Nanomaterial-based approaches for treatment of tympanic membrane perforations

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UNIVERSITA’ DI PISA

FACOLTA’ DI INGEGNERIA

Dipartimento di Ingegneria Civile e Industriale

Corso di Laurea Magistrale in

Materials and Nanotechnology

TESI DI LAUREA

Nanomaterial-based approaches for treatment of

tympanic membrane perforations

Relatore:

Candidato:

Serena Danti

Sara Munafò

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Aim of the work

This Master Thesis lies within the framework of the 4NanoEARDRM project, which stands for NANOfabricated NANOcomposite NANObioactive and NANOfunctional rEplacements of tympAnic membRane as advanced DRUg delivery and regenerative platforMs. As the project title reveals, it aims at synergizing different nanotechnologies for an optimal tympanic membrane restoration, including acoustic, regenerative and therapeutic cues, to ultimately achieve a durable and effective performance in implanted patients, which still represents an unmet medical need. A biomimetic tympanic membrane scaffold will be designed and fabricated, with features on the nano-to-microscale all the way to achieve its full function (regenerative, anti- infective, immunomodulatory, otocompatible and acousto-mechanic) thanks to the cooperation of academic and health experts in otology, biomaterials engineering, microbiology, nanotechnology, biofabrication, acousto-mechanics, chemistry, as well as industrial partners of the nano-biotech industry.

The new device will be “4 times” “nano”, as it will comprise nanofibers supportive for human mesenchymal stem cell (hMSC) differentiation into tympanic membrane fibroblasts, immunomodulatory nanofibrils, antibiotic-delivery nanoparticles (NPs), and will accomplish nanoscale vibration. At the same time, it will be “for” “nano”, thus meaning for enabling exploitable nanotechnologies and nanomedicine products in otologic surgery.

In this work, the tympanic membrane electrospun scaffold drug loading and the drug delivery from it are carried out. Two different parts can be highlighted.

The first part, carried out in the Institute for Technology-Inspired Regenerative Medicine (MERLN, Maastricht University), is centered on devising suitable fabrication strategies for the incorporation of PLGA-based NPs containing antibiotics within the electrospun network, which can serve as depots to locally deliver the drug over a prolonged period in

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a controlled manner after surgery. In order to reach this goal, the scaffold fabrication process parameters and the NPs concentration/type to be used have been optimized. Afterwards, the attention is put on ensuring the successful adhesion of the NPs previously mentioned to the PEOT/PBT nanofibers. This has been done by taking advantage of different approaches, including visual evaluation and release profile analysis of the fluorescent dyes loaded onto the PLGA NPs – with respect to time, temperature, and medium in which the NPs-loaded electrospun scaffold has been immersed. Moreover, the adhesion and proliferation of hMSCs on NPs-loaded electrospun scaffolds have been evaluated by seeding and comparing the growth of hMSCs on NP-loaded electrospun scaffolds with PEOT/PBT control groups.

The latter part has been carried out in the Chemical and Civil Engineering Department (DICI, University of Pisa), where the work has been focused on replicating the same analysis by developing innovative Ciprofloxacin-loaded PLGA NPs thanks to the electrospray technique. This involves a long optimization of the solvent mixture to be used, as long as the nanoparticles’ production parameters.

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1. Introduction

The human ear is a complex biomechanical system, devoted to sound perception, even if some of the hearing function is performed in the central nervous system.

Three parts are conventionally identified in the human ear: the outer, middle, and inner ear (Fig.1). The outer ear includes the auricle and external auditory canal. The middle ear is an air-filled space, which is normally sealed laterally by the tympanic membrane (TM) also called eardrum, including three small bones (the malleus, the incus, and the stapes) involved in sound conduction. The inner ear consists of the bony labyrinth, a system of passages mainly comprising two functional parts: the cochlea (a spiral-shaped organ responsible for the transduction of acoustic signals into neurological signals) and the vestibular system, devoted to balance. Sound is collected by the auricle and conveyed by the outer ear canal to the TM, which vibrates under sound pressure. The TM moves the ossicles, which transfer the vibrations to the cochlea, in the inner ear. The sensitive hair of the inner ear trigger the generation of nerve signals sent to the brain. 1, 2

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1.1 Tympanic membrane

1.1.1 Anatomy and structure

The tympanic membrane (TM), or eardrum, is a thin, semitransparent, flexible and tough membrane which forms a protective boundary between the outer ear and the middle ear. It is devoted to the transmission of the sound waves to the ear ossicles inside the middle ear, and then to the oval window in the fluid-filled cochlea.1

The TM is almost oval in shape, with a diameter about 8–10 mm, and conical in cross section, with the apex pointing medially towards the middle ear. Thus, its outer surface is slightly concave. In addition, it is placed in the ear canal with a particular orientation, which allows it to have a larger surface than the ear canal section itself. In physiological conditions, its curved conical shape has a cone angle of 132–137° with a cone depth included in the range 1.42–2 mm. The angle between the eardrum and the superior and posterior wall of the ear canal is 140°, while the angle between the eardrum and the inferior and anterior wall is 30° (Fig. 2).3

Figure 2. Section of the TM.

In the through-thickness section of the TM, three distinct layers can be identified, which vary in density, thickness, composition and arrangement, thus resulting in a tri-laminar tissue. Starting from the lateral side, three distinct layers can be identified: an outer epidermal layer continuous with skin of the external auditory meatus, a middle fibrous layer and an inner mucosal epithelial layer continuous with the lining of the tympanic cavity (Fig. 3).

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Figure 3. A schematic diagram of a typical TM cross-section.

The thin epidermal layer is a typical keratinizing epithelium composed of four strata, made of different cell types. The mucosal layer is the continuation of the mucosa of the middle ear cavity and it is a very thin layer of cells with presumably no effect on the mechanical properties of the eardrum. The fibrous layer consists of collagen fibrils arranged as outer radial and inner circular layers, with predominantly type III collagen in the inner layer and type II collagen in the outer layer.1

Macroscopic observation reveals that the periphery of the TM is firmly anchored to the wall of the tympanic cavity, around most of its circumference, by a fibrocartilaginous ring called the annular ligament (AL). This ligament is a fibrous thickening firmly attached to a sulcus in the bony tympanic ring, except superiorly where it separates the two main regions of the TM called the ‘‘pars tensa’’ (PT) and the ‘‘pars flaccida’’ (PF). The differences in the pars tensa and pars flaccida lie in the structure of their lamina propria. The PT, where the majority of perforations occur, is the inferior part and is also the most extended part; its lamina propria consists of two subepidermal connective tissue layers, between which there are two collagenous layers with radial (outer) and circular (inner) fiber orientation. The radial fibers become more packed as they converge on the manubrium, while the circular fiber layer grows thicker towards the periphery. These collagen fibers exhibit a viscoelastic and orthotropic behavior with membrane (or in- plane) properties different from through-thickness (or out-of- plane) properties.4_{Parabolic and transversal fibers have been identified in the PT,}

too5_{; they arise from near the lateral process of the malleus and extend downwards}

in a parabolic course to the anterior or posterior quadrant. The transversal fibers are short fibers running horizontally in the inferior quadrant. The PF is the

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superior part of the TM: its lamina propria is made up with loose connective tissue containing collagen and elastic fibers. The abundance of elastic fibers may account for its flaccid nature.3

It is clear that such a complex collagenous network reflects and is essential for the correct vibratory function of TM.

Figure 4. Schematic representation of TM (on the left) and of the TM fiber orientation.

1.1.2. Diseases and current treatments

The human tympanic membrane has a key role for hearing. For this reasons, changes in structure and mechanical properties of the TM due to middle ear diseases can deteriorate sound transmission and cause conductive hearing loss.6

Between all the middle ear diseases encountered by otolaryngologists, tympanic membrane perforations (TMPs), or holes in the eardrum (Fig. 5), are prevalent. Although the overall incidence is unknown, 7_{various specific populations have a}

higher prevalence; for example, a global survey has shown Australian aboriginals to have the highest prevalence of perforation ranging from 28% to 34%.8

Furthermore, it has been estimated that up to 40% of Australian indigenous children will have a perforated TM by 18 months of age, with cases reported as early as the first 6 weeks of life. 9, 10

While about the 80% of acute perforations heal spontaneously, perforations which fail to heal within 3 months are termed chronic.11_{Chronic TM perforations}

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can lead to significant morbidities such as hearing loss12_{, chronic ear discharge,}

recurrent middle ear infection (e.g., otitis media [OM]),11_{cholesteatoma.}13

Chronic TM perforations that occur in association with chronic suppurative OM may lead to significant intra- and extracranial complications, with mortality rates in this group of up to 18.6%.14

The World Health Organization estimated that the global burden of illness from chronic suppurative OM involves 65–330 million individuals, and accounts for 28,000 deaths and a disease burden of over 2 million disability-adjusted life years.15

a) b)

Figure 5. An otoscopic photo of a normal intact left TM (a), which is semitransparent, with external auditory canal (EAC) at the margins; a TM with medium-sized perforation (arrow

heads) (b).

Currently, the majority of patients with chronic TMPs require a surgical procedure known as myringoplasty or tympanoplasty to seal the perforation, with several approaches to incisions and techniques. Myringoplasty typically involves a general anesthesia, followed by surgery for donor tissue collection and grafting, together with their associated risks and costs. Myringoplasty can also lead to surgical complications such as blunting or lateralization of the TM, retraction pockets, squamous cysts, and cholesteatoma, which may require further interventions and long-term follow-up. Irrespective of the surgical technique used, normal conductive hearing is only achieved in 37%–84% of cases,16, 17

suggesting that current methods of myringoplasty do not always fulfill the aim of improved hearing. Moreover, today the most commonly used grafts, which serve as a scaffold to guide the migration of keratinocytes across the wound gap, are

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autologous and may include temporalis fascia (gold standard), cartilage, fat, and perichondrium. Despite the availability of these different graft materials, each has its own limitations. An autologous graft may be associated with donor-site morbidity, lack of graft material in revision cases, and longer operative times. On the other hand, the use of allografts and xenografts may carry the potential risk of infection transmission, and the use of synthetic materials requires further research before they can become viable alternatives. 18 More importantly, all these grafts

are unable to replicate the complex microanatomy and vibroacoustic properties of a native TM.19

It is important to underline that the healing of the TM is so difficult because big differences exist between TM wound healing compared to other parts of the body. Firstly, when other tissues heal in response to injury, they undergo sequential phases of hemostasis, inflammation, cellular proliferation, and finally cellular migration. In TM wound healing, the first two stages remain, but migration precedes proliferation.8_{In addition, during normal tissue healing, granulation}

tissue usually develops and serves as a platform for re-epithelialization. In contrast, in TM perforations, the squamous epithelium bridges across first, followed by the remainder of the epithelial layer, and concludes with the fibrous layer.20_{Occasionally, failure of the fibrous layer to migrate leaves a dimeric}

neo-membrane consisting of an epithelial and mucosal layer with few disorganized fibrils in between, resulting in a flaccid TM with poor sound conduction. Secondly, proliferation is shown to be greatest at a site distal from the injury. These TM regeneration centers have been identified to be in the annulus and handle of the malleus. 21_{Thirdly, the wound edge of TM perforations is suspended}

in air, hence, no underlying tissue matrix is present to support the regenerating epithelium and migrating vessels, which by necessity must then derive from the surrounding TM lamina propria.22

To overcome all these issues related with traditional therapies, a new approach to treat TMPs is needed.

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1.2 Tissue engineering of the tympanic membrane

Regenerative medicine and tissue engineering techniques, which are one of the major components of regenerative medicine, have been applied in both the laboratory and clinical settings to enhance the healing of TM perforations and to potentially replace autologous grafts in human patients. 22-24

Tissue engineering (TE), as viewed today, is ‘an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ’.25_{Most frequently, the ultimate goal is implantation of these tissue}

constructs into the body to repair an injury or replace the function of a failing organ. The major advantage of TE approach is that tissues can be reconstructed to closely match the needs of the patient’s requirements and can be transplanted into the patient’s body with a minimal surgical intervention, which eventually conquers several limitations encountered in the traditional tissue transplantation approaches.26_{It also reduces the problems faced with traditional donor organ}

transplantation, such as poor biocompatibility and bio-functionality, and immune rejection.27

Two main approaches are utilized in this area to produce engineered tissue. First, scaffolding materials can be used as a cell support device upon which cells are seeded in vitro; cells are then encouraged to lay down matrix to produce the foundations of a tissue for transplantation. The second approach involves using the scaffold as a growth factor/ drug delivery device. This strategy involves the scaffold being combined with bioactive molecules (BMs), such as growth factors or materials with particular properties, so upon implantation cells from the body are recruited to the scaffold site and form tissue upon and throughout the matrices. These two approaches are not mutually exclusive and can be easily combined. The manor in which a cell type and scaffolding are combined should be carefully matched for purpose as it has been demonstrated that composition, topography and architecture of scaffolds are able to interact and influence cell behavior.7

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In this regard, there are three key essential elements to be considered for the success of tissue development: (i) the cells that create tissue, (ii) the scaffold that gives structural support to cells, and (iii) BMs that can impregnate the scaffold (Fig. 6).28

Figure 6. Key factors contributing in the success of TM tissue engineering.

1.2.1 Cells

Cells are the prime determinant factor for the success of TE, because they are the basic units of living organisms. There are different types of cells, which are commonly categorized as stem (unspecialized) and non-stem (specialized) cells, that could be used for TE. Although the use of non-stem cells for tissue engineering dates far back, they are generally differentiated cells, which give rise to limited proliferative life span as compared to stem cells. This is because stem cells are specialized to divide but not differentiate, whereas other cell types are committed to form specific types of cells. In other words, by definition, stem cells are immature or undifferentiated cells that are able to renew by themselves and differentiate into more specialized, tissue-/organ-specific cells. This ability allows them to act as a good repair system for the defective tissues or organs of our body. Stem cells are generally classified into three types: totipotent (e.g., zygote), which are capable of producing any type of cells/tissues; pluripotent (e.g., embryonic stem cells), are capable of producing most of the cells/tissues, whilst multipotent (e.g., mesenchymal stem cells) produce a limited number of cells/tissues with specific functions. Stem cells are also categorized into embryonic or adult cells according to their source of origination.29 The second ones are typically harvested

from bone marrow, which is chiefly constituted of two distinct stem cells: (i) hematopoietic stem cells (HSCs), which are responsible for the formation of blood cells, and (ii) mesenchymal stem cells, also named mesenchymal stromal cells (MSCs), which can differentiate into a variety of cell types, including osteoblasts (bone cells), chondrocytes (cartilage

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cells), myocytes (muscle cells) and adipocytes (fat cells which give rise to marrow adipose tissue).

Figure 7. A schematic mesengenic process, showing stepwise cellular transitions from the putative mesenchymal stem cell to higly differentiated phenotypes.

In this work, human MSCs (hMSCs) from the bone marrow have been selected as a suitable cellular model to test the TM scaffolds. hMSCs are adult undifferentiated immature cells with fibroblast-like morphology and biological characteristics, originating from the mesoderm and capable of self-renewal and pluripotency.30, 31

The application of these cells for the treatment of TM perforations was first proposed by Rahman et al and showed good results in a rat model.32_Regenerating

a bioengineered TM with hMSCs is also legitimated by developmental biology.33

From an embryologic point of view, TM has a triple origin that includes endodermal, mesodermal and ectodermal components.34_{The fibrous layer}

originates from the branchial arch mesenchyme, an embryonic connective tissue derived from the mesoderm, which explains the extensive presence of collagen type II in the pars tensa as a product of TM fibroblasts.

Moreover, the potential advantage of using adult stem cells is that the patient’s own cells could be easily isolated, expanded in culture, and then transplanted into

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the patient’s body where tissue regeneration is required. By this approach, there is no (or very minimal) chance of immune rejection.35-37

Although the stem cells are believed to be the key factor for the future of TE, one of the major challenges associated with the use of these cells is to provide appropriate cellular environment cues that regulate cell growth and subsequent tissue formation in a controlled and efficient manner. As certain cells are anchorage dependent, they would not survive if delivered without a suitable scaffold. Engineering a perfect scaffold with all the qualities of natural extra-cellular matrix is therefore of great importance and a critical prerequisite for the characteristic growth of cells and subsequent tissue functions.

1.2.2 Scaffold

Scaffold is another important determinant factor for the success of TE and its success typically depends on the characteristics of the starting material and the fabrication methodology.

The scaffold, by definition, is a temporary supporting structure for growing cells and tissues. It plays a critical role in supporting the cells to accommodate, and thus it is also called synthetic extra-cellular matrix (ECM), which is the complex nano-featured environment in which cells live and grow. These cells then undergo proliferation, migration, and differentiation in three dimensions, which eventually leads to the formation of a specific tissue with appropriate functions as would be found in the human body. To facilitate these measures, the scaffold should possess a few basic characteristics (Table 1).

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Table 1. Basic characteristics of a perfect tissue scaffold.

An ideal scaffold for TE should possess all the qualities of a native ECM and should function in the same way as that of ECM under physiological conditions. Therefore, the characteristics of a scaffold vary according to the tissue types where the scaffold is to be applied.

However, irrespective of applications, any scaffold should be biocompatible; that is, it should not provoke any rejection, inflammation, and immune responses. It should provide a 3D template for the cells to attach and to guide their growth. It should have a porous architecture with a high surface area for the maximum loading of cells, cell-surface (scaffold) interaction, tissue in-growth, and transportation of nutrients and oxygen. It is worth pointing out that most TE applications require scaffolds that are biodegradable. It is desirable if the degradation rate of the scaffolds matches the rate of tissue regeneration but, in general, not faster, because that faster rate may lead to a decrease in tissue functionality. Degradation products, if produced, should be removed from the body via metabolic pathways at an adequate rate that keeps the concentration of these degradation products in the tissue as a tolerable level.38_{It should be}

mechanically strong to withstand in vivo biological forces,39_{and it should support}

the cells to synthesize specific proteins and other biochemical and biological processes required for a healthy tissue growth. Besides, it should be sterilizable to avoid toxic contaminations without compromising any structural and other related properties. Finally, the production process of scaffold with all the above

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unique characteristics must be accomplished in a reproducible, economical, and scalable manner.26, 40

1.2.2.1 Biomaterials for scaffolds

Generally, three classes of biomaterials have been utilized for engineering tissues: naturally derived materials (e.g., collagen, chitin, alginate), acellular tissue matrices (e.g., bladder submucosa and small intestinal submucosa), and synthetic polymers. The choice of materials depends on the type of tissue to be reconstructed. Naturally derived materials and acellular tissue matrices have the potential advantage of biological recognition and low toxicity, but depending on the source they can have impurities that can evoke undesirable immune responses.41_{However, synthetic polymers can be produced reproducibly on a}

large scale with controlled properties of their strength, degradation rate, and microstructure; moreover, these polymers have gained Food and Drug Administration (FDA) approval for human use in a variety of applications. Since the main requirement of a TM scaffold is to act as a support for the neo-formation of the collagenous network and to emulate the TM anatomic and histologic features, in order to find the best compromise between the final purpose of our scaffold and the techniques of production to be used, a commercial random block copolymer of poly(ethylene oxide terephthalate) and poly(butylene terephthalate) (PEOT/PBT), whose commercial name is PolyActiveÔ (PA), has been selected.

This segmented copolymer is constituted by two segments with very different characteristics: the hard and hydrophobic segment PBT, and the soft and hydrophilic segment PEOT. At high temperatures, the segments are well mixed, as the disordering Brownian effects prevail over the intersegmental interactions. As the temperature is lowered, PBT and PEOT recognize their molecular incompatibility, which results in the formation of microphase-separated domains of soft and hard segments. The typical morphology is made of amorphous or semi-crystalline hard domains dispersed into a soft matrix. By varying the amount and

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the length of these two different blocks, mechanical, biological and physicochemical properties can be tailored.

Figure 8. Chemical structure of PEOT/PBT segmented block copolymers.

As a polymer suitable for bioscaffolds, PA must possess some obvious requirements. Indeed, it is biocompatible, biodegradable and bioresorbable; thus, it is clinically applied and approved for many biomedical applications including the TM regeneration. Above all, it is able to host a bioinstructive system, that is, the chemical and physical microenvironment required for cell viability, differentiation and growth for the TM restoration.

Apart from the necessary biorelated properties, the copolymer is a thermoplastic elastomer which exhibits a low melt viscosity, to allow for an easy injectability and filament production. Furthermore, its solidification kinetics compromises between two simultaneous requirements: on the one hand, the necessity to quickly impart a sufficient consistency to allow for the scaffold to retain its shape and dimension. On the other hand, the somewhat opposite need to keep a liquid-like behavior for a sufficient time, in order to guarantee an optimal healing between two consecutive filament layers. The material rheological behavior in the melt state and during its phase transition stages, therefore, is crucial in determining the best manufacturing conditions. 42

1.2.2.2 Scaffolds biofabrication

In relation to the peculiar anatomic and histologic features of the human TM, some scaffolding techniques show theoretical advantages that can be exploited for whole TM regeneration via TE.

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Although the multiple methods have been employed to processing TM nano-fibrous scaffolds, they often encountered some difficulties, especially in controlling the fiber orientation of the scaffolds. For the success of TE, it is desirable to control not only the fiber diameter but also the fiber orientation of scaffolds. In this work, a multiscale approach based on electrospinning and additive manufacturing was investigated to fabricate scaffolds resembling the anatomic features and collagen fibers arrangement of the human TM.

1.2.2.2.1 Electrospinning

Electrospinning (ES) is one of the most popular and exploited techniques to process nano-fibrous thin scaffolds that mimic the structural features of native ECM using a variety of biomaterials, in particular polymeric materials. Indeed, ES is simple, cost-effective and allows the production of polymeric ultrafine fiber structures with spatial orientation, resembling the fibrous elements of the ECM. The high surface area to volume ratio and interconnected porous architecture are typical advantages of electrospun meshes, as they provide great many sites for cell adhesion.43_{The possibility of producing electrospun meshes able to induce}

cell alignment is one of the advantages of tuning fiber orientation.44_{This because}

preliminary investigations have suggested that the cells interact well with these scaffolds and the cells grow preferentially in the direction of the fiber orientation. ES essentially employs electrostatic forces to produce polymer fibers, ranging in diameter from a few microns down to tens of nanometers (40-200 nm). The basic configuration of ES is schematically illustrated in Figure 9, which consists of three major components: (i) a spinneret, (ii) a fiber collector, and (iii) a high-voltage power system. As shown in the figure, the spinneret is directly connected to a syringe, which acts as a reservoir for the polymer solution to be electrospun. This polymer solution can be fed through the spinneret with the help of a syringe pump at a steady and controllable feed rate. The feeding rate can be controlled correspondingly to the concentration of polymer solution. It is worth pointing out that the material to be spun by this technique must be viscous, but the viscosity may vary depending on the type of materials used. The fiber-collecting device is

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positioned right below the spinneret, with an appropriate gap (usually a few centimeters). A high-voltage/low-current power system is required for the conversion of polymer solution to a charged polymer jet. The electric voltage (in a range of kilovolts) is applied across the spinneret and the grounded metallic counter electrode (fiber collector) to facilitate the charged jet to eject from the spinneret tip toward the surface of the fiber collector.

Figure 9. A schematic of electrospinning system.

Although the experimental design and functional components of ES seem to be extremely simple, mechanism involved in the spinning of polymer nanofibers is rather complicated. Upon applying an optimized electric potential to the spinneret, a pendent droplet of the polymer solution at the tip of the spinneret gets electrified; thereby, inducing charge accumulation on the surface of the droplet subsequently allows the droplet to deform into a cone shape, known as Taylor’s cone.43 This

deformation is commonly caused by two electrostatic forces: (i) electrostatic repulsion between the surface charges of the droplet, and (ii) Coulombic force exerted by the strong external electric field applied.44_{Once the applied electric}

field surpasses a critical value (threshold), the electrostatic force tends to exceed the viscoelastic force and the surface tension of the polymer droplet; thereby, a fine charged polymer jet is forced to eject from the tip of the Taylor cone. Initially, the polymer jet travels toward the grounded collecting plate; however, while in transit, the different polymer strands in the jet get separated out due to the mutual

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repulsion, a phenomenon called ‘‘splaying,’’45 which gives rise to a series of

ultra-fine dry fibers. The distance between the spinneret and the collecting substrate is where the solvent within the ejected jet stream evaporates, resulting in a collection of non-woven small micron or submicron-sized fibers that form a highly porous scaffold on the grounded metallic collector. It is worth mentioning that different types of fiber collectors are available corresponding to the fiber’s spatial orientations (aligned and random). In this work, a rotatable cylinder collector has been used to obtain aligned nanofibers. The alignment of the fibers is rather complicated and numerous parameters influence the fiber orientation, which are generally divided into three groups: a) solution parameters (polymer solution concentration, polymer’s molecular weight, surface tension, conductivity, solvent dielectric constant); b) processing parameters (flow rate, applied electrical voltage, distance between the nozzle and the collector, rotational speed and shape of the collector; and c) ambient parameters, such as relative humidity and temperature. The effect of manipulating some of these key parameters and corresponding changes in the morphological appearances and sizes of electrospun polymer fibrous structures are depicted in Table 2.46 However, it is hard to

determine the effect of many of the processing parameters on the resultant fibrous assembly as they are interrelated.47, 48

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1.2.2.2.2 Co-electrospinning/electrospray

In addition to the ability and ease to form nanofibers, other significant advantages of electrospinning include high adaptability and ease for modification. Indeed, the function of the electrospun scaffolds can be modulated through the use of several innovative electrospinning techniques that permit the fabrication of nanofibrous multilayered electrospun products to meet specific requirements of different biomedical applications.49, 50

In this work, simultaneous co-electrospinning/electrospray (co-E/E) has been proposed for the deposition of biodegradable polymeric smart nanoparticles (NPs) containing antibiotics within biocompatible beaded fibers, so as to produce nanofibrous scaffolds embedded with homogeneously distributed electrosprayed NPs to ensure local and controlled delivery after surgery.

Electrospinning and electrospray are facile techniques governed by similar principles, which use electrostatic forces to overcome the surface tension of charged liquids, and which usually use identical apparatus. Compared to electrospinning, the degree of electrostatic stretch over the surface tension is relatively low during electrospray, leading to the formation of particulate products (nanoparticles or microparticles) instead of fibrous products. Consequently, a polymer solution with a relatively low polymer concentration (and hence low viscosity) is normally used in electrospray.51

Through concurrent electrospinning and electrospray using a rotating mandrel and two different capillaries through which the respective polymer solutions are fed, whose configuration is illustrated in Figure 10, poly(lactic-co-glycolic acid) (PLGA) NPs antibiotic-encapsulated nanospheres are embedded in nanofibrous PA scaffolds. This combination of electro-hydrodynamic techniques gives rise to a higher extent of exposed NPs, thus increasing the surface-dependent antibacterial properties of nanocomposite fibers.52

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Figure 10. Configuration of the co-electrospinning/electrospray setup.

Controlling the biodegradation of nanospheres enables controlled release of the drug from the scaffold, making it multifunctional for the TM membrane restoration. While the nanofibrous scaffold promotes local tissue regeneration, the released PLGA NP-based antibiotics perform anti-inflammatory properties after surgery. Such a comprehensive treatment holds great promise for postoperative TM pathologies patients.

1.2.2.2.3 Additive Manufacturing

In addition to this, additive manufacturing (AM) techniques allow customized forms to be produced via computer-assisted control also used for the fabrication of scaffolds in a number of TE applications.53

AM techniques, also known as solid freeform fabrication (SFF) techniques, are based on the construction of 3D objects built layer-by-layer. Basically, 3D models used in AM are generally treated or designed using Computer Aided Design (CAD) and commuted-aided manufacturing (CAM) software. After data treatment, from the major part of the computer-aided design CAD software, the information is generally converted to a stereolitography-file (STL-file) containing the information of the surface geometry of a 3D object. This neutral file is composed of different information regarding the 3D model.

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AM techniques may be divided into four categories: i) Stereolitography (SLA); ii) Selective Laser Sintering (SLS); iii) Three-Dimensional Printing (3DP); and iv) Fused Deposition Modeling (FDM). Although its application is limited to polymers that can be process in melt phase, FDM has already shown the potentialities of producing scaffolds for TE. Coined by Scott Crump in 1992, it fabricates 3D scaffolds by melting and extruding material through a moveable nozzle with a small orifice onto a substrate platform. The filament material is fed through two rotating rollers into the extruder head, where the material can be melted. The nozzle moves in the x and y directions so that the filament is deposited on a parallel series of material roads to form a material layer, and subsequently the build platform in the z direction is lowered to build the new layer on the top of the first one. After the extruded material cools, solidifying itself and bonding to the previous layer, a 3D structure is yielded.

Figure 11. Schematic representation of the FDM system.

The advantages of this technique include its low cost, the lack of use of organic solvent, the ability to form a fully interconnected pore network in complex 3D architecture, and rare or no requirement of cleaning up the finished objects. Even though there are also inherent limitations (raw material selection, the effect of high temperatures on raw material, and the lack of adequate resolution),53_{it is exactly the}

combination of FDM and ES that has interestingly been proposed in this work to obtain the manufacturing of multiscale scaffolds with improved mechanical and biological properties.54

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Ideally, the replication of the TM collagen fibers could only be achieved producing different layers of electrospun fibers, so as to resemble the different orientations of the natural ECM (i.e., radial on one side and circular on the other side). Despite the several advantages shown by ES, the necessity of minimal mechanical stability for handling and application on the affected site is a key requirement for a tissue-engineered TM.55

Thus, through the production of multiscale advanced scaffolds to obtain biomimetic substrates for a tissue engineered TM has been achieved thanks to the use of conventional ES onto FDM previous obtained substrates, in which radial and circular nanofibers patterns alternate.

1.2.3 Bioactive molecules

In the traditional design of biomaterials, bio-inert substrates are favored because of the reduced induction of immune responses. With advances in the understanding of cell-biomaterial interactions, the current design of biomaterial scaffolds has focused more on bioactive material systems. Bioactive systems can be established through the development of delivery systems for bioactive molecules, or the fabrication of bioactive scaffolds by blending those substances with different polymers.54_{From the}

moment that ear infections and related inflammatory processes greatly challenge the success rate of TM substitutes, nanosized chitin (CN) is a candidate of choice to be added to the biodegradable polymer in order to obtain anisotropic TM scaffolds with immunomodulatory and antibacterial properties.

CN is a green abundant nanocomponent approved in dermatology and cosmetics, that is high biocompatible with living tissues, biodegradable and non-toxic.55

Large amounts of this structural material can be found in animals, in exoskeleton shells of arthropods (crabs, shrimps and beetles), internal flexible backbone of cephalopods, worms, webs of spiders, cell walls of fungi and yeasts56, 57_{, from which}

it can be extracted with a variety of methods like acid hydrolysis,58-60_,

TEMPO-mediated oxidation,61_{ultrasonication}62_{and gelation,}63_{followed by an extensive}

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From a chemical point of view, chitin is a high molecular weight linear polysaccharide consisting of copolymer repeated units of N-acetyl-D-glucosamine and D-glucosamine, where the D-glucosamine content is dependent on the degree of deacetylation (DDA). This structure with two hydroxyl groups and an acetamide group makes chitin very crystalline with strong hydrogen bonding.64_{In fact, this}

semicrystalline biopolymer forms microfibrillar arrangements in living organisms that are tightly bonded to each other through a large number of hydrogen bonds. This percolating network based on hydrogen bonding forces is maintained also after its extraction, leading to a cooperative alignment of the fibrils and, as a consequence, to a reinforcement of the whole nanocomposite.65

Figure 12. Chitin structure.

Most importantly, CN exerts immunomodulatory, anti-inflammatory and antibacterial properties thanks to its cationic nature which originates from the protonated amino groups (-NH2) on the backbone of the molecule. The degree of protonation is a function of solution pH and the DDA, which can be controlled. This is an important feature that allows chitin also to recruit and bind negatively-charged molecules or NPs, protecting them from degradation and increasing local concentration efficacy.66, 67_{This physical entrapment can generate biologically active}

scaffolds.

1.3 Controlled release strategies in TM tissue

engineering

Although a lot of efforts have been done in order to optimize the previously mentioned nanofibrous electrospun composite scaffold, further adjustments were needed in order to further improve the final device for an optimal TM restoration. To

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have a durable and effective performance in implanted patients, immunomodulatory CN are not enough: drugs like antibiotics need to be incorporated in the device. In order to ensure local and controlled delivery of antibiotics after surgery, biodegradable polymer smart NPs need to be deposited in the nanofibrous composite mesh. The reasons behind this choice are presented here in detail.

1.3.1 Drug Delivery Systems (DDS): principles and

evolution

Drug delivery refers to approaches, formulations, technologies, and systems that enables the introduction of a therapeutic substance in the body and improves its efficacy and safety by controlling the rate, time, and place of release of drugs in the body.

The choice of the drug delivery method in the body can have a very important effect on the efficiency of the applied drug. Every drug molecule needs a delivery system to carry the drug to the site of action upon administration to the patient. Traditionally, delivery of the drugs can be achieved using various types of dosage forms including tablets, capsules, creams, ointments, liquids, aerosols, injections, and suppositories.

Figure 13. Traditional drug delivery systems.

Most of these conventional DDS are known to provide immediate release of the drug with little or no control over delivery rate.

By looking at the pharmacokinetic (PK) curve, that is a plot of drug concentration in the blood (plasma) versus time after delivery of the drugs, it is clear that to achieve and

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maintain therapeutically effective plasma concentrations, several doses are needed daily, which may cause significant fluctuations in plasma levels (Figure 14).

Figure 14. PK curves of plasma concentration of a drug versus time for two types of drug delivery systems: typical bolus PK for multiple dosing with oral tablets or injections, and "zero order" PK for one dose of controlleg delivery

from a specific formulation.

Because of these fluctuations in drug plasma levels, the drug level could fall below the minimum effective concentration (MEC) or exceed the minimum toxic concentration (MTC). Such fluctuations result in unwanted side effects or lack of intended therapeutic benefit to the patient.

Fortunately, 1960s led to the birth of the field of “controlled drug delivery” (CDD), which allows to control the level of the released drug inside the body. The major advantage of developing systems that release drugs in a controlled manner can be appreciated by examining again the PK curve. It is clear that controlled-released formulations can maintain a desired blood plasma level within the therapeutics index for long periods of time. These DDS are called “zero order systems”, since they release drug at a constant rate.

As a consequence, patient compliance is improved, and a short treatment period/less frequency of dosage can be achieved thanks to the maximum utilization of the drug. Moreover, despite the higher manufacturing costs, the drug bioavailability and the drug fraction accumulated in the required zone increase.

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Even though all the first DDS were macro-scale devices that could be held in the hand, it was in the 1980s, when scientists realized that the properties of DDS are size-dependent, that the interest and activity in nanoscale carriers rapidly grew. It was already known that these devices have a lot of advantages with respect to the other devices, and this is the reason why nanotechnology applied to regenerative medicine spreads rapidly.

1.3.2 Nanoparticles as drug nanocarriers

Nanoparticles (NPs) are solid colloidal particles with dimensions usually between 10 and 200 nm, which offer great versatility in terms of shapes, surface chemistry, and components.

The use of NPs in drug delivery brings many advantages with respect to other DDS: i) increased drug concentration at the desired site of action, called “drug targeting”, ii) appropriate release rate, iii) reduction in toxicity while maintaining therapeutics effects, iv) greater safety and biocompatibility, v) minimal loss of drug, vi) protection of unstable compounds against degradation, vii) drug administration in inaccessible regions of the body.

These nanodevices can be manufactured with different materials like metals, ceramics and natural/synthetic polymers, depending on the final application. For our purpose, the most attractive NPs type are biodegradable polymeric NPs.

1.3.2.1 Polymeric nanoparticles

Polymeric nanoparticles (PNPs) have attracted considerable interest over the last few years due to their unique properties and behavior resulting from their small size.68_As

asserted by different authors, these nanoparticulate materials show potential for a wide range of applications such as diagnostics and drug delivery.69, 70_{Advantages of PNPs as}

carriers include controlled release, the ability to combine both, therapy an imaging (theranostics), protection of drug molecules and its specific targeting, facilitating improvements in the therapeutic index.71, 72

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The technology of polymeric drug delivery relies heavily in the biodegradability and biocompatibility of the polymers. Biodegradable polymers have advantages since they are completely eliminated from the body by natural metabolic pathways.73_Natural

polymers are usually biocompatible and biodegradable; however their use has been limited due to batch-to-batch variations in properties and could be mildly immunogenic. On the other hand, synthetic polymers are well-known for their controlled chemical composition. Several synthetic or natural polymers have been used for the preparation of PNPs, such as proteins, sugars or other natural macromolecules, biodegradable polymers and non-biodegradable, but pharmaceutically acceptable polymers.74, 75

To date, many biodegradable polymers approved from the Food and Drug Administration (FDA) for therapeutics devices are used for the controlled release of drugs, but among them poly lactic-co-glycolic acid (PLGA) and polycaprolactone (PCL) play a pivotal role both in general and in the TM TE.

PLGA is a synthetic, biocompatible and biodegradable copolymer of polylactic acid (PLA) and polyglycolic acid (PGA). Depending on the ratio of lactic to glycolide used for the polymerization, different forms of PLGA can be obtained, and this reflects on the final properties and on the degradation time of the NPs. Indeed, the higher the content of glycolide units, the lower the time required for degradation as compared to predominantly lactide materials. In addition, polymers that are end-capped with esters (as opposed to the free carboxylic acid) demonstrate longer degradation lives. This flexibility in degradation has made it convenient for the fabrication of NPs.

Figure 15. Structure of poly(lactic-co-glycolic acid). x=number of units of lactic acid; y=number of units of glycolic acid.

However, PLGA degrades by hydrolysis of its ester linkages producing its original monomers in the body, that under normal physiological conditions are by-products of various metabolic pathways in the body. Consequently, they can be excreted easily with minimal systemic toxicity. However, it has been reported that the PLGA degradation

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reduces the local pH low enough to create an autocatalytic environment, and this could be one disadvantage when this biopolymer is used.

On the other hand, PCL is a semi-crystalline polymer composed of hexanoate repeat units, included in the class of aliphatic polyesters. PCL has been thoroughly investigated for its peculiar physical, thermal, and mechanical properties; however, the superior rheological and viscoelastic properties over many of its aliphatic polyester counterparts render PCL easy to manufacture and manipulate into a large range of biodegradable devices.

Figure 16. Structure of polycaprolactone.

PCL is strongly hydrophobic, highly soluble at room temperature, and easily processable due to the low melting temperature (Tm = 60ºC). Owing to its native biocompatibility and biodegradability, PCL has been extensively studied for the preparation of controlled drug delivery systems.76_{Moreover, its compatibility with a wide range of drugs and its}

high encapsulation efficiency enabled uniform drug distribution in the matrix, assuring a long-term release—up to several months—by a degradation mechanism. The advantages of PCL include its high permeability to small drug molecules, and its negligible tendency to generate an acidic environment during degradation as compared to other polyesters such as PLA and polyglycolic acid (PGAs).

Degradation times of PCL depend on its molecular weight, crystallinity degree, and morphology.77_{Faster degradation was observed in the amorphous phases. Nevertheless,}

the alteration of the PCL degradation pattern has been for years an important research field, with the objective of modulating the biodegradation kinetics of the polymer.

The active principle, whatever it may be, can be loaded either by adsorption, dispersion within the PLGA/PCL-NPs matrix, or encapsulation. In this light, an obvious distinction can be drawn between nanospheres and nanocapsules. Nanospheres are massive colloidal particles (whose shape is not necessarily spherical) that can adsorb drug molecules on the

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particle surface or confine them within the particle matrix both by physical entrapment or chemical bonding. Nanocapsules can instead be seen as vesicular systems made up of a polymer shell surrounding a core cavity which typically contains either an aqueous or an oily core where the nanoparticle payload can be dissolved.

Figure 17. Two different NPs formulation: nanocapsule (left) and nanosphere (right).

1.3.3 Nanoparticles preparation techniques

To self-assemble these materials into PNPs, several preparation techniques has been successfully developed. The choice of a specific method plays a vital role in order to obtain PNPs with the desired properties for a particular application. Indeed, it is usually determined by the type of polymer, the drug's physicochemical properties and the final desired characteristics of the PNPs.78_{Nevertheless, all these methods share a common}

step which is polymer precipitation. This can occur either through addition of a non-solvent or after a decrease of polymer solubility. Effective post-synthesis purification of NPs is also an important step for controlling their quality and characteristics and therefore their suitability for a biomedical application. Filtration, centrifugation and dialysis techniques are commonly used as purification methods.79

NPs preparation methods can be divided into two groups, namely, those based on the polymerization of monomers and those taking advantage of preformed polymers (Figure 18). These methods can be further classified into two categories: two- step procedures involving the preparation of an emulsification system followed by formation of NPs in the second step of the process, and one-step procedures where emulsification is not required for the formation of NPs.

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Figure 18. Schematic representation of numerous techniques for the PNPs preparation.

For the polymerization methods, the monomers are polymerized to form the encapsulating polymer. This process can be carried out in two ways, either using emulsion polymerization techniques or interfacial polymerization.74_{Some drawbacks have been}

reported which have limited the use of polymerization methods for the synthesis of PNPs. Not only are most PNPs formed from slowly biodegradable or nonbiodegradable monomers, but also non-biocompatible byproducts may be generated with these methods. Toxic residues such as monomers and initiators may persist which require extensive purification work to result in a pharmaceutically acceptable product. Considering the limitations of polymerization techniques and the final goal of this project, attention is focused on describing and using the methods involving preformed polymers, as many of the problems involved in the former method can be avoided. In particular, three methods have been exploited and compared to produce biodegradable smart NPs: emulsification-solvent evaporation and nanoprecipitation.

1.3.3.1 Emulsification-solvent evaporation

The term emulsion is defined as a mixing of one liquid phase into another totally or partially immiscible, through the use of amphiphilic surface-active molecules (surfactants) that reduce the interfacial tension between the two liquids in order to achieve stability.

Emulsions can also be classified based on their composition (oil, water, surfactants) or morphology. Generally, emulsions may be of the oil-in-water (O/W) or water-in-oil (W/O) types depending on whether the oil is dispersed as droplets in water, or vice versa.

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In addition, more complex systems such as (water-in-oil)-in-water (W/O/W) can also be obtained. Depending on the droplet size, the emulsion formed can be classified into three main categories: nanoemulsions, miniemulsions and macroemulsions (Figure 18). A common mistake reported in the scientific literature is to describe nanoemulsions as “microemulsions”, but the latter is a thermodynamic phase and not an emulsion system.

80

Figure 19. Schematic diagram of emulsions fabricated from water, oil, and surfactants.

In two-step emulsification/solvent removal methods the polymer organic solution is emulsified in an aqueous phase. Low- and high-energy emulsification techniques can be used to produce nanodroplets and consequently nanoparticles. In emulsion method, the droplet formation step is fundamental because it determines the size distribution of the resulting PNPs. Polymer precipitation on preformed nanodroplets is achieved by removing the organic solvent by different methods such as solvent evaporation, fast diffusion after dilution or salting out. A similarity between these techniques is the drug encapsulation process in which the drug is generally added in the polymer solution. Solvent evaporation has been chosen in this work (Figure 20). It was the first method developed to prepare PNPs from a preformed polymer. For this method, the polymer is first dissolved in a volatile solvent. Dichloromethane and chloroform have been widely used in the past. However, due to their toxicity they have been replaced by ethyl acetate which displays a better toxicological profile and therefore more suitable for biomedical applications. The resulting organic solution is emulsified in the aqueous phase and the mixture is typically processed using a surfactant and high-speed homogenization or ultrasonication, yielding a dispersion of nanodroplets. A suspension of NPs is formed by evaporation of the polymer solvent, which is allowed to diffuse through the continuous phase of the emulsion.81_{The solvent is then evaporated by continuous magnetic stirring}

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solidified NPs can be washed and collected by centrifuging, followed by freeze-drying for long term storage.82

Figure 20. Schematic representation of the emulsification-solvent evaporation method for the production of nanospheres.

The emulsification-solvent evaporation method has been applied in this work to prepare PLGA-loaded NPs with the desired characteristics by adjusting different experimental parameters.

1.3.3.2 Nanoprecipitation

The nanoprecipitation (NP) method, also called solvent displacement, is a one-step procedure that was firstly developed by Fessi et al.83_{The basic principle of this technique}

is based on the interfacial deposition of a polymer after displacement of the organic solvent from a lipophilic solution to the aqueous phase (Figure 21). The polymer is dissolved in a water-miscible solvent of intermediate polarity and this solution is added into a stirred aqueous solution in one shot, stepwise, dropwise or by controlled addition rate.84_{Due to the fast spontaneous diffusion of the polymer solution into the aqueous}

phase, the NPs form instantaneously in an attempt to avoid the water molecules. This process appears to be governed by the Marangoni effect, wherein a decrease in the interfacial tension between the two phases, increases the surface area due to the rapid diffusion and leads to formation of small droplets of organic solvent. As the solvent diffuses out from the nanodroplets, the polymer precipitates in the form of nanocapsules or nanospheres. In general, the organic phase is added to the aqueous phase, but the protocol could also be reversed without compromising the NPs formation.

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Figure 21. Schematic illustration of the nanoprecipitation method for the preparation of nanospheres. The most common used organic solvent is acetone, because it is miscible with water and easy to remove by evaporation. Moreover, smaller particles were obtained using acetone, which could be related to a more efficient solvent diffusion and faster polymer dispersion into water. It is also possible to use either two organic phases or two aqueous phases as long as solubility, insolubility and miscibility conditions are satisfied. Usually, surfactants could be included in the process to guarantee the stability of the colloidal suspension, but their presence is not required to ensure formation of NPs. The obtained NPs are typically characterized by a well-defined size and a narrow size distribution, which is better than those produced by the emulsification-solvent evaporation procedure. The key variables that are conditioning the final NPs properties are those related with the experimental design. By carefully adjusting the nature and concentration of the components, organic phase/aqueous phase ratio, organic phase injection rate, fluid dynamics and mixing speed, it is possible to control the PNPs physicochemical properties.

This method is widely used due to its simplicity, quickness and reproducibility, and sometimes it is preferred to the emulsion-solvent evaporation method. However, one of the difficulties associated with this technique is the mixing process during NP, that can be overcame by using a microfluidic platform where the hydrodynamic flow ensures a fast and tunable mixing of solvent/non-solvent in the microfluidic channels.85_Moreover,

by using the NP method, only hydrophobic drugs can be encapsulated into the PNPs, and this limits its applicability.

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1.3.3.3 Electrospray

A number of different techniques for the manufacturing of nano/micro polymeric NPs have been reported to date, where each of them presents its own critical shortcomings (low encapsulation efficiencies, poor control over size distribution, challenging up scaling capacity, modest reproducibility).

Electrospray (ES) has been showed to be a one-step, inexpensive and innovative approach to produce unique nanoscale drug-encapsulating NPs with great capabilities by comparison with other manufacturing techniques. Indeed, it allows a tight control over size with quasi-monodisperse size distributions (which provides better control over drug release improving therapeutic effect), the generation of particles with virtually no drug loss, high level of reproducibility, and high encapsulation efficiencies for hydrophilic as well as for hydrophobic drugs without the need of surfactants or elevated temperatures. Moreover, it permits the direct collection of particles on the collector in their dried form. Compared to the applied shear stresses and interfacial adsorption during solvent evaporation processes, the electrospray can serve as a gentler encapsulation method. 86-88

The principles of electrospray are based on the ability of an electric field to deform the interface of a liquid drop. The setup essentially consists of a high voltage supplier, syringe pump and a grounded collector (Figure 22). A solution of polymer/drug in a sufficiently conductive solvent is usually employed and pumped through a charged needle. Under the application of a high electric field between the tip of the needle and a metallic collector, the solution jet at the end of the metallic needle stretches; when the electrostatic force acting on the liquid stream exiting the needle overcome the surface tension of the liquid, a conical shape, known as the Taylor cone, is formed which then breaks up into charged, monodisperse droplets. Once the droplet travels towards the collector, the solvent gets evaporated and solid particles are deposited on the collector. 89

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Figure 22. Schematic representation of particle preparation by electrospray process.

Electrospinning is based on the same principles but differs from electrospray process in that a more concentrated polymer solution (with stronger chain entanglement density) applied with a much higher voltage results in the production of nano/microfibers instead of particles. Optimization of electrospray involves not only the selection of an appropriate solvent for dissolving the polymer and the drug for proper electrospray to take place, but also the factors like the polymer concentration and electrospray conditions which are critical.

Yet, electrospray is a complex process in terms of controlling the process of particle formation as the underlying mechanisms are influenced by many variables including flow rate, electric field strength, solution properties and external conditions.90_{Many of these}

variables have an interdependent influence on the resulting particle properties and drug release profile, and it is thus challenging to fully optimize the final output.

In this work the production of uniformly sized, antibiotic-loaded PLGA nanoparticles using the ES route by electrospraying polymer solutions over a solid substrate is reported, with the mean size of the particles in the remarkably broad 80–200 nm range.

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2. Materials and Methods

2.1 Materials

PolyActive® (PA) was provided by PolyVation BV (Groningen, The Netherlands). This commercial random block copolymer is composed of PEOT and PBT and is also named PEOT/ PBT (300PEOT55PBT45). The commercial nomenclature aPEOTbPBTc represents: (a) the molecular weight (Mw, g mol−1) of the poly(ethylene glycol), (b) and (c) the weight ratios of PEOT and PBT, respectively. It was stored in a dry place at room temperature.

Poly (DL-lactide-co-glycolide) 50:50 DLG 5A (acid-terminated) Resomer® and Poly(e-caprolactone) (100 CL 4E-HD, 31 kDa, ester-terminated) Resomer® were used to produce polymeric nanoparticles, both provided by Evonik Industries AG (Birmingham, AL, USA). Lumogen Red 305 and NHS-Rhodamine B were used as fluorescent dyes to track the release profile from the polymeric nanoparticles. They were both supplied by Thermo Fisher Scientific (Waltham, Massachusetts, Stati Uniti). Ciprofloxacin HCl was purchased from AppliChem (Darmstad, Germany) and used as antibiotic drug to be released from the polymeric nanoparticles.

Chloroform (CHCl3), hexafluorisopropanol (HFIP), dichloromethane (DCM) and methanol (MeOH) were used as combined solvents for the polymeric solutions to produce fibers and nanoparticles respectively. CHCl3, DCM and MeOH were supplied by Merck KGaA (Darmstadt, Germany), while HFIP by Biosolve BV (Valkenswaard, The Netherlands).

Human mesenchymal stem cells (hMSCs) were supplied from Merck Millipore S.A.S., (Burlington, MA, US). Fetal Bovine Serum (FBS) and LIVE/DEAD kit were bought from Thermo Fisher Scientific (Waltham, MA, USA). Resazurin, Dulbecco’s Minimal Essential Medium (DMEM), phosphate-buffered saline (PBS) were supplied by Sigma (Milan, Italy).