The essence of CMDF involves passing a polymeric filament through a saturated solution of the filament material and the substance to be embedded. Specifically, we demonstrate the scope of this approach by coating a PLA filament with Rhodamine B, ZnO NPs, and Cip, followed by their 3D printing.
3.2. ZnO NP Coating
Nanomaterials are particles with at least one dimension below 100 nm. The most studied nanomaterials are NPs, benefiting medicine, energy harvesting and storage, and sensing. The inclusion of nanomaterials within 3D objects can bring unique advantages. Metal oxide NPs, such as ZnO, are among the most promising materials for medical implants because of their potent antimicrobial activity, including antibiotic-resistant bacteria [
36]. ZnO NPs are low-toxicity, FDA-approved materials, with broad applications, as highly efficient antibacterial and antifungal materials [
37]. Therefore, we evaluated the applicability of our system for ZnO NPs. The resulting printed objects should have a uniform coating around and along the filament, and provide an antibacterial activity without affecting the mechanical properties of the PLA. Accordingly, we applied CMDF to coat the PLA filament by passing it through a dispersion of ZnO NPs, as described in section 2.3.2. The amount of ZnO around the filament (
Figure 5) and the printed samples (
Figure 6) was analyzed by a spectrophotometric method based on the complexation by Zincon, for samples obtained by dissolving the PLA in NaOH and adjusting the acidity (see
Section 2.3.5).
The amount of ZnO NPs was evaluated along the filament length and as a function of ZnO concentration in the coating dispersion. As seen in
Figure 5, the amount of Zn in all sections within the first meter of the filament decreases, until reaching a constant value. We expect this trend to continue for long filaments, changing only after a noticeable reduction of the concentration of ZnO NPs in the coating solution. For example, 35 m of filament coating would cause a decrease of only 5% ZnO NPs in the dispersion. This indicates that CMDF holds great promise for the treatment of long filaments. A second interesting finding concerns the significant increase of Zn in the printed samples as a result of increasing the concentration of dissolved PLA in the coating dispersion, as appears in
Figure 6. This alludes to an interaction between the PLA and the ZnO NPs, which is supported by the fact that the addition of PLA in the ZnO NPs solution increases the dispersion stability and prevents the aggregation of the NPs (
Figure S5).
Obtaining long-lasting antibacterial surfaces based on metal oxide NPs requires minimal leaching of the inorganic material. Hence, we examined the leaching of Zn(II) ions over time from the coated printed samples placed in a PBS solution (120 rpm at 37 °C) for 9 weeks. Less than 5 μg of Zn(II) ions leaching out of every gram of printed samples was detected after 9 weeks (less than 2.5% loss), implying very high durability of the additive NPs within the polymer.
Tensile strength testing was carried out to ensure that the mechanical properties of the filaments did not change during the coating process. Hence, the following filaments were tested after being printed doggybones in two orientations, XY and Z (Fig. S1): filaments that were not coated, filaments coated using 10 and 20 mg/ml of dissolved PLA without any added material, and filaments coated with up to 15 mg/ml ZnO NPs. XY samples were printed with the large face of the structure on the heated bed, thus enabling testing the PLA strands melted one onto the other at 90° layers. Z samples were printed upwards, applying the pressure of the Ingstrom machine during the measurement against the layers’ connection. It was found that the addition of the additives, i.e., PLA and ZnO NPs, did not affect the Young modulus (E) of all printed structures, at the two orientations (EXY= 530 ± 40 MPa. EZ= 510 ± 10 MPa). This lack of changes indicates that the bulk properties after coating and printing are unaffected.
ZnO NPs are often used for biological applications due to their effective antibacterial properties and structure-dependent physicochemical properties [
38]. Therefore, we conducted antibacterial tests to assess the effectiveness of the coating on the printed samples (
Figure 7). The test involved measuring the optical density of the solution containing the bacteria left after being exposed for four hours to the printed samples, and an additional 20 hours of incubation inside Lysogeny broth (LB). Hence, the lower the
O.D., the higher the antibacterial activity (see section 2.3.4 for more details).
Three important trends can be seen in
Figure 7: i) As expected, the more ZnO NPs present in the coating process, the stronger the antibacterial effect is. ii) Unexpectedly, the gram-negative
E.Coli showed stronger viability than the gram-positive
S.Aureus, requiring more ZnO NPs to prohibit its extermination. This stands against numerous studies and theories regarding the weaker resistance of gram-negative bacteria to metal oxide NPs in general, and the activity of ZnO NPs against
E.Coli in comparison to S.Aureus in particular [
36,
39]. iii) A stronger or equal antibacterial activity against E.Coli was found for all ZnO NP concentrations higher than 5 mg/ml when 10 mg/ml of PLA was used. We recall that the amount of ZnO NPs increases with that of PLA in the coating solution (
Figure 6). For this reason, we expected to find a greater eradication of bacteria on samples made with 20 mg/ml of PLA (as seen for
S.Aureus) against both bacteria.
The two surprising trends can be explained through possible compatibilities between E.Coli and FDM-printed PLA samples. We hypothesize that the cracks and pores on the sample’s surface are compatible with the growth pattern of E.Coli, thus acting as an optimal breeding site for the pathogen. Coating made using a high concentration of PLA could have resulted in a greater abundance of defects in the filament and print or a looser layer, providing additional optimal sites for bacteria growth. This claim is supported by the higher O.D. found of bacteria upon 20 and 10 mg/ml of dissolved PLA samples where no ZnO NPs were suspended (1.7±0.3 and 1.5±0.1 a.u. after 20 hours of incubation, respectively). However, an in-depth comparison of the growth speed of the different bacteria colonies on FDM-printed samples is beyond the scope of this research.
In summary, ZnO NPs dispersed in a hydrophobic phase for a long period were used to homogeneously coat a PLA filament. The embedded ZnO NPs were stable in the printed objects for nine weeks under physiological conditions, and showed high antibacterial activity against both gram-negative and gram-positive bacteria, indicating their potential use in biological implants and prostheses.
3.3. Cip Coating
To further test other capabilities of CMDF, we loaded small organic molecules and followed their rate of release and kinetics involved. The model material was Ciprofloxacin (Cip), known for its broad spectrum of antibacterial activity, possible anticancer activity, and relative ease of detection [
40,
41]. Cip is a proper candidate for such tests, as it has already shown in-vivo promise as a medical additive for preprinting surgical implants in rabbits [
22].
The release profile of the Cip was followed through UV light absorption measurements for an incubation time in PBS for up to 35 days (
Figure 8), for Printed samples coated in a solution containing 20 mg/ml PLA and 5 to 50 mg/ml dissolved Cip.
It was found that the release profile closely matches the Ritger-Peppas theoretical and empirical model for material diffusion out of a reservoir-type device (
Figure S6). In that model, the diffusing materials are non-covalently embedded within a polymeric structure and are released under a rate-controlling polymeric membrane through pores [
23]. It should be noted that we also carried out antibacterial tests and found full eradication of both bacteria by the printed samples containing Cip. As the applied method consists of prolonged direct exposure of highly concentrated bacteria suspension in small volumes and the surface, and clear direct Cip release was found, the result is as expected.