Incorporation of an Antibiotic in Poly(Lactic Acid) and Polypropylene by Melt Processing

Introduction

Poly(lactic acid) (PLA) can be considered a valid alternative to conventional petroleum-based polymers for many applications, from food packaging to biomedical applications, because of its chemical and physical properties and its decreasing, although still high, production cost (1–2–3–4–5–6). Nevertheless, the potential for massive use of PLA is limited due to the need for improvement of some of its functional properties, including mechanical characteristics and the gas/vapor barrier. However, it has been found that the above drawbacks can be overcome by the addition of an appropriate filler (7–8–9–10–11–12–13–14). Many studies have been conducted to provide PLA with antimicrobial properties that might encourage its use in active food packaging as well as in specific biomedical applications (15–16–17–18–19–20). Controlled drug delivery systems are attracting increasing attention in the biomedical and pharmaceutical sectors (21–22).

Providing a polymeric device with antibacterial properties can be achieved by different routes, including the modification of the polymer structure. The incorporation of antimicrobials, or other molecules, into a polymer matrix by melt processing is a method that has been widely adopted in the recent past since it has the advantage of using equipment already commonly used to process thermoplastic materials (23–24–25–26–27–28–29–30–31–32). This method also ensures large production volumes and solventless systems with obvious positive implications for environmental and economic factors. Nevertheless, the high temperatures involved in melt processing may lead to the melting and/or decomposition of the antimicrobial agent, with consequent deactivation. Thus, it is mandatory to know both the melt and decomposition temperatures of the biocide additives in order to avoid their degradation.

In our previous works, we successfully prepared different antimicrobial polymeric systems by melt compounding (24–25–26–27, 29–30–31–32). In particular, we incorporated different antibacterial additives into an appropriate polymeric matrix, taking both the final application and the processing temperature of the polymeric sample into account when choosing the polymer-antibacterial pairing.

Ciprofloxacin (CFX) is a wide-spectrum antibiotic belonging to the fluoroquinolone family. It is active against many strains of bacterial pathogens responsible for urinary tract, respiratory, abdominal and gastrointestinal infections, including both Gram-negative and Gram-positive ones (33–34–35–36). Its melting temperature is about 268°C (37).

In this work, PLA and PP samples incorporating CFX by melt compounding were prepared and characterized. The effect of the CFX amount on the antimicrobial properties, the release kinetics and the mechanical performances was tested by using microbiological and chemical-physical methodologies. Particular attention was paid to the degradation phenomena occurring during the processing of the PLA/CFX systems.

Materials and Methods

Materials and preparation

The polymers used in this study were a sample of PLA (PLA 2002D; supplied by Natureworks) and a sample of PP (Moplen X30G; supplied by Basell). Ciprofloxacin (CFX, chemical formula: C₁₇H₁₈FN₃O₃ T_m = 253-257°C) was supplied by Sigma Aldrich and used as received without further purification.

The materials were processed by melt mixing using a batch mixer (Brabender PLE330) at 200°C and a rotational speed of 80 rpm. In detail, for both matrices, the polymer was first fed to the mixer and compounded for 4 minutes. Thereafter, the CFX was added and the blend was processed for no longer than 1 minute in order to avoid eventual degradation phenomena of the additive. CFX was added to both polymers at 1%, 2% and 4% (w/w). For comparison, both the pure matrices (PP and PLA) were processed under the same conditions. Table I shows the composition of all the samples and their identification codes.

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Table I

Composition of samples and their codes

Sample code	Poly(lactic acid) (PLA) [wt %]	Polypropylene (PP) [wt %]	Ciprofloxacin (CFX) (wt %)
PLA	100	–	–
PLA/CFX 1	99	–	1
PLA/CFX 2	98	–	2
PLA/CFX 4	96	–	4
PP	–	100	–
PP/CFX 1	–	99	1
PP/CFX 2	–	98	2
PP/CFX 4	–	96	4

In order to prevent hydrolytic scissions during processing, the PLA was dried overnight in a vacuum oven at 90°C. Films were prepared by compression molding using a Carver Laboratory press. The material was preventively ground, placed in a mold between 2 Teflon sheets and pressed at 200°C and 100 bar for about 2 minutes to obtain a film with a thickness of 200 μm.

Characterizations

The morphology of the materials was studied by using a scanning electron microscope (SEM) (Quanta 200F ESEM™; FEI). The samples were fractured under liquid nitrogen and then sputter-coated with a thin layer of gold to avoid electrostatic charging under the electron beam.

Tensile mechanical measurements were carried out using a dynamometer (model 3365; Instrom) on rectangular-shaped specimens (10 × 90 mm) cut off from films prepared as described above. The grip distance was in all the cases 30 mm and the crosshead speed 5 mm/min. Eight samples for each material were tested and data are reported as means ± SD.

The antimicrobial activity of the materials was determined by the agar diffusion method, evaluating the presence of inhibition zones. In particular, Micrococcus luteus ATCC 10240 was used as tester strain in order to study the antimicrobial property of the prepared materials. A bacterial suspension of ∼10⁹ colony forming units (CFU) was inoculated into 5 mL of LB soft agar to obtain an uniform bacterial overlay on LB agar plates. Polymer discs (diameter 12 mm) containing CFX at 1%, 2% and 4% (w/w) were placed over bacterial tester overlay. Neat polymer samples were used as controls. Bacterial growth inhibition halos were observed after overnight incubation at 37°C.

A series of CFX solutions of distilled water containing 0.1 thru 5 mg/L of CFX was used to obtain a calibration curve correlating the absorbance peak intensity and the CFX concentration using a UV/vis spectrophotometer (model UVPC 2401; Shimadzu Italia). In the concentration range investigated for this study, the calibration curve was found to be a line. The maximum absorbance peak of CFX was detected at 276 nm. The release of CFX from the films was investigated by immersing a preweighed sample (a square of 10×10 mm, approximately 0.4 mm thick) in 5 mL of distilled water. At specific time intervals, for 3 weeks, the absorbance peak intensity at 276 nm of the storage solutions was measured and converted to the quantities of CFX released, based on the calibration line previously calculated.

Rheological measurements were performed using a plate-plate rotational rheometer (HAAKE MARS; Thermo Scientific) operating at 200°C. The instrument was set to operate in the frequency sweep mode in the range 0.1 to 500 rad/sec with a strain of 5%.

Intrinsic viscosity measurements were carried out in a Ubbelohde capillary viscometer at 35°C. The solution (concentrations 0.2 wt%) were prepared by solubilizing the samples in tetrahydrofuran (THF) for 3 hours under agitation at 50°C. The intrinsic viscosity was calculated using a single point measurement and applying the Solomon-Ciuta equation (38):

[ η ] = 2 c η s p − ln ⁡ η r e l

[1]

where [η] is the intrinsic viscosity, η_sp and η_rel are, respectively, the specific and the relative viscosity and c the concentration of the polymer in the tested solution. By processing the viscosimetric data the viscosity average molecular weight was calculated by using the Mark-Houwink equation:

[ η ] = K M V a

[2]

where M_v is the viscosity average molecular weight, K = 1.74 × 10^-4 dl/g and a = 0.736 (39).

Results and discussion

The SEM micrographs of neat CFX powder (Fig. 1a, b), show that CFX is visible under the form of irregular crystalline aggregates formed by needle-like crystals. These aggregates are clearly visible in the cross section of the films containing CFX, as shown by SEM micrographs reported in Figure 1 (C–H). In particular, the CFX is well dispersed in both the polymeric matrices (PLA and PP) and, as expected, on increasing the antibiotic concentration, a larger extent of aggregates is visible in the sections. Nevertheless, the cross sections of PLA-based materials show a lesser amount of CFX crystals when compared to the corresponding PP-based materials.

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Fig. 1

SEM micrographs of neat ciprofloxacin powder and of cross sections of the polymeric systems incorporating CFX: (A) and (B) CFX at 2 different magnifications (C) PLA/CFX 1; (D) PP/CFX 1; (E) PLA/CFX 2; (F) PP/CFX 2; (G) PLA/CFX 4; (H) PP/CFX 4.

In order to verify if CFX incorporation caused some modification of the mechanical performance of the materials, thus inhibiting or reducing its practical use, tensile mechanical tests were performed. Figure 2 (A–C) reports the elastic modulus (E), the tensile strength (TS), and the elongation at break (EB), respectively, of all the materials prepared for this study.

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Fig. 2

Tensile properties of all the materials prepared in the frame of this work: (A) elastic modulus (E); (B) tensile strength (TS); (C) elongation at break (EB).

The results show that for all the PP-based materials, the variations of the tensile properties, E, TS and EB, are very small even at the highest CFX concentration used. In particular, the increase of CFX content leads to a slight increase in E and a slight decrease in EB. Indeed, CFX acts as a microfiller for PP- based materials at this low concentration, causing a slight increment in the rigidity and a decrement in the ductility. In any case, these variations are small and have poor significance in terms of mechanical performance variations. On the contrary, the PLA-based materials showed significant variations for all the tensile properties in comparison with those of the neat matrix. In particular, the elastic modulus of PLA samples containing CFX strongly decreases as the antibiotic amount is increased up to 2% (w/w) and then increases with the highest concentration of CFX, i.e. 4% w/w. Moreover, the final properties, TS and EB, drastically decrease with the addition of CFX. In detail, the trend decreases up to 2% of CFX and thereafter remains almost constant.

These results suggest that probably some interactions between PLA and CFX occur during processing that could lead to some degradative phenomena of this polymeric matrix in contrast with the slight reinforcing effect evidenced with the materials based on PP.

To verify that the incorporation of CFX conferred antimicrobial activity to the polymeric matrix, agar diffusion tests were performed using the compounded films against a Gram-positive strain, M. luteus, that is routinely used as tester strain to evaluate the growth inhibition zone around the sample. Figure 3 reports the bacterial inhibition halos observed around all the materials prepared in this work after an overnight incubation at 37°C.

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Fig. 3

Agar diffusion test performed on M. luteus overlay for: (A) PLA-based materials and (b) PP-based materials.

As expected, both the neat PLA (Fig. 3a) and the neat PP (Fig. 3b) show no antibacterial activity, while the PP/CFX systems, even at the lowest antibiotic content, show antimicrobial activity (see Fig. 3b). Moreover, the bacterial growth inhibition halos around the samples increase on increasing the amount of CFX incorporated and a net enlargement of the inhibition zone was observed for PP containing the 4% CFX. On the contrary, the PLA-based materials incorporating 1% and 2% of CFX produced no inhibition zone and only a slight antibacterial activity was observed when the 4% of CFX was added to PLA. Considering the positive results achieved with the PP-based materials, this result was somewhat surprising. It cannot be attributed to the high processing temperature since both the polymeric systems were compounded at the same temperature, i.e. 200°C. Probably some physical or chemical interaction between the PLA and CFX can be suggested to explain this result.

The antimicrobial properties of the films are dependent on their release of CFX. Therefore, the release kinetics in distilled water was evaluated. In Figure 4 the release kinetics of CFX from PLA- and PP-based materials are reported. For PP- based materials, the increase in the amount of CFX in the compounded films led to an increased CFX release during the time. These results are in good agreement with the inhibition halos showing in the samples. Moreover, the release of CFX from the films is characterized by an initial phase with a rapid solubilization of CFX micro-crystals, followed by a second phase characterized by a slow kinetics achieving a plateau.

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Fig. 4

Cumulative CFX release as a function of the time in distilled water of the polymeric systems incorporating CFX.

According to the results of the agar diffusion test, the PLA-based materials released a negligible amount of CFX during the 3 weeks of immersion and only the PLA + CFX 4% released a detectable amount of CFX, comparable to that of PP + CFX 1%. Again, these results corroborate the assumption that some interaction occurred between the PLA and the CFX.

The proposed mechanism of reaction between the CFX and PLA is shown in Figure 5. Very probably, the high processing temperature gives enough energy to the system to allow an amide formation reaction. Protonation of the carbonyl group by acid (the monomer of PLA is lactic acid, moreover the CFX has a carboxylic function) makes the carbonyl group electrophilic enough for the piperazine ring of CFX to be attacked by the nitrogen, creating a tetrahedral, instable intermediate (1). Indeed the intermediate undergoes an elimination reaction, losing an alcohol (3) from one side and adding an amide (2) with the breaking of the main polymer chain. This reaction would explain the supposed degradation phenomena of PLA occurring during the processing. Moreover, the formation of compound #4 is in good accordance with the negligible CFX release because the chemical bonding would prohibit its release.

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Fig. 5

Proposed mechanism of reaction between the CFX and PLA occurring during the melt mixing.

However, in order to further investigate the hypothesis of significant PLA degradation phenomena occurring during processing, the torque of the PLA/CFX 4 system was recorded during the mixing and rheological measurements were performed.

Figure 6 reports the torque as a function of the time for neat PLA and PLA/CFX 4. It is worth recalling (see the Materials and methods section) that the CFX is added in the batch mixer after 4 minutes of mixing, only for 1 minute. Consequently, for the first 4 minutes the 2 torque curves are almost identical. After a rapid decrease during the first 2 minutes of mixing, the torque reaches almost a plateau during the 4^th minute. Afterwards, the neat PLA finally reaches the plateau in the last minute of mixing. Converseily, the addition of CFX caused a drastic decrease in the torque, thus suggesting that a reaction between the antibiotic additive and the PLA had occurred and inducing degradation phenomena of the polymeric matrix.

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Fig. 6

Torque as function of the time of PLA and PLA/CFX 4.

This assumption was corroborated by rheological measurements reported in Figure 7. Indeed, the viscosity as function of the frequency of PLA/CFX 4 is drastically lower than the viscosity exhibited by the pure matrix. As reported in the scientific literature, this decrease in the viscosity can be explained by considering that relevant degradation phenomena of the matrix occurred during processing (7, 40). In order to definitively confirm this hypothesis, measurements of intrinsic viscosity ([η]) were carried out for PLA and PLA/CFX4 and the respective viscosity average molecular weights (M_v) were calculated. The results clearly show that the presence of CFX dramatically decreases the intrinsic viscosity of PLA mixed with CFX, from 0.93 dL/g for PLA to 0.49 dL/g for PLA/CFX4. Consequently the viscosity average molecular weight dropped from 116 kDa for PLA to 49.9 kDa for PLA/CFX4, thus corroborating the assumptions previously reported and the proposed mechanism of reaction.

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Fig. 7

Complex viscosity as function of the frequency of PLA and PLA/CFX 4.

Conclusions

The incorporation of CFX into PLA by melt compounding leads to a material with negligible antimicrobial activity. Conversely, although the same compositions and processing conditions were used, the PP-based systems incorporating CFX show a clear antimicrobial activity. These results were found to be in good agreement with the CFX release kinetics. Indeed, for PP-based materials, the increase of the CFX amount in the compounded films led to an increased CFX release during the measured time, while the PLA-based materials released a negligible amount of CFX during the 3 weeks of immersion. Only PLA/CFX 4 released a detectable amount of CFX comparable to that of PP/CFX 1. Moreover, the mechanical performance of PLA/CFX systems dramatically worsened.

These results were correlated to degradation phenomena occurring during the processing, demonstrated by the dramatic decrease in the melt and intrinsic viscosity of the PLA/CFX system in comparison with that of the neat PLA.

The negligible release of CFX with a consequent weak antimicrobial activity and the degradation of PLA are explained by assuming that a chemical reaction between PLA and CFX occurred during the melt compounding.