Keywords

11.1 Matrix-Assisted Pulsed Laser Evaporation Technique: General Approach

Thin films are defined as layers of material with a thickness between nanometers to micrometers, while the thin film deposition is a term which refers to the technique of applying a film onto a substrate. The main techniques used for thin film deposition are classified as: (1) laser assisted deposition techniques and (2) non-laser assisted deposition techniques. Laser assisted techniques used for obtaining thin film depositions on different substrates have multiple advantages compared to other techniques given the following facts: (1) control of the monolayer thickness; (2) strong adhesion of the thin film to the surface of the monolayer; (3) low substrate temperature; (4) ensuring the stoichiometry of precursors; (5) economical consumption of precursors [1]. The main laser-assisted techniques used for the deposition of thin films are: (1) pulsed laser deposition [2, 3]; (2) matrix-assisted pulsed laser evaporation technique [4–6]; (3) spin coating technique [7, 8]; (4) drop casting technique [9].

The interest for laser deposition techniques is given by the fact that the resulted thin films have a controlled topography, which can be manipulated at nanometric level [10]. These materials provide several important applications in the biological field, such as: (1) drug delivery systems [11–13], (2) tissue engineering [14], (3) implants with improved biocompatibility [4, 12], (4) improved adherent surfaces [15, 16], (5) antibacterial surfaces [17, 18], (6) gas sensors [19, 20], etc.

Matrix-assisted pulsed laser evaporation (MAPLE) technique is derived from pulsed laser deposition (PLD) technique, where the target is represented by a frozen homogenous solution of the material of interest, which is diluted in a volatile solvent (matrix solvent). The temperature at which the target solution is frozen is given by the liquid nitrogen temperature. The target is placed in a vacuum chamber and a high energy laser is directed trough it. The pulsed laser energy is absorbed by the solvent in the target and it is converted in thermal energy, which determines the evaporation of the solvent. The evaporating molecules of the solvent collide with the solute (material) molecules, which are transformed in a gas phase, because of the transferred kinetic energy. The advantages of MAPLE compared to PLD are given by the fact that this technique avoids the photochemical damage and the decomposition in PLD technique, which is determined by high energy of the laser pulses [10] (Fig. 11.1).

Fig. 11.1
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Schematic representation of the principle of matrix-assisted pulsed laser evaporation technique (MAPLE)

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11.2 The MAPLE Deposition Apparatus

The MAPLE technique was developed as an improvement for the PLD technique, in order to be used for thin film depositions of organic materials. It was implemented for the first time in the 1990s by the US Naval Research Laboratory to obtain functionalized polymer films [21].

The MAPLE apparatus, as shown in Fig. 11.2, consists of a sealed chamber which presents a cryogenically cooled rotating target holder and a substrate holder, also having a rotation movement. The target holder is connected to a liquid nitrogen tank. The chamber has an input for a background gas and for vacuum. It also presents the laser beam focusing system, which directs pulsating laser beam at a 45° angle, on the target surface [21].

Fig. 11.2
figure 2

Schematic representation of matrix-assisted pulsed laser evaporation (MAPLE) system; Important parameters in the technological process of MAPLE thin film deposition are the following: (1) the fluency of the laser, (2) the repetition rate, (3) the number of pulses, (4) the target rotation rate, (5) the angle at which the laser beam scans the target surface, (6) the background pressure, (7) the distance between substrate and target [22, 23]

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Usually the technique uses an excimer laser such as KrF, with λ = 248 nm, or ArF, with λ = 193 nm, and a pulse width between 10 and 30 ns, focused on the target in a 1–10 mm2 spot. The repetition rate is usually set between 1 and 20 Hz and the laser fluence is between 0.01 and 1.0 J/cm2, being set according to the type of material (solute) and solvent used to make the target. The process can be held at different pressures, from vacuum, to 70 Pa, in the presence of an inert gas or a background gas [21].

11.3 Thin Films Based on Magnetite Nanostructures

Magnetite nanoparticles are intensively used in different biomedical applications especially due to their magnetic properties [24], biocompatibility [25], and easy obtaining methods [26–28]. Thus, Fe3O4 nanoparticle-based materials are used as: (1) drug delivery systems [29–31]; (2) antimicrobial materials [32–34]; (3) hyperthermia applications for cancer treatment [35, 36]; (4) contrast substance for magnetic resonance imaging techniques [35, 37]; etc.

11.3.1 Preparation

Magnetite can be found as a natural mineral, but it can also be artificially obtained using different chemical methods. Fe3O4 nanoparticles were obtained for the first time as a ferrofluid in 1981, by Massart [38] using a co-precipitation method, based on the combination of ferric and ferrous salts in an alkaline medium (sodium hydroxide).

Regarding the composition of magnetite, it is an iron oxide consisting of Fe3+ and Fe2+ ions, with a characteristic molar ratio of Fe3+:Fe2+ = 2:1 [39].

The main preparation methods are presented in Table 11.1, where the methods’ principle and the implied factors are briefly summarized.

Table 11.1 The main chemical methods for obtaining Fe3O4 nanoparticles
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11.3.2 Functionalized Magnetite Nanostructures

The functionalization of nanomaterials consists in modifying the surface of nanoparticles by means of attaching different type of molecules, in order to improve the properties of the inorganic structure: (1) the biocompatibility [50, 51], (2) stability [52, 53], (3) targeting properties [31, 35, 54, 55], (4) carrier properties [56–58] (Fig. 11.3).

Fig. 11.3
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Applications of functionalized magnetite nanoparticles

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Magnetite surface chemistry depends on the pH, acting like Lewis acids in aqueous systems: at low pH values the surface of Fe3O4 is positively charged, while at high pH values the magnetite surface is negatively charged [59–61]. The main classes of magnetite functionalization methods are: (1) covalent bonding and (2) non-covalent bonding. The non-covalent bonding between the functionalizing molecules and the magnetite nanoparticle surface is commonly encountered by means of hydrogen bonding with HO− groups in Fe3O4.

The functionalizing agents which can interact with Fe3O4 nanoparticles are classified as follows: (1) organic functionalizations and (2) inorganic functionalizations. In the first class are included small molecules and surfactants (dehydroascorbic acid [37], silane compounds [62], folic acid [35], carboxyl [36]) generally applied to reduce the aggregation phenomena of magnetite nanoparticles in suspension; polymers (PEG [63], chitosan [64], PVA [65]), used to improve biocompatibility, stability, or to modify the character of the nanoparticle surface; enzymes (pullulanase [66], porphyrin [67], glucose oxidase [68]), with sensing properties; respectively, therapeutic molecules (docetaxel [31], usnic acid [32], danorubicin [69], umbelliprenin [70], rotavirus capsid surface protein [57]), used to obtain drug delivery systems. Magnetite functionalization with inorganic coatings is generally applied for different reasons, like: (1) enhancing the magnetic properties of the nanoparticles [71–73]; (2) enhancing the antioxidant properties of magnetite [74]; (3) inducing antibacterial properties [75]; (4) improving the biocompatibility of the system [76, 77].

11.3.3 Thin Films

Magnetite-based thin films can be obtained by several techniques, as follows: (1) pulsed laser deposition technique [78], (2) matrix-assisted laser evaporation technique [79], (3) ultrasound-enhanced ferrite plating [80], (4) chemical vapor deposition [81], (5) DC reactive magnetron sputtering [82] and reactive sputtering [83].

Previously obtained Fe3O4 functionalized nanoparticles are prepared as a diluted suspension in the matrix solvent (chloroform 1 % wt./vol.) [84] and then put into a precooled target holder and frozen in liquid nitrogen. For example, Cristescu et al. [84] used the following experimental parameters for all of the Fe3O4@oleic acid@antibiotic MAPLE deposited thin film samples: a laser fluence between 65 and 300 mJ/cm2, a repetition rate of 10 Hz, 7,200–20,000 laser pulses, a target rotation rate of 0.4 Hz, an angle of 45° between the laser beam and the target surface, a distance of 4 cm between the substrate and target, and a background pressure of 30–100 Pa [84].

11.3.4 Biological Applications

Nosocomial infections, or hospital-acquired infections, are a current problem of the medical system, over 1.7 million hospital-associated infections contributing and causing over 99.000 deaths every year [85]. In Europe, gram-negative associated infections cause the most numerous untreatable infections [86], therefore combating the antibiotic resistance being an important subject of the latest scientific studies in the biomedical field. Biofilms are microbial communities included in a polysaccharide matrix, attached to a substrate. These are commonly encountered in unsterile prosthetic devices, contributing to a large number of infectious cases. Thus, there are several studies conducted in order to obtain anti-biofilm surface coatings for medical devices, and matrix-assisted pulsed laser evaporation technique offers great solutions; Table 11.2 gives a summary of the latest examples regarding this aspect.

Table 11.2 Matrix-assisted pulsed laser evaporation technique for anti-biofilm surface coatings
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Table 11.2 presents several examples of MAPLE deposited thin films based on modified magnetite nanoparticles, which exhibit antibacterial properties, which can be used as a growth support for cells. The Fe3O4@oleic acid/antibiotic thin films are excellent candidates which can be used as surface modification methods for medical devices and implants, with anti-adherence and antimicrobial properties [17]. However, the anti-adherence property refers only to the microbial colonies, as it was proved that human epithelial carcinoma HeLa cell monolayers can grow on these modified surfaces. The antimicrobial properties of the obtained samples were tested against both gram-negative (Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli) and gram-positive (Staphylococcus aureus, Bacillus subtilis) bacteria, using the antimicrobial activity assay (API 20E biochemical tests, VITEK I automatic system), to compare the substrate effect with several antibiotic substrates and the microbiological assay investigation procedure to measure the percent of viable bacterial cells attached to the substrates (compared to a control, represented by glass substrate). The in vitro biocompatibility of the obtained samples was evaluated after the addition of the microbial suspensions over the HeLa cell monolayer cultivated on the MAPLE modified substrates. The samples were prepared by Giemsa staining and evaluated using an inverted microscope to conclude over the degree of cell confluency, the cytotoxic effect of nanoparticles, the number of adherent bacteria, and the adherence pattern (localized adherence, where bacteria form microcolonies, diffuse adherence, where bacteria adhere to the whole cell surface and aggregative adherence, where aggregates of bacteria attach to the cells, forming an overlapped arrangement. The cell morphology was not affected by the presence of the nanoparticles, neither was the adherence pattern or the adherence index, compared to control samples.

Our group obtained Fe3O4@eugenol nanoparticles by co-precipitation method, which were embedded in poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid)–polyvinyl alcohol (P(3HB-3HV)–PVA) microspheres by oil-in-water microemulsion method; these resulted microspheres were used as modifying material for inert substrates [79]. The P(3HB-3HV)–PVA–Fe3O4@eugenol thin films were obtained by MAPLE deposition from 1 % (w/v) microsphere suspension in DMSO using a KrF* laser source (248 nm, 25 ns laser pulses, 300–500 mJ/cm2 laser fluence and a repetition rate of 15 Hz, with 45,000–160,000 laser pulses). The in vitro biocompatibility was evaluated using human endothelial cells EAhy929; the proliferation and viability of the cells was tested using commercial kits, resulting in high viability of the endothelial cells, the cells’ proliferation being increased at 24 h after incubation and being maintained at 48 and 72 h (compared to control). The obtained samples were also tested against biofilm formation for Staphylococcus aureus and Pseudomonas aeruginosa bacteria using the microbial biofilm assay, which demonstrated the anti-biofilm antibacterial growth effect of the resulted biomaterial.

The same experimental procedure was used by Holban et al. [90] to obtain polylactic acid (PLA)–chitosan (CS)–Fe3O4@eugenol microsphere thin films depositions. The in vitro biocompatibility was tested for human endothelial cells EAhy926, using a commercial cell proliferation assay and a fluorescence long term-tracking method. The tests showed that the obtained thin films offer biocompatible support for endothelial cells growth, their morphology and proliferation capability being normal [90]. For the anti-biofilm evaluation, Staphylococcus aureus and Pseudomonas aeruginosa strains were cultured in Luria Broth medium and put in contact with the resulted biomaterials. The biofilm formation is affected after 24 and 48 h of incubation compared to uncoated magnetite embedded in microspheres control.

Our research group also obtained polylactic-co-glycolic acid (PLGA)–polyvinyl alcohol (PVA)–Fe3O4@usnic acid thin film depositions by MAPLE using a KrF* laser source (248 nm, 25 ns laser pulses, 200–400 mJ/cm2 laser fluence and a repetition rate of 10 Hz, with 10,000–20,000 laser pulses) [87]. The in vitro biocompatibility was evaluated for human mesenchymal stem cells from human bone marrow. The viability of the cultured cells was over 92 %, proving that the obtained thin films can support the normal development of the cells. Also, the normal morphology of the cells showed that the obtained materials have biocompatible properties. To evaluate the antibacterial effect, a minimal inhibitory assay and a microbial adherence and biofilm assay were employed for S. aureus bacteria. The obtained thin film inhibited the formation of bacterial strains for 3 days under static conditions, diminishing S. aureus adherence and biofilm formation.

Anghel et al. [89] obtained Fe3O4@Cinnamomum verum MAPLE thin film depositions on gastrostomy tubes, having antibacterial activity against gram-positive (S. aureus) and gram-negative (E. coli) bacteria [89]. Cinnamomum verum is a natural oil with anti-inflammatory, antiseptic, antifungal, and antiviral properties, which can stimulate the immune system and have antioxidant properties. The functionalized magnetite nanoparticles were obtained by co-precipitation method and dispersed in DMSO (1.5 % w/v solution) and frozen in liquid nitrogen. The MAPLE deposition was held using a KrF*laser (248 nm, 25 ns laser pulses, 300–500 mJ/cm2 laser fluence and a repetition rate of 0.4 Hz, with 30,000–60,000 laser pulses). Regarding the antibacterial effect of the modified tubes, the most inhibitory effect was proved for S. aureus (compared to control). The in vitro biocompatibility effect, tested using the MTT assay on human endothelial cells, proved a normal development at 24 and 48 h after incubation, and an improved proliferation at 72 h, compared to control. The fluorescence microscopy images obtained at 5 days after incubation showed a normal growth and morphology of the cells.

11.4 Thin Films Based on Inorganic–Organic Hybrid Nanomaterials

Hybrid organic–inorganic nanomaterials have been intensively used in different biomedical applications due to the combination of properties from both organic and inorganic moieties [91]. Examples of such applications are: (1) tissue engineering [15], (2) antibacterial and anti-biofilm effect [92], (3) drug delivery systems [93].

There are several reasons for developing such materials, excelling in the improvement of properties like: (1) increased biocompatibility of the designed nanomaterials, by applying several organic functionalizing agents; (2) antibacterial properties of the organic material; (3) increased stability; (4) modifying the surface character; (5) drug loading.

11.4.1 Preparation

The preparation of hybrid nanomaterials can be done in several ways, which are grouped in two main classes, depending on the interactions that take place between the organic and inorganic phases: (1) methods where no covalent bond is formed between the two phases, (2) methods where covalent bonds are formed between the two phases. Table 11.3 [94] summarizes the main methods for obtaining organic–inorganic hybrid nanomaterials (Fig. 11.4).

Table 11.3 Methods for obtaining organic–inorganic nanomaterials [94]
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Fig. 11.4
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Schematic representations of non-covalent bonding methods for obtaining hybrid nanomaterials

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11.4.2 Thin Films

Birjega et al. [119] obtained layered double hydroxide (LDH)–polyethylene glycol (PEG)/ethylene glycol (EG) thin films deposited by MAPLE technique [119]. The interest for LDH is given by the fact that it is an artificial clay, which consists of positively charged layers, arranged parallel one to another. It acts as a host material for anions located in the interlayer regions, which can be easily replaced by other negatively charged molecules of biological interest. The main application of these thin film coatings consists in modifying the surface character and controls its surface wetting. The Mg-Al LDH (Mg/Al = 3) was obtained using a co-precipitation method (at suprasaturation, pH = 10) from aqueous solutions of Mg and Al nitrates, sodium hydroxide and carbonate, resulting in a gel, which underwent a drying process (85 °C, 24 h), followed by a calcination process (460 °C, 18 h, nitrogen atmosphere). LDH-polymer (PEG/EG) composites were prepared by immersing Mg(Al)O mixed oxides powders immersed in aqueous polymer solutions (200 amu/1,450 amu, where Mg(Al)O/PEG and Mg(Al)O/EG = 1.76/1), separated by vacuum filtration and dried (vacuum, 30 °C, 24 h) [119]. For MAPLE deposition of the thin films, a Nd:YAG laser (266 nm, 5 ns pulses, with a repetition rate of 10Hz, a laser fluence of 1–2 J/cm2) was used. Other important parameters are: (1) a 45° angle between the laser and the target; (2) a laser spot size between 0.6 and 0.8 mm2; (3) 40,000–60,000 laser pulses.

Predoi et al. obtained γ-Fe2O3@dextran thin films deposited by MAPLE technique using a UV KrF* excimer laser (248 nm), with 25 ns pulses and a repetition rate of 10 Hz. 25 × 103 laser pulses were applied and a fluence of 0.5 J/cm2 was assured [120]. The target was prepared using a solution of 0–25 wt.% iron oxide nanoparticles obtained by co-precipitation method, 10 wt.% dextran (2,500 Da) and distillated, frozen in liquid nitrogen solution. The surface morphology of the obtained samples was investigated by scanning electron microscopy technique, which proved an aggregated aspect of the films, consisting of micrometer sized grains. Also, by other investigations, the authors concluded that the resulted thin films have a crystallinity, chemical composition, and molecular structure identical to the materials used for target preparation.

In the experiment described by Miroiu et al. [15], hydroxyapatite–silk fibroin thin films were obtained by MAPLE deposition. The target was prepared using polymer solutions (2 wt.% and 4 wt.%, respectively) and adding hydroxyapatite (HA) in order to obtain a HA–fibroin weight ratio of about 3:2 and 3:4 respectively. The HA–fibroin solutions were mechanically stirred and several drops of NaOH or NaCl were added in order to adjust the pH to 7.4 (physiological value). Then, the solutions were frozen in liquid nitrogen to obtain the targets. For the deposition process, a KrF* excimer laser (248 nm, with 25 ns pulses, a repetition rate of 10–15 Hz and a laser fluence of 0.4–0.5 J/cm2) was used; 20,000–50,000 pulses were applied for each film [15].

11.4.3 Biological Applications

Miroiu et al. obtained hydroxyapatite–silk fibroin thin films deposited by MAPLE on the surface of metallic prosthesis. The aim of the study was given by the fact that the biomimetic modifies surface display enhanced properties like bioaffinity and osteoconductivity. The in vitro biocompatibility test using SaOs2 osteosarcoma cells cultured for 72 h on the surface of the modified implants showed an improved viability and spreading of the cells. The elongated morphology of the cells proved that the resulted hydroxyapatite–silk fibroin coatings have good performances as bone implants, assuring an optimal interface between the living tissue and the metallic surface of the prosthesis. The best results were given by the HA3-FIB4 sample (3 wt% hydroxyapatite–4 wt% fibroin) [15].

The γ-Fe2O3@dextran thin film depositions obtained using MAPLE technique by Predoi et al. [120] were investigated as biocompatible structures used for implant modification coatings in locoregional cancer treatment by hyperthermia after a surgical intervention. Thus, human hepatocarcinoma cells HepG2 were cultivated on the obtained thin films, the viability investigated by MTT colorimetric assay, resulting in a good biocompatibility of the materials. Regarding the morphological aspect of the cells, the cells cultured on the 5 wt.% iron oxide samples grew into larger multicellular aggregates [120] (Table 11.4).

Table 11.4 Classes of organic–inorganic thin film depositions using MAPLE
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11.5 Conclusions and Perspectives

Matrix-assisted pulsed laser evaporation is the most frequently used method to obtain thin film nanoarchitectonics for biomedical applications, because of its numerous advantages, like assuring control of the monolayer thickness, a strong adhesion of the thin film to the surface of the monolayer, low substrate temperature, ensuring the stoichiometry of precursors, and economical consumption of precursors. This technique has been applied to obtain magnetite modified surfaces with antibacterial properties, used for implants and medical devices, in order to prevent the nosocomial infections, frequently associated with improper sterilization or surgical procedures. However, these systems do not affect the adherence and biocompatibility of tissue cells. Hybrid organic–inorganic nanomaterials are preferred because they combine properties from both components, resulting in an increased biocompatibility of the designed nanomaterials, by applying several organic functionalizing agents, antibacterial properties of the organic material, increased stability, a modified surface character, drug loading. Such thin films have been applied for modified surface prosthesis with antibacterial properties and improved biocompatibility and cellular adherence. Some systems have been designed for delivery action, in order to improve some properties, or for therapeutic effects.