease treatment delivery systems, new tools for molecular and cellular biology, the security of agricultural and food systems, new materials for pathogen detection, and protection of the environment [25]. Scientists are engaged to develop new antimicrobial products, using conventional or natural actives, in order to improve the therapeutic success, especially in chronic infections caused by intracellular microorganisms or pathogens which are hard to eliminate, like Brucella, Mycobacterium bovis, Streptococcus aureus, Rhodococcus equi, etc. On the other hand, researchers are also very concerned about tissue distribution of nanoparticles in animals, and respective antimicrobial residues in food and environment, and it is toxicity. Then, the new developed products are being


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intensively tested at important researches institutions in world, in order to clarify the real impact of nanoparticles for environment, animals and Public Health.

5.1. Conventional antimicrobials

The use of PLGA (acid lactic-coglycolic polymer) nanospheres composed by povidine iodine was purposed for bovine mastitis intramammary treatment [47]. Povidine iodine is a potent and unspecific antiseptic, frequently used for skin disinfection. In vitro evaluation showed good results against the main mastitis pathogens, but in vivo study was not done. On the other hand, considering the possibility of overcomes the limitations of conventional treatments used for staphylococci mastitis, Brazilian researchers from EMBRAPA Gado de Leite (Dairy Cattle) and UFOP (Universidade Federal de Ouro Preto) developed a new formulation of nanostructured cloxacillin for intramammary administration. These clinical trials are in Phase II with cows and ewes [48]. Partial results have showed that the nanoparticles are able to direct the antimicrobial active to the mammary gland epitheliums’ superficial and to the polymorphonuclear neutrophil compartment (i.e. inside the cells), increasing the concentration of iodine in intracellular compartments. The same group develops mucoadhesive nanoparticles to improve the efficacy of the ocular topical treatment in infections with Moraxella bovis.

5.2. Natural actives

The use antimicrobial nanoparticles produced with natural actives is showing important safety and efficacy results against bacterial infections in animals, without causing residues in animal products, with no need for withdrawal. The antimicrobial action of propolis is worldwide recognized. The bacterial sensitivity indices ranging from 85.2% to 100%, according to antibiotic susceptibility tests using Staphylococcus and Streptococcus sp. strains isolated from bovine mastitis cases (49-53). The nanostructured propolis (Figure 2), whose efficacy in vitro presented comparatively higher than that of natural intact propolis against Staphylococcus aureus ATCCs. During the production and stabilization of the nanoparticles were used amphiphilic molecules (both polar and nonpolar) biocompatible and special conditions to obtain a product alcohol free. In this context, nanopropolis can potentially be less irritating to the breast tissue as compared to alcoholic extracts used routinely for bovine intramammary formulations, which generally determine inflammatory processes in the gland [54]. Thus, it appears that the process of nanostructuring enable the optimization of the therapeutic efficacy and safety of propolis, when administered to animals. In order to meet the demand of industry organic milk production EMBRAPA Dairy Cattle (Brazil), together with partners, nanopropolis, which is a promising prospect for the control of mastitis in herds. Pilot studies have shown that propolis nanostructured has excellent bioavailability and antimicrobial activity associated with the absence of adequate safety and residues in milk, because it is a compound of natural base. The nanostructured propolis presented in vitro efficacy comparatively higher than that of natural intact propolis against strains of Staphylococcus aureus reference. During the production and stabilization of the nanoparticles were used amphiphilic molecules (both polar and nonpolar) biocompatible and special conditions to obtain a product alcohol free. In this context, nanopropolis can potentially be less irritating to the breast tissue as compared to alcoholic extracts used routinely for bovine intramammary formulations, which generally determine inflammatory processes in the gland [54]. Thus, it appears that the process of nanostructuring enable the optimization of the therapeutic efficacy and safety of propolis, when administered to animals. An important scientific partnership was established between two research institutions in Brazil: EMBRAPA Gado de Leite (Juiz de Fora-MG) and Faculdade de Medicina Veterinária e Zootecnia (FMVZ UNESP Botucatu-SP), in order to conduct safety and efficacy trials using the nanopropolis formulation in commercial dairy herds, for mastitis control. This project is being supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). If product be efficient as expected, it may be produced by commercial companies in industrial scale.


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Fig. 3 Photomicroscopy of nanopropolis atomic force which helps its bioavailability and antimicrobial action. Source: Gern & Bernardes-Filho, 2012 [55]. According to EMBRAPA Gado de Leite (Dairy Cattle) researchers, all components of the nanoparticle formulation are biocompatible, biodegradable and present accessible costs for pharmaceuticals industry. The importance of this new technology is that nanoparticles can act synergistically with antibiotics This has great importance in the mastitis treatment, particularly those caused by multidrug resistant Staphylococcus aureus. This pathogen often survives to conventional treatments and determines chronic infections.. The use of this technology also applies to the treatment of infectious bovine keratoconjunctivitis. Researchers from EMBRAPA Dairy Cattle (Minas Gerais, Brazil) verified that the herd of the Experimental Field of Coronel Pacheco (CECP) was affected by an outbreak of this disease. A nanopropolis batch was experimentally applied in a pilot treatment, with few animals. All animals treated were cured, indicating good antimicrobial results of the nanoparticle.

6. Nanothecnology aspects in Pets

The creation and manipulation of new synthetic molecules can provide us with new therapeutical compounds to treat diseases in our pet population. These new compounds for example would protect animals from viral or bacterial infections and accelerate wound healing. Also these new compounds could carry drugs and genes into cells, making treatment of diseases more efficacious [56]. Most of pet diseases one day will be addressed by the use of nanopharmaceuticals [13].

6.1. Nano antimicrobials

Considering the specific use of nano antimicrobials for treatment of infectious diseases in dogs and cats, there are no scientific reports available. The main efforts of pet care industry seems to be concentrated in develop products for surface deodorization and disinfection. Shampoos composed by silver colloidal nanoparticles are recommended for dogs and cats as antimicrobial cleanse and coat softener. Some specific companies are producing antimicrobial and deodorant sprays recommended for pet clothing (composed of nano TiO2 particles and de-ionized water); for cat and/or dog owner’s furniture; for kitty litter; and to keep dog cleaning. Producer companies state that these products are effective against bacteria, virus and fungi; deodorize odor and smell; have long-term effect and are Green and Eco friendly.

6.2. Antitumor nanodrugs

Scientists at the University of Missouri developed a new formulation of gold nanoparticles for prostate cancer treatment in dogs [57]. This new treatment would require doses that are thousands of times smaller than chemotherapy and do not travel through the body inflicting damage to healthy areas. Researchers found remarkable results in mice, which showed a significant reduction in tumor volume through single injections of the radioactive gold nanoparticles. According to the responsible researchers, these findings have formed a solid foundation, and they hope to translate the utility of this novel nanomedicine therapy to treating human cancer patients.


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6.3. Nano vaccines

A study indicated that synthetic oligodeoxynucleotides and antigens in biodegradable nanospheres can be used as an alternative approach for immunization [58]. Controlling the synthesis process of nanoparticles it is possible to obtain particles of different sizes from the same material. The control of parameters of the particle such as size, charge and surface of the particle allows the selective targeting to specific cell groups, or even cellular regions. Thus, is possible a selective modulation of specific immune responses such as MHC class I, MHC class II , Th1 and Th2 cells [59, 60]. A better immune response seems to be obtained with biodegradable nanospheres vaccines produced by conventional methods. These new perspectives for vaccines development are contributing with better efficacy and safety results, both in pets and livestock animals.

7. Nanoparticles toxicity

The introduction of nanomaterials into clinical and other applications highlights the so-called ‘nanomaterial paradox’: the very same properties that are desirable and potentially useful from a technological or biomedical perspective, such as the ability to cross biological barriers and the high degree of surface reactivity, are also the properties that may give rise to unexpected and hazardous toxicities. It can be noted, however, that the nanomaterials paradox is not unique to nanomaterials or indeed to nanomedicine; for example the principle applies also to pharmaceuticals [61]. The growing use of nanomaterials also reveals a need for risk assessment regarding the environment and even food. As with any new technology, scientific progress must go hand-in-hand with risk assessment. Any risk assessment should include the development of new measuring methods, standardization initiatives, scientific monitoring and communication efforts [62]. Some studies have demonstrated that the use of standard toxicological screening assays may not be appropriate for all nanoparticulate systems [3]. The key issues for hazard assessment are: dose; standardization, differences in individual susceptibility, studies at cellular level, and studies at organ and system levels [61]. More active research is necessary to apply this technology to the real needs of animal health by taking into account the economic constraints of this sector, to assess their effects on the target animal species and to thoroughly evaluate the risks for the environment and consumers. Considering that pharmaceuticals and devices that contain nanomaterials, when considered for use in food-producing animals, may raise questions related to human food safety, there were created some Nanotechnology Programs in research reference institutions like Center for Veterinary Medicine (CVM) from U.S. Food and Drug Administration. As informed in the website, the goal of CVM research is “to better understand the biodistribution of various nanomaterials in food animal edible tissue, including milk and eggs. This information will help the Center to develop study protocol recommendations and to address other human food safety related issues, and provide the Center with food safety data for animal drug products that contain nanomaterials or otherwise use nanotechnology” [63]. Governmental initiatives for nanosafety reports elaboration and regulatory standardization are being also verified in several countries [9,61, 64-66,68]. In Brazil there is a highly organized research group - Rede de Nanotecnologia para o Agronegócio (Agronano) (Nanotechnology Network for Agribusiness). This scientific group, internationally recognized by FAO, is one of the most important on the binomial nanotechnology / livestock. Agronano’s researchers contribute with important safety and efficacy studies regarding the use of nanoparticles in animals and its impact on animal health, Public Health and environment [17].

8. Final comments

Nanotechnology represents an important scientific advance and can contribute with several benefits for human and animal’s health. The high variety of nanoparticles engineering has allowed the development of a great number of new antimicrobial products, with excellent efficacy results; quick and specific action, and also high bioavailability and biodegradability levels. In Veterinary Medicine, these advances impact on less production/economic losses (livestock animals) and on better health conditions for pets. However, nanoproducts’ safety is a worldwide concern, and continues studies must be conducted in order to better comprehend the nano interactions with organisms and environment.

Acknowledgements The support by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) – Process number 2012/03212-6 –; Agronano (Nanotechnology Network for Agribusiness); and by FAPEMIG (NANOBIOMG and Pronex CBB – APQ-04334-10)- is gratefully acknowledged.


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