Poly (ε-caprolactone) microspheres for prolonged release of selenium nanoparticles

Poly (є-caprolactone) (PCL) microspheres as a carrier for sustained release of antibacterial agent, selenium nanoparticles (SeNPs), were developed. The obtained PCL/SeNPs microspheres were in the range 1-4 m with the encapsulation efficiency of about 90 %. The degradation process and release behavior of SeNPs from PCL microspheres were investigated in five different degradation media: phosphate buffer solution (PBS), a solution of lipase isolated from the porcine pancreas in PBS, 0.1M hydrochloric acid (HCl), Pseudomonas aeruginosa PAO1 cell-free extract in PBS and implant fluid (exudate) from the subcutaneously implanted sterile polyvinyl sponges which induce a foreign-body inflammatory reaction. The samples were thoroughly characterized by SEM, TEM, FTIR, XRD, PSA, DSC, confocal microscopy, and ICP-OES techniques. Under physiological conditions at neutral pH, a very slow release of SeNPs occurred (3 and 8% in the case of PBS or PBS+lipase, respectively and after 660 days), while in the acidic environment their presence was not detected. On the other hand, the release in the medium with bacterial extract was much more pronounced, even after 24 h (13%). After 7 days, the * Corresponding author: Magdalena Stevanović; Tel.: +381-11-2636-994; Fax:+381-11-2185-263 Institute of Technical Sciences of the Serbian Academy of Sciences and Arts Knez Mihailova 35/IV, 11000 Belgrade, Serbia E-mail address :magdalena.stevanovic@itn.sanu.ac.rs; magir@eunet.rs ACCEPTED MANUSCRIPT


INTRODUCTION
Degenerative and inflammatory diseases of the bones and joints make half of all chronic diseases in middle age people or older population in developed countries [1]. As a result, millions of medical devices are used every year. However, a significant quantity of these devices becomes colonized by microorganisms leading to implant-related infections, following implant damage and foreign-body reaction [2]. Despite the great progress in the field of biomaterials, this is still a major problem in orthopedics and soft tissue augmentation which causes implants failure. In a study conducted by Mittal et al. [3], more than 90 % of investigated patients stated that this is the largest drawback of metal implants. Implant accompanied infections are the consequence of bacterial adhesion to implant surfaces triggering the biofilm formation at the implantation site [4]. The formation of biofilms occurs in several phases leading to several major problems. The first problem is that bacterial populations on implant surfaces may become a reservoir of bacteria that can spread through the whole body. Furthermore, biofilms are highly resistant to antibiotic therapy so it is extremely hard to eliminate these bacteria by conventional antimicrobial therapies. Because the immune system and antimicrobial therapies are often inefficient to eliminate bacteria forming the biofilm, a chronic infection may take place [5]. It is a great challenge to manage orthopedic and soft-tissue augmentation implant infections that can cause implant replacement and also, in severe cases, can lead to amputation and death. About two-thirds of all orthopedic implant

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A C C E P T E D M A N U S C R I P T infections are initiated by different strains of staphylococci [6]. Actually, the most serious problems could be caused by Staphylococcus aureus and Pseudomonas aeruginosa infections [7]. Staphylococcus aureus bacteria are the major contributing agents of two main infections affecting bone: arthritis and osteomyelitis, which are associated with inflammation and bone destruction [8]. Pseudomonas bacteria usually do not cause severe infections in healthy people [9]. However, infections caused by Pseudomonas aeruginosa are commonly associated with other infections, i.e. they often appear in already immunocompromised patients. Pseudomonas is the most frequent pathogen find in patients who have been hospitalized for an extended period of time, and it is a frequent cause of nosocomial infections [10].
Pseudomonas infections often occur, for example, when there is already an existing infection caused by Staphylococci.
With the aim to prevent postsurgical infection, systemic antibiotic therapy is commonly applied to patients after the implantation [11,12]. However, there are many weaknesses, such as relatively low antibiotic concentration at the target site, as well as potential toxicity [13]. Also, consistent usage of broad-spectrum antibiotics may trigger resistant microorganism infections, which are associated with worse outcomes and higher costs [14,15]. Taking all this into account, coating/impregnation of implants with non-antibiotic antimicrobial substances emerged as a promising approach. However, many currently used non-antibiotic antimicrobial coating materials have been shown to be insufficiently efficient. For example, silver ions, well-known antimicrobial agent, are readily precipitated by chloride ions in human tissues [16]. Also, it should be mentioned that coating devices with silver, gold, and platinum is expensive [17].
On the other hand, SeNPs recently gained attention as a material which possesses antibacterial, as well as antiviral activities [18][19][20][21]. Selenium is an essential trace element in human bodies, required for their normal functioning [22]. Furthermore, recent findings indicate that selenium has critical roles in different physiological processes, including the modulation of immune responses [23] and it is necessary for bone health [24]. The effects of selenium on bone and the underlying mechanisms are well described in the review of Zeng et al. [25]. Selenium deficiency can retard growth, modify bone metabolism and increase the risk of bone disease [25][26][27]. However, selenium can be also toxic at concentration levels not much higher than the beneficial requirement [28,29]. Conversely, compared to elemental selenium, SeNPs have shown a reduced risk of selenium toxicity, but same bioavailability and efficacy compared with other seleno-compounds [30,31].

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Herein, we describe for the first time, the synthesis and characterization of SeNPs encapsulated within PCL microspheres with the aim to establish a system which will be capable to slow release the SeNPs from PCL matrix on site and when it is needed, i.e. when infection or inflammation occurs.
PCL is chosen since it is FDA-approved aliphatic polyester which belongs to a group of slowdegrading polymers. Some of its numerous applications in the field of biomedicine are summarized in a few review papers [32][33][34]. Although the majority of researches done with PCL in the past few years are focused on tissue engineering [35], this polymer still has a great potential in drug delivery systems thanks to its excellent biocompatibility [36] or ability to provide sustained release [37]. Also, the slow degradation and prolonged release of active components from PCL could be very beneficial in implant coatings approach. In this case, it is possible to provide antimicrobial protection, prevent disease remission or alter regeneration in a reasonable long period. For such system, degradation rate and release behavior are parameters that must be thoroughly investigated. The degradation rate is highly dependent on several factors including the degree of crystallinity, hydrophilicity, copolymer composition, molecular weight, molecular architecture, size and geometry of the samples, and the conditions in the degradation environment.
In this study, degradation and release behavior of newly synthesized PCL/SeNPs microspheres were investigated in five different media: phosphate buffer solution (PBS), solution of lipase isolated from the porcine pancreas in PBS, 0.1M hydrochloric acid (HCl), Pseudomonas aeruginosa PAO1 cellfree extract in PBS and implant fluid (exudate) from in vivo implanted sterile polyvinyl sponges which induce a foreign-body inflammatory reaction. Samples were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), particle size distribution analysis (PSD), differential scanning calorimetry (DSC) and inductively coupled plasma optical emission spectrometry (ICP-OES). The influence of PCL/SeNPs on cell viability, ROS generation and formation of DNA strand breaks in HepG2 and phagocytic Raw 264.7 cells was investigated. The antibacterial activity of the samples was determined against Gram-positive bacteria: Staphylococcus aureus (ATCC 25923) and Staphylococcus epidermidis (ATCC 1228).

Synthesis of SeNPs
SeNPs were synthesized by simple chemical reduction, using sodium selenite as a source of selenium ions, ascorbic acid as a reducing agent and BSA as a stabilizer. Droplets of 20 mM solution of sodium selenite (12.5 ml) and 8.6 % solution of BSA (w/v, 5 ml) were simultaneously added to 0.125 M solution of ascorbic acid (10 ml). The reaction vessel with ascorbic acid (also where the reduction takes place) was covered with aluminum foil in order to prevent interaction with light. The obtained brick red colloidal solution of SeNPs was homogenized for 30 min on a magnetic stirrer (1000 rpm) and then filtered through the 0.24 μm syringe filter (Millipore). The final solution was stored in a refrigerator. The amount of SeNPs in colloidal solution was determined by ICP-OES. For the purpose of characterization by FTIR, XRD and antibacterial testing, obtained colloidal solution of SeNPs was lyophilized.

Encapsulation of SeNPs within PCL microspheres
The obtained colloidal solution of SeNPs was further used for encapsulation within PCL microspheres using a solvent/nonsolvent method. Briefly, 300 mg of commercial PCL granules was dissolved with mild heating to 50 °C in 30 ml of acetone. After that, 0.5 ml of a solution containing SeNPs was dropwise added to the organic phase. A high-speed homogenizer was used for 5 min at 21 000 rpm to homogenize this mixture. The obtained mixture was poured into a non-solvent system ethanol (75 ml) followed by addition of 0.05 % PGA solution (10 ml). This instantly resulted in precipitation of PCL microspheres loaded with SeNPs. Homogenization was then carried on a magnetic stirrer for 30 min.
The encapsulation efficiency EE% of SeNPs was determined based on the following equation: where W e is the amount of incorporated SeNPs within PCL microspheres, determined experimentally by ICP-OES, and W i is the total quantity of SeNPs added initially during the preparation procedure. In order to thoroughly investigate some properties of obtained PCL/SeNPs, blank PCL microspheres were produced by the same procedure without the addition of SeNPs and high speed homogenization.

Morphology studies
The morphology of as-synthesized SeNPs and PCL/SeNPs was analyzed by SEM (JEOL JSM-639OLV) and TEM (2100 microscope, Jeol Ltd., Tokyo, Japan). For SEM analysis, the samples were coated with gold using the physical vapor deposition (PVD) process. The covering was performed by a Baltec SCD 005 sputter coater, using 30 mA current from the distance of 50 mm during 180 s. For TEM analysis, samples were prepared by placing drops of suspension-containing particles onto a lacey carbon film supported by a 300-mesh-copper grid.

Fourier-transform infrared spectroscopy (FTIR)
The quality analysis of the samples was performed by FTIR spectroscopy. FTIR spectra of samples were obtained on MIDAC M 2000 Series Research Laboratory FTIR Spectrometer, using the KBr pellet technique. Measurements were performed in a spectral range of 400-4000 cm −1 at room temperature.

X-ray diffraction (XRD) measurements
X-ray diffraction spectra were obtained on an X-ray diffractometer, Philips PW 1050 diffractometer with Cu-Kα radiation (Ni filter). The samples were scanned in the 2θ range of 10° to 60°, with a scanning step width of 0.05°, and 2 s per step. Crystallite size determination was carried out using a variant of the Scherrer equation: where D is the apparent crystallite size, b is the full-width at half maximum FWHM of the X-ray diffraction line (peak broadening) in radians, λ is the wavelength used 1.5406 Å, and θ is the angle between the incident ray and the scattering planes. The constant of 0.9 is a shape factor and its value depends on crystallite morphology.

Particle size analysis (PSA)
The particle size distribution of SeNPs and PCL/SeNPs samples was determined by the PSA

Biocompatibility study
First, the investigations regarding cell viability (MTT assay), the formation of reactive oxygen species (DCFH-DA assay) and genotoxicity (comet assay) were conducted on the HepG2 cell line. Cell viability after the exposure to PCL/SeNPs was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) according to Mosmann [39] with minor modifications [40]. The formation of intracellular reactive oxygen species (ROS) was measured spectrophotometrically using a fluorescent probe, dichloro-dihydro-fluorescein diacetate (DCFH-DA) as described elsewhere [41]. In were suspended in 7.5ml of the above-mentioned media and placed in water bath at 37°C. (sodium azide was added to the first two media). At exact times, samples were collected, centrifuged (10 min at 7000 rpm) and sediments and supernatants separated by decantation. In order to remove a residue from media, sediments were washed several times with distilled water, filtered through the quantitative filter paper and left to dry at room temperature for two days. All samples were stored in a refrigerator before analysis.

Study on degradation efficiency of PCL by a Pseudomonas aeruginosa PAO1 cell-free extract
We investigated the ability and the role of this bacterium on PCL degradation, using a microcosm approach in order to mimic bacterial infections conditions. For this purpose, Pseudomonas aeruginosa Bradford method [44]. CFE was used in two different experimental setups including semi-solid agarbased medium and aqueous PBS medium.

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Experiments on agar plates were conducted in order to prove degradability potential of CFE toward PCL. Firstly, a polymer suspension was prepared by dissolving 100 mg of PCL polymer in 2ml of dichloromethane and water up to 20ml, followed by sonication (60Hz, 5 pulses of 1 min) [45]. This suspension was further warmed at 65 o C to evaporate dichloromethane and then mixed with agar (final concentration 1% w/v in 200 mMTris-HCl buffer pH 8.5) in 1:1 ratio and poured into a glass Petri dish.
After solidification at ambient temperature, agar plugs (diameter = 3 mm) were taken out of plates for the addition of cell-free extracts. For the purpose of this experiment, P. aeruginosa PAO1 was grown in MSM medium using either glucose or olive oil as carbon sources. Cell-free extracts (50 µl) from both growth media were applied into wells and plates and incubated for 24 h at 30 o C when another aliquot of cell-free extracts (50 µl) was added to wells and plates and further incubated for 3 days at 30 o C.

Influence of P. aeruginosa PAO1 CFE toward PCL/SeNPs in aqueous PBS medium
For experiments in an aqueous medium, 85 mg of PCL/SeNPs powder was suspended in a mixture of 20 mM PBS (8 ml) and P. aeruginosa CFE. The experiment was carried out for three weeks at 37 o C. Cell-free extracts (2 ml; 1.8 to 2 mg of total protein/ml) were added in regular periods, three times throughout the duration of the experiment. After 24 h, and at the beginning of the second and third week, before the addition of fresh cell-free extracts, aliquots of 1ml were taken from the reaction mixture, centrifuged (5 min, 13000 rpm, Eppendorf Centrifuge 5417C, Hamburg, Germany) and pellets and supernatants stored at -20 o C for further analysis.

Degradability potential of implant exudate on PCL/SeNPs.
To observe whether SeNPs could be released from PCL microspheres during an inflammatory process accompanying implantation, a rat model of sterile inflammation to foreign-body was applied. Namely, Albino Oxford (AO) rats, both sexes, 12 weeks old, were bread at the Institute for Medical Research of the Military Medical Academy (MMA). All animal experiments were approved by the Ethical Committee for Protection of Experimental Animals of MMA. A sterile inflammation to foreign body was induced by subcutaneous implantation of polyvinyl sponges (1cm x 1.5cm x 0.25cm), as described previously [46].
Two sponges per animal were implanted at the dorsal site of the skin, under the general ketamine/xylazine anesthesia. To collect the exudate, animals were sacrificed by the anesthetics overdose, and the sponges were harvested 2 days after implantation. The exudate was squeezed with a syringe and the cells were pelleted by centrifugation (2000 RPM for 10 minutes). The in vitro release of SeNPs from PCL microspheres in cell-free exudate was carried out by incubating PCL/SeNPs (0.5mg/ml) in the exudate at 37 o C and 5% CO2 for 11 days, followed by the measurement of released SeNPs with ICP-OES. For the ICP-OES analysis, the exudate was filtered and then the measurements were done directly.
To assess the degradability of PCL/SeNPs in vivo, PCL/SeNPs were injected into subcutaneously implanted sponges (totally 4 mg/animal), whereas the control groups received the equivalent amount of sterile PBS. The sponges from control and treated animals were extracted after 3 hours, 4 days or 11 days (2 animals per group per time point). The exudate was used for ICP-OES analysis. In addition to standard ICP-OES analysis used to detect released SeNPs, the samples were prepared without filtration, to include SeNPs within PCL in sponges (Total Se) in the exudate. The infiltrating cells isolated from sponges were placed on microscope slides using the cytocentrifuge (Shandon 4, ThermoFisher Scientific), and analyzed by confocal microscopy, as described for Raw 264.7 cells.

Antibacterial activity
The antibacterial effects of PCL/SeNPs, as well as SeNPs alone, were examined against

Physicochemical characterization of as-prepared samples SeNPs and PCL/SeNPs
The XRD pattern of lyophilized SeNPs revealed that Se was in amorphous form ( Figure 1a). The only peak which can be noticed on the XRD pattern ( Figure 1a) is peak which belongs to BSA which is used in the synthesis of SeNPs as a stabilizer. The concentration of SeNPs in the colloidal solution was estimated by ICP-OES to be 600±61 μg/ml, as calculated from three different batches. Morphology and size of nano-and microparticles along with surface chemistry are one of the most influential parameters that determine their fate within biological systems [48][49][50]. One of the major requirements for the controlled and well-balanced release of the drugs in the body is its ideal spherical shape of the particles and narrow distribution of their sizes [17]. A report from particle size distribution measurement of SeNPs indicates that 50 % of particles have a radius below 57 nm, while 90 % of them are smaller than 97 nm ( Figure 1b). The Figure 1c, TEM image obtained from Se colloidal solution, shows that SeNPs are quite uniform and spherical with a diameter below 100 nm. The stability of the colloidal solution, stored in a refrigerator, is estimated to be at least four months, based on the appearance of turbidity. matrix. Based on equation 1 given above, the EE% was calculated to be 90.2% while the loading amount of Se in the PCL/SeNPs system was determined to be 0.0946%. Controlled release systems such as PCL/SeNPs can be an effective means for local drug delivery. In local drug delivery, the main goal is to supply therapeutic levels of an active substances at a physical site in the body for a prolonged period. A second goal is to reduce systemic toxicities [51]. Although selenium is needed for the normal functioning of the body, the problem is that it displays a narrow spectrum between favorable and toxic effects [28,29]. According to the U.S. Food and Drug Administration (FDA) recommended dietary allowances (RDAs) of selenium is 55 μg for adults [52] while the National institutes for health (NIH) consider 400 μg of selenium as the tolerable upper intake level (ULs) i.e. maximum daily intake unlikely to cause adverse health effects [53]. In the case of our system, this amount will be accomplished with roughly 420 mg of PCL/SeNPs powder. Also, the additional aspect of SeNPs loading amount which we have considered is the fact that, in this case, there is no significant amount of Se adsorbed on the surface of PCL microspheres and hence no accompanying burst release. This is very important since eliminates the possibility of a potential initial local toxic effect of SeNPs so the system can be considered as safe for size of the particles. In our previous work, PGA has proved to be a good stabilizing agent for obtaining PCL submicron particles [54]. It is characterized by good biocompatibility and adhesive properties which allow its application in the food and pharmaceutical industry, even as a component of surgical glues [55,56].

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The XRD patterns of blank and loaded PCL microspheres are given in Figure 3b. On both diagrams, two dominant peaks come from diffraction from (110) and (200) crystalline planes [59]. It is noticed a slight shift in 2θ values from 21.55° and 23.85° for the blank polymer to 21.65° and 23.95° for polymer loaded with SeNPs, respectively. Besides these diffraction peaks, a peak of small intensity at 26.1° 2θ, that originates from PGA was also noticed (SI Figure 2). One can also observe that the baseline for the sample containing SeNPs, is slightly lifted (

Biocompatibility study
The biocompatibility and safety of PCL/SeNPs were tested with the combination of several methods, namely MTT and comet assay, respectively, while the influence on the formation of reactive oxygen species was assessed with DCFH-DA method. The PCL is one of the synthetic polymers recognized as biocompatible and bioresorbable by leading world organizations such as FDA. On the other hand, as it was already mentioned above, besides its beneficial effects, selenium is considered as a very toxic material so its biomedical application is often limited and required great precautions. In our previous work, we reported that blank PCL particles have no toxic effects toward HepG2 cells [57].  since there was no difference between them and those obtained for the solvent control; c) The induction of ROS formation after 5h exposure to a different concentration of PCL/SeNPs. The data for the control were excluded since there was no difference between them and those obtained for the solvent control; d) Relative fluorescence units RFU measured at every 30 min over 5h forPCL/SeNPs concentrations from 0 to 1.

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The first cells to arrive at the site of implantation and infection are professional phagocytes, such as granulocytes and macrophages [2]. Thereby, the intracellular PCL/SeNPs could behave differently and induce cytotoxicity upon their internalization. Therefore, we also tested whether PCL/SeNPs can induce cell death of Raw 264.7 murine macrophages after 24 h in vitro. Expectedly, Raw 264.7 cells were able to internalize smaller PCL/SeNPs, whereas the larger ones were surrounded by Raw 264.7 cells and were located extracellularly (Figure 5a). Due to their surface plasmon resonance at 520 nm [62], SeNPs could be detected by confocal microscopy as strongly scattering nanoparticles upon 546 nm laser excitation.
The analysis confirmed that smaller PCL/SeNPs were indeed localized within Raw 264.7 cells, as well as outside the cells. SeNPs were largely confined within the PCL microspheres irrespective of their intracellular localization.  (25,50 or 100 μg/ml). After that, the cells were harvested and the viability was determined by Tripan blue exclusion test.
The results are shown as mean ± SD (n=3 measurements) of viability relative to control non-treated cells (100%), from a representative experiment out of two with similar results. **p<0.01 compared to control (0 μg/ml).
Moreover, the viability of Raw 264.7 cell was not decreased for more than 20% after 24h cultures at the highest concentration used 100 μg/ml (0.01 v/v%) in these experiments, suggesting a lack of significant cytotoxicity for phagocytic Raw 264.7 cells (Figure 5b). These results suggested that there is no significant release of SeNPs from PCL within 24h, even upon the internalization. However, to confirm this hypothesis, more sensitive techniques should be applied to study the intracellular release of SeNPs from PCL.

Calorimetric studies
Before starting our discussion, it should be emphasized that all samples were taken from the same batch so all of them had same starting morphology, microstructure, percent of crystallinity, and the same amount of SeNPs (assuming a homogeneous distribution of SeNPs). Therefore, we can conclude that the sample degradation rate and release of SeNPs was influenced only by the media nature. DSC diagrams of the samples suspended in phosphate buffer solution (PBS), PBS with lipase isolated from porcine pancreas and 0.1M HCl are given in Figure 6.

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Although DSC is not very precise and is often mistakenly considered as a technique for routine measurements of phase transitions, it provides useful data for interpretation of polymer structure and interactions based on the shape of the melting/crystallization curves. Generally, the more imperfect polymer crystals are and the wider distribution of lamella thickness is, the more irregular shape of the melting curve will be recorded (broadness of the peak, asymmetrical profile, shifting of melting point, etc.). If we compare diagrams obtained for each medium, we could observe the difference in shapes for samples taken after one week and at the end of experiments. Melting endotherms measured after one week were quite symmetrical, while those obtained at the end of experiments were irregularly shaped in a lower temperature range. This irregularity slowly evolves with degradation time in pH-neutral media (Figures 6a and 6b) while in the acidic medium (Figure 6c), it appears only at the end of the experiment and is less pronounced. The appearance of shoulders in lower temperature parts of the peaks could be related to the bimodal distribution of lamella thickness. The melting peak temperatures and corresponding heat of fusions are given in SI Table 1. It is evident that melting temperature and heat of fusion increase with degradation time since degradation first takes place in amorphous regions. By comparing the values of crystallinity slightly higher values were noticed in samples taken from PBS+lipase between 5 th and 15 th week. The overall increase in crystallinity is almost the same for all media, about 15 % in total.

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Compared to Figure 3b, the first notable change in all XRD patterns (Figure 7 a-c) is a disappearance of PGA diffraction peak which is probably due to PGA desorption from PCL surface during the degradation process. The stabilization effects of PGA on the PCL surface were previously determined in a similar system [54]. However, the interaction between these two polymers seems to be insufficient to overcome PGA desorption due to hydrophilic interactions with water. The second conclusion based on observation of all XRD patterns is that there is no formation of new crystalline phases or a significant change in crystallinity during the degradation period. As shown by Pinto et.al.
[63], amorphous selenium spontaneously crystallizes during storage at room temperature. However, this behavior is not observed in our system presumably because a stabilization effect of BSA is preserved in hydrophobic PCL environment. In order to closer investigate changes in the crystalline structure of PCL, the Scherrer equation was employed and results for crystallite size are presented in Figure 7. Calculations were made for both peaks present in XRD patterns ( (110) and (200) networks during degradation in medium with Pseudomonas lipase [59]. Conversely, in the same study, it is observed a constant decrease of crystallite size in PBS.

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Degradability potential of P. aeruginosa PAO1 CFE toward PCL and PCL/SeNPs
In order to examine the influence of P. aeruginosa PAO1 CFE on the degradation of PCL, experiments on agar plates were conducted. Zone of clearance around wells in the agar plate indicates the enzymatic degradability of the PCL polymer (Figure 8a.). In case of the CFE of culture grown on olive oil used as carbon source, a greater clearance zone was obtained (radius 10.5 mm) compared to the case when glucose was carbon source because higher esterase titer was induced under these growth conditions.

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The change of melting endotherms of PCL/SeNPs with degradation time is most clearly notable for the medium which contains P. aeruginosa PAO1 CFE (Figure 8). The evolution of shoulder in the lower temperature segment of the peak was visible even after 7 days of degradation, while in the media which simulate physiological conditions this phenomenon was prominent only for the samples taken after 108 and 660 days from degradation media. The overall increase in melting peak temperatures was 2.6 °C, which is comparable with experimental results for previous media, but with about 2 °C lower temperatures (SI Table 2). When it comes to crystallinity degree, the value obtained for the sample taken after one day was 53 % and afterward increased to 68% or 15% in total. These results are in a good agreement with those obtained for the first three media. Based on the DSC measurements, degradation effects such as an increase in crystallinity and bimodal distribution of lamellar thickness were achieved in the medium which contained P. aeruginosa PAO1 CFE after 3 weeks, while same effects were noticed in the first three media only after 660 days of degradation.

In vitro release behavior of SeNPs from PCL microspheres
The release of versatile drugs from PCL microspheres have been thoroughly investigated in the past. Details of these studies are summarized in a few excellent review papers [32][33][34]. Diffusion of drugs from the microsphere matrix is recognized as a dominant mechanism of drug release from PCL [34]. For this reason, drug distribution within polymer microspheres has a significant influence on the drug diffusion rate. For instance, drug molecules distributed closer to the microsphere surface diffuse out faster from the polymer matrix. Encapsulation of a hydrophilic drug within a hydrophobic polymer, such is PCL, usually results in drug molecules distributed closer to the polymer surface [64].
Another important aspect in drug release is degradation medium. Accelerated degradation could be achieved using an acidic/basic medium, or medium with adequate enzymes, which would enhance the hydrolysis of polyesters and better mimic physiological conditions than temperature degradation, for instance. The surrounding conditions, such as neutral or low pH, had different effects on SeNPs release during the degradation period in such manner that acidic environment inhibited SeNPs release (Figure 9.).
To the best of our knowledge, such behavior has not been reported so far in the literature. A possible explanation for this phenomenon is related to BSA conformation under the acidic conditions. Generally, a low pH causes unfolding of the BSA molecule. These conformational changes can further prevent passage of BSA with SeNPs through diffusion channels and release from the polymer matrix. Suppression of BSA release in the acidic environment was already shown with PLGA microparticles [65]. As expected, the presence of porcine pancreas lipase accelerated SeNPs release, but not in significant

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A C C E P T E D M A N U S C R I P T amount, from 2 to 8 %. On the other hand, a two-fold increase in SeNPs release was noticed in Pseudomonas extract after only 24 h and proceeded to its maximum value of around 30 % after 7 days.
During the following two weeks, the concentration of SeNPs in supernatant did not increase further. On the opposite, a small drop in SeNPs concentration was noticed, probably due to SeNPs adsorption on the polymer surface. Lipase isolated from Pseudomonas are well known for their ability to significantly accelerate the degradation of PCL [66,67]. For the first time in this work, a CFE, instead of a single isolated enzyme was used to better mimic the bacterial environment. Enzymatic biodegradation occurs mainly on the surface because it is difficult for these high molecular weight molecules to diffuse into a hydrophobic polymer. A reasonable explanation for the existence of the release profile plateau in Figure   9b is that all of the released SeNPs originate from regions that are amorphous and close to microsphere surface, which allows them to diffuse out faster from the polymer matrix. Conversely, the remaining amount of SeNPs (around 70 %) is deeply incorporated and located closer to crystalline phases. In order to release the remaining amount of SeNPs degradation process had to reach its final stage, i.e. breaking the polymer chains to soluble oligomers. Another possible explanation could be that some amount of SeNPs coated with BSA formed agglomerates which are not capable to diffuse out from the polymer matrix.
Bearing in mind results obtained from DSC and XRD measurements some correlations can be made regarding SeNPs release in different degradation media. Degradation processes such as the formation of diffusion channels probably produce some crystallite defects which further cause alteration of FWHM on XRD patterns. On the other hand, DSC technique is not sensitive enough to detect those initial degradation changes but it is still useful for detecting the change in crystallite size distribution as an evidence of advanced degradation when diffusion channels are well formed. In addition, the higher values of crystallinity noticed between 5 th and 15 th weeks in the medium PBS+lipase, as a consequence of higher degradation rate of amorphous regions, resulted in an increase in SeNPs release.
A C C E P T E D M A N U S C R I P T Figure 9. The release of SeNPs from different degradation media. a) Parallel view of release from PBS and PBS with lipase; b) Release from a medium which contains P. aeruginosa PAO1 CFE. For better comparison, the same scale was used for "y" axes in both graphics.

The release of SeNPs in implant fluid (exudate)
The release of SeNP from PCL was measured in implant fluid (exudate) extracted from the subcutaneously implanted polyvinyl sponges which induce a sterile inflammation as a part of foreign body reaction, as we described previously [46]. To test the release of SeNPs in vivo, 4 mg of PCL/SeNPs were injected in the sponges implanted subcutaneously into rats, followed by the extraction of sponges after 3 hours, 4 days or 11 days from the animals. At 3h time point, the total concentration of Se detected by ICP-OES analysis was shown to be 9%., and the presence of brightly scattering particles sized around 1-4 µm (PCL/SeNPs) was also detected by confocal microscopy within the infiltrating CD45 + cells or extracellularly (SI Figure 3).
The levels of total Se (released or contained within PCL) were undetectable in the extracted sponges after 4 and 11 days post injection. These results suggest that the PCL/SeNPs were not contained within the sponges, and where probably distributed by lymphatics throughout the body. Therefore, to better study the release of SeNPs from PCL in vivo, a kind of scaffold biomaterial would be a better model than the porous polyvinyl sponges, so additional investigations are required to resolve the dynamics of SeNPs release form PCL microspheres in vivo.
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Antibacterial activity
Although SeNPs are not recognized as a strong antibacterial agent, an increased scientific interest on this topic was noticed in the last several years. One of the main reasons for this growing interest are findings that elemental nano-Se expresses lower toxicity compared to other Se compounds and that this microelement is normally present in our bodies and very important for our health compared to other popular antimicrobial agents, such as Ag. Results of antibacterial activity of SeNPs as well as for PCL/SeNPs are presented in Figure 10. Minimum inhibitory concentration (MIC) is defined as the lowest concentration of a compound at which a microorganism does not demonstrate any visible growth. One can see that the SeNPs antibacterial effect against Staphylococcus aureus was twice stronger compared to that against Staphylococcus epidermidis. These results are similar to those obtained by other authors [21,68] and show that SeNPs have a good potential for the prevention of infections caused by investigated bacterial strains. Results of this study (Fig. 10) also provided evidence of a considerable antibacterial activity against both bacterial strains, in the presence of PCL/SeNPs as well. This probably can be explained also due to the fact that the PGA used for the stabilization of the PCL/SeNPs also has antimicrobial properties since it is a polyelectrolyte. The potent bactericidal activity of polyelectrolytes could be explained by their strong interaction with the charged cell membrane of bacteria [69].

A C C E P T E D M A N U S C R I P T
Also, it was found that particles possessed good biocompatibility since they showed no cytotoxicity, a low potential for ROS generation and a low genotoxicity potential. All these results imply that these designed particles could be a highly attractive and efficient platform for preventing infection on implants.  The degradation and release processes were investigated in five different media.  The release is triggered in the bacterial environment as well as by foreign body inflammatory reaction to implant.

A C C E P T E D
 PCL/SeNPs can be considered as biocompatible.  Considerable antibacterial activity against S. aureus and S. epidermidis was exhibited.