Bottom-Up Development of Nanoimprinted PLLA Composite Films with Enhanced Antibacterial Properties for Smart Packaging Applications

: In this work, polymer nanocomposite ﬁlms based on poly(L-lactic acid) (PLLA) were reinforced with mesoporous silica nanoparticles, mesoporous cellular foam (MCF) and Santa Barbara amorphous-15 (SBA). PLLA is a biobased aliphatic polyester, that possesses excellent thermomechanical properties, and has already been commercialized for packaging applications. The aim was to utilize nanoparticles that have already been established as nanocarriers to enhance the mechanical and thermal properties of PLLA. Since the introduction of antibacterial properties has become an emerging trend in packaging applications, to achieve an e ﬀ ective antimicrobial activity, micro / nano 3D micropillars decorated with cone- and needle-shaped nanostructures were implemented on the surface of the ﬁlms by means of thermal nanoimprint lithography (t-NIL), a novel and feasible fabrication technique with multiple industrial applications. The materials were characterized regarding their composition and crystallinity using Fourier transform infrared spectroscopy (FT-IR) and X-ray di ﬀ raction (XRD), respectively, and their thermal properties using di ﬀ erential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Their mechanical properties were examined by the nanoindentation technique, while the ﬁlms’ antimicrobial activity against the bacteria Escherichia coli and Staphylococcus aureus strains was tested in vitro. The results demonstrated the successful production of nanocomposite PLLA ﬁlms, which exhibited improved mechanical and thermal properties compared to the pristine material, as well as notable antibacterial activity, setting new groundwork for the potential development of biobased smart packaging materials.


Introduction
Scientific research has been directed towards biobased polymers, as the environmental impact of petroleum-based materials has become a matter of great concern in modern societies [1]. To this end, renewable resources like proteins, oils, and carbohydrates have been employed to produce biodegradable and sustainable polymeric and nanocomposite materials [2,3]. In this context, packaging industries have recently focused on the development of environmentally-friendly, "smart" packaging materials which, at the same time can include aspects like antimicrobial protection and/or hydrophobicity (tamper-proofing), providing extended shelf life and fulfilling consumer demands and therefore maximizing profits [4]. Such properties are usually achieved by utilization of nanofillers, which can serve both as reinforcing agents and nanocarriers of antimicrobial substances [5].
Poly(L-lactic acid) (PLLA), the polymer of interest here, is a biobased and compostable aliphatic polyester, and its monomer, DL-lactide is produced on a mass scale by the microbial fermentation of agricultural by-products [6][7][8][9]. Owing to its beneficial characteristics, such as good mechanical properties, transparency, biodegradability, and biocompatibility, PLLA is widely used in many industrial fields e.g., in the pharmaceutics, biomedicine, and textile industries [2,8,[10][11][12][13]. Having been classified as a safe (Generally Recognized as Safe-GRAS) material by the Food and Drug Administration-FDA, it is also currently used in food packaging applications [14], and also has strong potential as a "smart" packaging material. PLLA nanocomposites can play a significant role in this sector, because, in addition to possessing improved features compared to the neat polyester, such as heat sealability and gas barrier properties [1], they also introduce new characteristics such as antioxidant and antimicrobial properties, when encapsulated with the respective agents [2,15,16].
In order to load a polymer matrix with active agents, porous nanomaterials of determined structure are preferred. Among those, ordered mesoporous silica nanoparticles (MSNs) maintain a major position, as they have been screened successfully for potential use in drug delivery systems [17][18][19]. Lately, nanoparticles of this class with different geometries have been used to create smart packaging materials with prominent results. In particular, Santa Barbara amorphous (SBA-15) and mesoporous cellular foam (MCF) nanoparticles, with 2D hexagonal arrangement of cylindrical pores and 3D porous system of spherical structure, respectively, [17,20,21] were used as nanocarriers for chemical preservatives to develop polymeric matrices with antimicrobial properties [22][23][24]. Ideally, the next step towards more consumer-friendly materials would be the incorporation of natural ingredients that exhibit such properties, such as essential oils [25][26][27][28]. However, their volatile nature requires further optimization of such procedure, as their effective concentration decreases during high temperature processing [29][30][31].
An alternative approach to developing substrates with antibacterial properties lies in the formation of topographical features in micro-or nanoscales [32]. Such nanosized topographies can easily be fabricated by t-NIL [33,34]. Nanostructured polymeric films can exhibit hydrophobic and antibacterial characteristics, making them suitable for applications in electronics, optics, and especially biomedicine, where such properties pave the way for novel therapeutic strategies [24,[35][36][37]. For example, Nerantzaki [32] et al. showed that the synergistic effect of proper nanoinclusions combined with nanotopography can result in materials with superior antimicrobial properties. Based on the above, it is only natural to assume that applying this strategy to materials intended for packaging applications would be beneficial for this industry as well.
In this work, we examine the possibility of developing a fully biobased nanocomposite material with the prospect of using it in smart packaging. To this end, MCF and SBA-15 nanoparticles, which due to their porous system can potentially act as nanocarriers and mechanical properties enhancers, were prepared and incorporated in PLLA biobased polyester. The nanocomposites were then formed into films and t-NIL was used to implement micro-and nanosized topographies. Different concentrations of the nanoparticles were used to prepare materials of different properties, namely 1, 2.5, and 5 wt%, and the produced films were then characterized as to their structural, thermal, and mechanical properties. Moreover, their antibacterial activity against E. coli and S. aureus was also examined, with promising results.

Synthesis of MCF and SBA-15 Silica
The mesostructured cellular foam (MCF) silica was synthesized via a self-assembly method using non-ionic surfactant pluronic P-123 as the structure directing agent, 1,2,3-trimethylbenzene (TMB) as co-surfactant, ammonium fluoride (NH 4 F) as mineralizing agent, and tetraethyl orthosilicate (TEOS) as the silica source, in acidic pH conditions [24,35]. Typically, P-123 was diluted in aqueous HCl 1.6 M followed by the addition of NH 4 F and TMB. After 1 h at 40 • C, TEOS was added and the reacting mixture was further stirred for 20 h at 40 • C. The mixture was then heated in an autoclave at 100 • C for 24 h. Finally, the solid products were recovered by filtration and washed with deionized water. The organic templates were removed by calcination in air at 500 • C, for 8 h, with a heating rate of 1 • C/min.
The SBA-15 (Santa Barbara amorphous) mesoporous silica was prepared according to previously reported methods [20,36,37]. In a typical procedure, pluronic P-123 was dissolved in aqueous 1M HCl solution at pH ≤ 1 at 35 • C. Subsequently, TEOS was added and the solution was stirred for another 1 h. The resulting suspension was aged under static conditions at 35 • C for 48 h, while the molar ratio of the synthesis mixture was 1 TEOS:0.018 P123:3.3HCl:97.5 H 2 O. The solid products were isolated by filtration and washed with deionized water. Calcination was carried out in air at 550 • C, for 6 h, with a heating rate of 1 • C /min, to combust the organic template and produce the mesoporous silica.

Nanocomposite Preparation
A total of six PLLA nanocomposites, containing 1, 2.5, and 5 wt% of MCF and SBA-15, respectively, were prepared via melt mixing [38]. PLLA and the respective nanoparticles were mixed in a twin-screw corotating melt mixer for 10 min at 180 • C with a rotating speed of 30 rpm.

Preparation of Films by Spin-Coating
Thin films of neat PLLA and the prepared nanocomposites were produced by spin coating [39,40]. Specifically, each material was dissolved in chloroform, in order to obtain a 10% w/v solution. A drop of the solution was placed on silicon substrates, previously cut into 2 cm × 2 cm pieces, and spin-coated at a speed of 500 rpm/min for 1 min. With this procedure, seven polymeric films were fabricated-neat PLLA, PLLA/MCF 1 wt%, PLLA/MCF 2.5 wt%, PLLA/MCF 5 wt%, PLLA/SBA-15 1 wt%, PLLA/SBA-15 2.5 wt%, and PLLA/SBA-15 5 wt%.

Patterning of PLLA Nanocomposite Films by NIL
Before imprint, the spin-coated silicon substrates were heated at 100 • C for 15 min to ensure solvent evaporation. Afterwards, thermal NIL was performed using a commercial desktop 4-inch Compact Nanoimprint (CNI) tool, that was supplied from NIL Technology ApS. Polydimethylsiloxane (PDMS) stamps were used to achieve a soft release and a successful replication. The polymeric substrate and the stamp were heated to 210 • C, under a pressure of 5 bars for 10 min. After cooling to below 40 • C, the PDMS mold was separated from the polymeric material [32]. FT-IR spectra of the silica nanoparticles and the nanocomposite PLLA films were obtained on a PerkinElmer FT-IR spectrometer (PerkinElmer, Waltham, MA, USA), model SPECTRUM 1000 (Spectrum 1). The silica nanoparticles' spectra were obtained using KBr pellets, while the nanocomposite materials' measurements were conducted using thin films. Infrared (IR) absorbance spectra were obtained between 450 and 4000 cm −1 at a resolution of 4 cm −1 using 16 co-added scans. All spectra presented are baseline corrected and normalized.

Thermogravimetric Analysis (TGA)
Thermogravimetric analysis of the mesoporous silicas and the prepared films was carried out with a SETARAM (Lyon, France) SETSYS TG-DTA 16/18. Samples (6.0 ± 0.2 mg) were placed in alumina crucibles. An empty alumina crucible was used as reference. The samples were heated from ambient temperature to 600 • C in a 50 mL/min flow of N2 at heating rate of 20 • C/min.

Scanning Electron Microscopy (SEM)
The morphology of the imprinted materials was examined using a scanning electron microscope (SEM), type FEI Quanta 650 FEG. All the studied samples were coated with carbon black to avoid charging under the electron beam.

Differential Scanning Calorimetry (DSC)
For the DSC measurements a Perkin-Elmer, Pyris Diamond DSC differential scanning calorimeter, previously calibrated with indium and zinc standards, was employed. To determine the thermal properties of the nanocomposite films after t-NIL, a sample of about 5 mg was used for each test, placed in sealed aluminum pan and heated to 200 • C at a heating rate of 20 • C/min.

Nanoindentation
Experiments were carried out using a dynamic ultra-micro-hardness tester (DUH-211; Shimadzu Co., Kyoto, Japan) fitted with a triangular pyramid indenter tip (Berkovich indenter) with a tip radius of 100 nm. The indentations made on the surface of the nanocomposites under study appeared as an equilateral triangle. After contact of the indenter with the surface, this was driven into the surface until a peak load of 3 mN was reached. The peak load was held for 3 s (in order to minimize the effect of viscoelastic deformation of the specimen, notably creep, on property measurements) and then the indenter was unloaded, to a load of zero. Five measurements were conducted on each specimen and the results were expressed as mean ± standard deviation.

Antibacterial Activity
The antibacterial activity of PLLA nanocomposite films, before and after patterning, was evaluated. The protocol used was the swab assay that is a method based on touch transfer assay [41]. This assay allows for evaluation of the attachment of viable bacteria directly from the surface of films and achievement of a homogenous and well-distributed inoculum on the surface of the films.
Bacterial strains of E. coli (BL21) and S. aureus (ATCC 25923) were used as the model Gram-negative and Gram-positive bacteria, respectively. Bacteria (E. coli and S. aureus) were grown at 37 • C, 200 rpm, 18-24 h in 50 mL of sterile tryptic soy broth (TSB) medium. Prior to inoculation, strains were sub-cultured into fresh TSB at 1:50 dilution and incubated for approximately 2 h at 37 • C, 200 rpm. Bacteria were collected by centrifugation (2000 rpm, 10 min) and resuspended in PBS. The number of bacteria was spectrophotometrically adjusted to O.D.600 nm = 0.5 (equivalent to 5 × 10 8 CFU/mL). Then, a cotton swab soaked on the bacterial suspension (5 × 10 4 CFU/mL) was used to spread (vertically and horizontally) the bacterial suspension. The films were allowed to dry for 10 min. Finally, the contact plates were pressed against the films for 10 s, incubated at 37 • C for 18-24 h, and the resulting colonies were counted.

FT-IR
As observed in Figure 1, both MCF and SBA-15 exhibited one broad band at 3453 cm −1 which can be attributed to -O-H stretching vibrations of absorbed water molecules, while the respective bending vibration mode could also be identified at 1633 cm −1 . The intense band at 1090 cm −1 is attributed to Si-O bonds, while the one at 802 cm −1 to Si-O-Si, all being characteristic bands of MCF and SBA-15, which proves that the corresponding mesoporous nanoparticles had been successfully prepared [19,24].

Porosity
The porous characteristics of the parent MCF and SBA-15 silica were studied by N 2 porosimetry at −196 • C and the respective data are presented in Figure 2 and Table 1. The adsorption isotherm of the parent MCF silica is of type IV according to the IUPAC classification [42], typical for such types of ordered mesoporous materials. It is well known that MCF exhibits cellular pore morphology of relatively large size pores giving a foam-like texture [43]. MCF exhibits a specific surface area (BET method) of 837 m 2 /g and a high total pore volume of 1.56 cc/g. Its average pore size is 18.5 nm and it could be ideal for an antimicrobial agent adsorption.
As observed in Figure 1, both MCF and SBA-15 exhibited one broad band at 3453 cm −1 which can be attributed to -O-H stretching vibrations of absorbed water molecules, while the respective bending vibration mode could also be identified at 1633 cm −1 . The intense band at 1090 cm −1 is attributed to Si-O bonds, while the one at 802 cm −1 to Si-O-Si, all being characteristic bands of MCF and SBA-15, which proves that the corresponding mesoporous nanoparticles had been successfully prepared [19,24].

Porosity
The porous characteristics of the parent MCF and SBA-15 silica were studied by N2 porosimetry at −196 °C and the respective data are presented in Figure 2 and Table 1. The adsorption isotherm of the parent MCF silica is of type IV according to the IUPAC classification [42], typical for such types of ordered mesoporous materials. It is well known that MCF exhibits cellular pore morphology of relatively large size pores giving a foam-like texture [43]. MCF exhibits a specific surface area (BET method) of 837 m 2 /g and a high total pore volume of 1.56 cc/g. Its average pore size is 18.5 nm and it could be ideal for an antimicrobial agent adsorption.
The adsorption isotherm of the parent SBA-15 silica is of type IV according to the IUPAC classification [42], typical for such types of ordered mesoporous materials. The specific surface area of SBA-15 (BET method) is 619 m 2 /g, while its total pore volume 0.5 cc/g and its pore diameter 4 nm.

TGA Analysis
The synthesized silicas' thermal stability was evaluated, using TGA analysis. The results demonstrate that both MCF and SBA-15 nanoparticles showed only a slight mass loss in the range of temperature of 40-100 °C which corresponds to the removal of the absorbed humidity ( Figure 3) [44]. The weight loss recorded at this stage, was about 4% and 6% for MCF and SBA-15 nanoparticles, respectively. It was clearly exhibited that up to 500 °C, the silicas do not present any significant mass loss, indicating that their decomposition starts at higher temperatures [24].   The adsorption isotherm of the parent SBA-15 silica is of type IV according to the IUPAC classification [42], typical for such types of ordered mesoporous materials. The specific surface area of SBA-15 (BET method) is 619 m 2 /g, while its total pore volume 0.5 cc/g and its pore diameter 4 nm.

TGA Analysis
The synthesized silicas' thermal stability was evaluated, using TGA analysis. The results demonstrate that both MCF and SBA-15 nanoparticles showed only a slight mass loss in the range of temperature of 40-100 • C which corresponds to the removal of the absorbed humidity ( Figure 3) [44]. The weight loss recorded at this stage, was about 4% and 6% for MCF and SBA-15 nanoparticles, respectively. It was clearly exhibited that up to 500 • C, the silicas do not present any significant mass loss, indicating that their decomposition starts at higher temperatures [24].

Morphological Study of Micro-and Nanosized Topographies
Hierarchical nanostructures were formed on the surface of the spin-coated nanocomposite films by means of thermal NIL, aimed at providing them with antibacterial properties. In order to confirm the successful formation of the nanostructures, the films' surface morphology was examined with scanning electron microscopy (SEM). In Figure 4 we demonstrate three-dimensional (3D) hierarchical domains on the surface of PLLA films. It can be seen that micropillars with 5 μm width and 5 μm height, decorated with cone-and needle-shaped nanostructures on the top surface, and with 600 nm width, were successfully fabricated on the surface of the nanocomposite PLLA films.
As SEM observations confirm, all nanocomposite films had fine morphological features while the nanostructures were homogenously dispersed throughout the surface. In addition, the presence of MCF and SBA-15 did not affect the surface morphology and the topographical features of the PLLA films since the hierarchical domains were perfectly shaped and homogenously distributed in the polymer matrix.

Fourier Transform-Infrared Spectroscopy (FT-IR)
FT-IR was used to determine the chemical composition of the prepared materials. In Figure 5, all the characteristic bands of PLLA were present-two bands at 3000 and 2850 cm −1 owing to the stretching vibrations of -C-H and one sharp, characteristic band at 1760 cm −1 due to stretching vibration of -C=O bonds. The bands at 1500-1400 cm −1 and at 1100-1000 cm −1 are associated with the bending and stretching vibrations of -C-H and -C-O bonds, respectively [32]. In the spectra of the nanocomposites, there is an increase in the intensity of the signal at 1090 cm −1 , derived from the mutual absorbance of PLLA and the nanoparticles at the specific wavenumber. This is evidence of the successful incorporation of the silicas into the polymer matrix. The nanocomposites' films obtained FT-IR spectra are presented in detail in Supplementary Materials ( Figure S1).

Morphological Study of Micro-and Nanosized Topographies
Hierarchical nanostructures were formed on the surface of the spin-coated nanocomposite films by means of thermal NIL, aimed at providing them with antibacterial properties. In order to confirm the successful formation of the nanostructures, the films' surface morphology was examined with scanning electron microscopy (SEM). In Figure 4 we demonstrate three-dimensional (3D) hierarchical domains on the surface of PLLA films. It can be seen that micropillars with 5 µm width and 5 µm height, decorated with cone-and needle-shaped nanostructures on the top surface, and with 600 nm width, were successfully fabricated on the surface of the nanocomposite PLLA films.  As SEM observations confirm, all nanocomposite films had fine morphological features while the nanostructures were homogenously dispersed throughout the surface. In addition, the presence of MCF and SBA-15 did not affect the surface morphology and the topographical features of the PLLA films since the hierarchical domains were perfectly shaped and homogenously distributed in the polymer matrix.

Fourier Transform-Infrared Spectroscopy (FT-IR)
FT-IR was used to determine the chemical composition of the prepared materials. In Figure 5, all the characteristic bands of PLLA were present-two bands at 3000 and 2850 cm −1 owing to the stretching vibrations of -C-H and one sharp, characteristic band at 1760 cm −1 due to stretching vibration of -C=O bonds. The bands at 1500-1400 cm −1 and at 1100-1000 cm −1 are associated with the bending and stretching vibrations of -C-H and -C-O bonds, respectively [32]. In the spectra of the nanocomposites, there is an increase in the intensity of the signal at 1090 cm −1 , derived from the mutual absorbance of PLLA and the nanoparticles at the specific wavenumber. This is evidence of the successful incorporation of the silicas into the polymer matrix. The nanocomposites' films obtained FT-IR spectra are presented in detail in Supplementary Materials ( Figure S1).

X-ray Diffraction (XRD)
To evaluate if the thermal treatment of t-NIL affected the crystallinity of the prepared films, XRD was employed. The diffractograms are presented in Figure 6, and they exhibit one broad peak at 16.8 • indicative of their amorphous nature. Considering that the materials were prepared via spin coating, their initial amorphous character was expected, as the solvent's rapid evaporation does not favor the polymer chains' arrangement and therefore hinders its crystallization [32]. Moreover, t-NIL does not seem to have an impact on the materials' crystallinity, since they remain amorphous after their imprinting treatment. Finally, reflections derived from the silica nanoparticles are not to be expected in the measurements' range, since MCF is amorphous [45] and SBA-15 reflects at lower angles [45,46]. indicative of their amorphous nature. Considering that the materials were prepared via spin coating, their initial amorphous character was expected, as the solvent's rapid evaporation does not favor the polymer chains' arrangement and therefore hinders its crystallization [32]. Moreover, t-NIL does not seem to have an impact on the materials' crystallinity, since they remain amorphous after their imprinting treatment. Finally, reflections derived from the silica nanoparticles are not to be expected in the measurements' range, since MCF is amorphous [45] and SBA-15 reflects at lower angles [45,46]. 10 20 30 40 2θ (⁰)

Differential Scanning Calorimetry (DSC)
In addition to XRD analysis, DSC measurements were also conducted, to further elaborate on the thermal properties of the nanopatterned materials. The obtained results are presented in Figure  7. The α-relaxation observed as an endothermic phenomenon right after Tg, along with the strong cold crystallization further support the XRD findings. The addition of the nanoparticles decreased the mobility of the polymer chains in the nanocomposites, but their effect was not very pronounced as the glass transition temperatures increased by 2-3 °C. However, the fillers acted as crystallization nuclei as the lower cold crystallization temperature (Tcc) recorded clearly demonstrates [47]. Overall, the thermal properties of the nanocomposites were improved compared to the pristine material, with an increased glass transition and a lower crystallization temperature. A more detailed analysis of the thermal properties and the effect of the silicas on the crystallization of the materials can be found on our recent publication [38]. The values of the samples' characteristic temperatures are summarized in Table 2.

Differential Scanning Calorimetry (DSC)
In addition to XRD analysis, DSC measurements were also conducted, to further elaborate on the thermal properties of the nanopatterned materials. The obtained results are presented in Figure 7. The α-relaxation observed as an endothermic phenomenon right after T g , along with the strong cold crystallization further support the XRD findings. The addition of the nanoparticles decreased the mobility of the polymer chains in the nanocomposites, but their effect was not very pronounced as the glass transition temperatures increased by 2-3 • C. However, the fillers acted as crystallization nuclei as the lower cold crystallization temperature (T cc ) recorded clearly demonstrates [24]. Overall, the thermal properties of the nanocomposites were improved compared to the pristine material, with an increased glass transition and a lower crystallization temperature. A more detailed analysis of the thermal properties and the effect of the silicas on the crystallization of the materials can be found on our recent publication [38]. The values of the samples' characteristic temperatures are summarized in Table 2.  The thermal stability of the materials was also assessed, by means of TGA. From the results presented in Figure 8, it is clear that the materials' degradation was impeded by the nanoinclusions, since the mass loss of the nanocomposites started at higher temperatures compared to neat PLLA and the Tmax also increases [48,49]. It is worth noting that all the nanocomposite films presented an increased mass residue compared to neat PLLA films. This increase can be associated with two different facts-firstly, after the nanofillers' addition into the polymeric matrix, it is more difficult for

Thermogravimetric Analysis (TGA)
The thermal stability of the materials was also assessed, by means of TGA. From the results presented in Figure 8, it is clear that the materials' degradation was impeded by the nanoinclusions, since the mass loss of the nanocomposites started at higher temperatures compared to neat PLLA and the T max also increases [47,48]. It is worth noting that all the nanocomposite films presented an increased mass residue compared to neat PLLA films. This increase can be associated with two different facts-firstly, after the nanofillers' addition into the polymeric matrix, it is more difficult for the macromolecular chains to move while some of them are even immobilized in the so-called rigid amorphous fraction (RAF) [6,49,50], resulting in a larger final mass residue and secondly, MCF and SBA-15 nanoparticles' degradation does not start in the temperature range where the measurements were conducted [24], as previously showed from the nanoparticles' TGA analysis. Therefore, part of the final residual mass corresponded to that of the incorporated nanoparticles, leading to a total mass increase. Taking the above-mentioned facts into consideration, it is clear that the thermal stability of the produced materials was improved. The values for the characteristic temperatures and mass residues at 500 • C are summarized in Table 3, while DTG curves are presented in Supplementary Materials ( Figure S3).

Nanoindentation Tests
To investigate the mechanical properties of the prepared materials, nanoindentation tests were conducted to compare their elastic modulus and hardness. This method is based on measuring the load as a function of the penetration depth [52]. The loading-unloading curves show no discontinuities or steps indicating the absence of any cracks during indentation, while PLLA/MCF 1 and 2.5 wt% showed smaller creep at the peak force. The indentation depths at the peak load range was between approximately 0.6 and 1.1 μm for all nanocomposite films. The loading-unloading

Nanoindentation Tests
To investigate the mechanical properties of the prepared materials, nanoindentation tests were conducted to compare their elastic modulus and hardness. This method is based on measuring the load as a function of the penetration depth [51]. The loading-unloading curves show no discontinuities or steps indicating the absence of any cracks during indentation, while PLLA/MCF 1 and 2.5 wt% showed smaller creep at the peak force. The indentation depths at the peak load range was between approximately 0.6 and 1.1 µm for all nanocomposite films. The loading-unloading nanoindentation curves can be found in Supplementary Materials ( Figure S2). Results showed that the addition of 1 and 2.5 wt% MCF nanoparticles led to a dramatic increase in indentation hardness and elastic modulus, since silica nanoparticles are materials of increased hardness compared to polymers [52] and consequently their dispersion into the polymer matrix resulted in materials of increased mechanical strength. Regarding the SBA-15 nanoparticles, while the elastic modulus was increased for both 1 and 2.5 wt% concentrations, the hardness increase was only significant in the case of 2.5 wt%. When filler incorporation increased to 5 wt%, in both materials the mechanical properties deteriorated compared to those of 2.5 wt%, which is probably an indication of agglomerate formation. The nanoindentation results are presented in detail in Table 4.

Antibacterial Activity Analysis
While trying to develop materials for smart-packaging, the most fundamental challenge is to eliminate or substantially reduce the ability of bacteria to adhere to material surfaces and to prevent biofilm formation. The t-NIL technique is a viable strategy to create micro-or nanometer-scaled topographies like slopes walls, columns etc., on materials surfaces, that drastically reduce the available area for the bacteria to attach compared with their smooth unpatterned equivalents [53].
The bactericidal properties of the nanopatterned films were examined by incubating two species of bacteria on each film surface for periods up to 24 h and assessing their bacterial viability. Specifically, Gram-negative E. coli and Gram-positive S. aureus were separately grown on the surface of the nanocomposite films PLLA/MCF 2.5 wt%, and PLLA/SBA-15 2.5 wt%.
As shown in Figure 9, lower cell densities were measured on the nanostructured surfaces (PLLA/SBA-15 2.5 wt% and PLLA/MCF 2.5 wt%) compared to the unpatterned films. Moreover, S.aureus presented the lowest growth on the surfaces with hierarchical structure (PLLA/SBA-15 2.5 wt% TOP and PLLA/MCF 2.5 wt% TOP) compared to its respective ones without topography (PLLA/SBA-15 2.5 wt% NO TOP and PLLA/MCF 2.5 wt% NO TOP). Similar results were obtained by studying the bacterial growth of E. coli on the surface of the above-mentioned films, with nanopatterned PLLA/MCF 2.5 wt% presenting the best antibacterial activity. Taking these results into consideration, it can be concluded that the nanocomposite PLLA films' surfaces enhanced with micro/nano-topographical features inhibited significant bacterial growth compared to those with flat or unpatterned surfaces, as the available area for bacteria attachment w reduced [32,33,54,55].
available area for the bacteria to attach compared with their smooth unpatterned equivalents [54].
The bactericidal properties of the nanopatterned films were examined by incubating two species of bacteria on each film surface for periods up to 24 h and assessing their bacterial viability. Specifically, Gram-negative E. coli and Gram-positive S. aureus were separately grown on the surface of the nanocomposite films PLLA/MCF 2.5 wt%, and PLLA/SBA-15 2.5 wt%.
As shown in Figure 9, lower cell densities were measured on the nanostructured surfaces (PLLA/SBA-15 2.5 wt% and PLLA/MCF 2.5 wt%) compared to the unpatterned films. Moreover, S.aureus presented the lowest growth on the surfaces with hierarchical structure (PLLA/SBA-15 2.5 wt% TOP and PLLA/MCF 2.5 wt% TOP) compared to its respective ones without topography (PLLA/SBA-15 2.5 wt% NO TOP and PLLA/MCF 2.5 wt% NO TOP). Similar results were obtained by studying the bacterial growth of E. coli on the surface of the above-mentioned films, with nanopatterned PLLA/MCF 2.5 wt% presenting the best antibacterial activity. Taking these results into consideration, it can be concluded that the nanocomposite PLLA films' surfaces enhanced with micro/nano-topographical features inhibited significant bacterial growth compared to those with flat or unpatterned surfaces, as the available area for bacteria attachment w reduced [32,33,55,56].

Conclusions
In this study, biobased nanoimprinted films of PLLA nanocomposites were prepared and studied. Home-made porous silica nanoparticles were prepared and melt-mixed with pristine PLLA, and the materials were spin-coated to form thin films containing 1, 2.5, and 5 wt% MCF and SBA-15 nanoparticles, respectively. To introduce antibacterial activity to their surfaces, the films were nanopatterned with t-NIL. Evaluation of the films' properties showed that the combination of techniques used, led to the successful synthesis of amorphous nanocomposite materials. The thermal and mechanical properties of the materials could be altered on-demand, depending on the amount of filler incorporated in the matrix, as they were found to act both as reinforcing and nucleating agents. In addition, all nanocomposite films exhibited improved thermal stability, as degradation started at increased temperatures and higher mass residues were achieved with increased nano silicate concentration. Finally, the nanostructures created from t-NIL were found to significantly inhibit the attachment of E. coli and S. aureus on the PLLA surfaces. This suggests that t-NIL is a viable and promising technique for designing materials with antibacterial properties. The fact that mesoporous home-made nanoparticles, that are known carriers for antimicrobial agents, can also act Figure 9. Number of CFU formed on the surface of the films prepared in this work (ns; non susceptible, *; low CFU number, **; high CFU number, ***; very high CFU number).

Conclusions
In this study, biobased nanoimprinted films of PLLA nanocomposites were prepared and studied. Home-made porous silica nanoparticles were prepared and melt-mixed with pristine PLLA, and the materials were spin-coated to form thin films containing 1, 2.5, and 5 wt% MCF and SBA-15 nanoparticles, respectively. To introduce antibacterial activity to their surfaces, the films were nanopatterned with t-NIL. Evaluation of the films' properties showed that the combination of techniques used, led to the successful synthesis of amorphous nanocomposite materials. The thermal and mechanical properties of the materials could be altered on-demand, depending on the amount of filler incorporated in the matrix, as they were found to act both as reinforcing and nucleating agents. In addition, all nanocomposite films exhibited improved thermal stability, as degradation started at increased temperatures and higher mass residues were achieved with increased nano silicate concentration. Finally, the nanostructures created from t-NIL were found to significantly inhibit the attachment of E. coli and S. aureus on the PLLA surfaces. This suggests that t-NIL is a viable and promising technique for designing materials with antibacterial properties. The fact that mesoporous home-made nanoparticles, that are known carriers for antimicrobial agents, can also act as reinforcing agents for a biobased packaging material, indicates that novel technological pathways are available to reach sustainable smart packaging.

Conflicts of Interest:
The authors declare no conflict of interest.