enzymatic degradation of poly(l-lactide) nanoparticles followed by the release of octenidine and...

BASIC SCIENCE

Nanomedicine: Nanotechnology, Biology, and Medicine10 (2014) 131–139

Research Article

Enzymatic degradation of poly(L-lactide) nanoparticles followed bythe release of octenidine and their bactericidal effects

Grit Baier, PhDa, Alex Cavallarob, Kathrin Friedemann, PhDa, Beate Müllera,Gunnar Glassera, Krasimir Vasilev, PhDb, Katharina Landfester, PhDa,⁎

aMax Planck Institute for Polymer Research, Mainz, GermanybMawson Institute, University of South Australia, Adelaide, Australia

Received 30 January 2013; accepted 5 July 2013

nanomedjournal.com

Abstract

The enzyme-triggered release of the antimicrobial agent octenidine out of poly(L-lactide)-based nanoparticles (PLLA-NPs) and their invitro antibacterial activities in the presence of gram-positive and gram-negative bacteria are presented. The formation of the nanoparticleswas achieved using a combination of the solvent evaporation and the miniemulsion approach. For the stabilization of the polymericnanoparticles, non-ionic polymers (polyvinylalcohol [PVA], hydroxyethyl starch [HES], human serum albumin [HSA]) were successfullyused for enzymatic degradation; ionic surfactants such as sodium dodecyl sulfate and cetyltrimethylammonium chloride inhibited theenzymatic degradation. The change in pH, size, size distribution and morphology during the degradation process of PLLA-NPs and therelease of the antimicrobial agent was studied. The influence of the different amounts of octenidine and of the different stabilizers on theNPs' stability, size, size distribution, morphology, zeta potential and on the surface group's density is discussed. Fluorescently labeled HES-stabilized PLLA-NPs are immobilized by colloidal electrospinning. The observed data from HPLC measurements show that octenidine isreleased out of PLLA-NPs which are stabilized with PVA, HES or HSA. In bacteria tests the PLLA nanoparticles showed a greater ability toinhibit the growth of Staphylococcus aureus compared to Escherichia coli.

From the Clinical Editor: This article discusses the enzyme-triggered release and antibacterial effects of octenidine from poly(L-lactide)-based nanoparticles demonstrating the viability of this approach for potential future antibacterial therapy.© 2014 Elsevier Inc. All rights reserved.

Key words: Poly(L-lactide) nanoparticles; Octenidine; Bactericidal effects

Reduction of the risk of bacterial contamination and infection,for example, in hospitals, medical devices, healthcare systems, isone of the biggest challenges due to the strong abilities of thebacteria to survive even in hostile conditions. The embedding ofantimicrobial molecules such as antibiotics,1,2 quaternaryammonium compounds3,4 and silver particles or ions5–7 intonanocarriers has been used to inactivate bacteria. Octenidine issimilar in its action compared to quaternary ammoniumcompounds. However, it has a broader spectrum of activityand it is currently increasingly used as a substitute for quaternaryammonium compounds or chlorhexidine in skin, mucosa and

This study was financially supported by FP7 project: BacterioSafe GrantNo. 245500 and KOALA project Grant No. 295155.

The authors thank Anke Kaltbeitzel for her help in cLSMmeasurements.⁎Corresponding author: Max Planck Institute for Polymer Research, D-

55128 Mainz, Germany.E-mail address: [email protected] (K. Landfester).

1549-9634/$ – see front matter © 2014 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.nano.2013.07.002

Please cite this article as: Baier G, et al, Enzymatic degradation of poly(L-lactideffects.... Nanomedicine: NBM 2014;10:131-139, http://dx.doi.org/10.1016/j.na

wound antiseptics. Octenidine has two cationic active moleculesnot interacting with each other due to the fact that they areseparated by 10 CH2 groups. Due to the lack of amide or estermoieties, octenidine is stable in a broad pH range between 1.6and 12.2. Octenidine is not sensitive to light and not prone tohydrolysis.8 It is typically used for skin, mucous membrane andopen wound decontamination. It shows properties of a cationicchemical compound and could be considered as a uniqueantimicrobial agent through its non-cytotoxic groups on the sideof action. In addition it exhibits a broad spectrum ofantimicrobial efficacy against gram-positive and gram-negativebacteria and fungi. Due to its excellent tissue compatibility,octenidine is often used for the treatment of chronic and acutewounds which are locally infected by pathogenic bacteria.Another application is the decontamination of skin, colonizedwith MS(R)SA (methicillin-sensitive [resistant] Staphylococcusaureus). Furthermore, it can be used for daily skin cleanings ofcatheter insertion sites or for daily wound treatments.9 Recently,

e) nanoparticles followed by the release of octenidine and their bactericidalno.2013.07.002

http://dx.doi.org/10.1016/j.nano.2013.07.002




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132 G. Baier et al / Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 131–139

octenidine was used with self-manufactured poly(methylmetha-crylate) (PMMA) bone cement pellets impregnated with differentconcentrations (5%, 6%, 7% and 8%) of octenidine for use inorthopedic surgery.10

Due to its biodegradability and biocompatibility, poly(L-lactide) (PLLA) is often used in different biomedical applica-tions, for example, as a drug carrier system such as fibers,particles, nanospheres, blends and films in the biomedical,pharmaceutical and drug delivery environment.11–14 Thehydrolytic degradability of PLLA in buffer solutions has beenextensively studied, especially for biomedical applications in invivo and in vitro experiments.15,16 Further investigations on thehydrolytic degradation of poly(DL-lactide) (PDLLA) were donein order to study the effect of temperature and acidity on thedegradation characteristics.17 Tokiwa and Jarerat18 reviewed thehydrolytic, microbial and enzymatic biodegradation of PLLA. In1981, Williams19 published the degradation with proteinase K,since then the enzymatic degradation of PDLLA polymers wasinvestigated by several groups.20–24 It was shown that differentenzymes have significant effects on the degradation of poly(L-lactide) and that esterase-type enzymes21 and proteinase K20

accelerate the degradation. When incubating PLLA withproteinase K in Tris buffer (pH 8.0) a remarkable drop in pHfrom 8.0 to 5.3 and a considerable loss of the molecular weightcould be observed after 1 week. In addition, the enzymaticdegradation of PLLA using proteinase K preferentially occurredin the amorphous region rather than in the crystalline one.18 Inanother study, the enzymatic degradation of the low-molecular-weight PDLLA was performed using an esterase-type enzyme,21

whereas the hydrolysis of the high-molecular-weight PDLLAwas achieved by proteinase K.22

Different preparation methods are described in the literaturefor the formulation of particles from preformed polymers.25 Thenano-precipitation, emulsification/solvent evaporation or diffu-sion, double emulsion and the salting out procedure are the mostpromising methods.26–30 The physical–chemical properties ofthe molecules which should be encapsulated are determiningfactors for the selection of an encapsulation method. Further-more, the size, size distribution and the synthesis of particleswith a high solid content are a big challenge. Our goal was tocombine miniemulsion and emulsion/solvent evaporation tech-niques in order to achieve an efficient and controllableencapsulation of hydrophobic compounds with high loadingefficiency into the polymeric matrix.

Here, we report the formation of stable narrowly size-distributed PLLA nanoparticles formed by the combination ofthe miniemulsion/solvent evaporation process. The antimicrobialagent octenidine was encapsulated within PLLA nanoparticles (upto 21 wt%) and then released by enzymatic degradation triggeredby esterase or proteinase K. Using different stabilizing moleculesallows the modification of the nanoparticles surface even thoughthe formation of nanoparticles with sodium dodecyl sulfate (SDS)and cetyltrimethylammonium chloride (CTACl, Aldrich) occur.31

These surfactants cannot be used for the enzyme-triggered releasesince they deactivate enzymes.32 Therefore poly(vinyl alcohol)(PVA) and for the first time hydroxyethyl starch (HES) and humanserum albumin (HSA) were used as stabilizing agents. Differentamounts of the antimicrobial agent octenidine were encapsulated

within the PLLA-NPS and its influence on the nanoparticlesstability, size, size distribution, zeta potential andmorphology wasstudied. To investigate the enzymatic degradation of the differentstabilized PLLA-NPs, the pH changes within 40 days, the changesin size and size distribution and in morphology were monitoredafter enzyme exposure. For a possible later application in wounddressing, the immobilization of fluorescent-labeledHES-stabilizedPLLA-NPs was performed using the colloidal electrospinningprocess and analyzed by scanning electron microscopy (SEM) andconfocal laser scanning microscopy (cLSM).The antibacterialactivities of the NPs were evaluated against gram-positive bacteria(S. aureus, ATCC 29213 and ATCC 43300) and gram-negativebacteria (Escherichia coli, ATCC 25922) by determination of theminimal inhibitory concentration (MIC) in vitro.

Methods

For details see supporting information (SI).

Results

Biocompatible and biodegradable PLLA nanoparticles werecreated by the combination of miniemulsion and the solventevaporation method. Using the miniemulsion process allows theformation of stable nanoparticles with a narrow sizedistribution.33 The choice of the stabilizing molecule plays animportant role when designing initially stable droplets andnanoparticles later. In a previous publication of our group it wasshown that ionic surfactants like SDS and CTACl are goodcandidates for the stabilization of PLLA-NPs.31 The disadvan-tage of both surfactants is that in aqueous solutions, anionic andcationic surfactants generally have denaturing effects onenzymes due to electrostatic and hydrophobic interactionsinherent to the system.34 For this reason the biocompatiblemolecules PVA, HES and HSA were used as stabilizingmolecules. The hydrophobic antiseptic agent octenidine wasembedded and the antimicrobial properties were tested in thepresence of S. aureus (ATCC 29213 and ATCC 43300) and E.coli (ATCC 25922) bacteria. The formulation process of thePLLA-NPs is schematically depicted in Figure 1.

The PLLA nanoparticles were synthesized with differentamounts of octenidine (see Table 1). Furthermore, low-molecular-weight ionic surfactants such as SDS and CTACland polymeric stabilizers as PVA, HES and HSA were used tostabilize the initial droplets and after polymerization the polymernanoparticles in order to study the influence of these moleculeson the hybrid nanoparticles size, size distribution, stability, andrelease properties.

Ionic stabilizers were mainly chosen as they are known to beeffective in formulations of small-size PLLA nanoparticles.However, ionic stabilizers are also known to reduce the activityof enzymes due to their denaturing effects, and therefore non-ionic polymers such as PVA or HES, or a protein as HSA werealso investigated in the current study as stabilizing moleculeswhich do not interact with the enzymes.

Figure 1. Schematic illustration of the formulation process of PLLA nanoparticles with octenidine as antimicrobial and of the electrospinning process. (A) SEMmicrograph and cLSM micrograph are shown obtained from immobilized HES-stabilized fluorescent-labeled PLLA-NPs in PVA fibers.

Table 1Effect of different octenidine amounts and types of stabilizers on the physico-chemical properties of the polymeric nanoparticles.

PLLA-NPs stabilizingmolecules (mg)

Octenidine(mg)

Diameter (nm)/standard deviation (%)

Zeta potential(mV)

Carboxylicgroups per nm2

PLLA-PVA – 280/21 −3 1.25PLLA-SDS – 160/26 −40 0.96PLLA-CTACl – 150/24 +42 1.18PLLA-SDS-80 80 150/22 −32 0.72PLLA-SDS-60 60 125/28 −36 0.86PLLA-SDS-40 40 120/27 −36 0.87PLLA-CTACl-80 80 140/22 +36 0.98PLLA-CTACl-60 60 115/25 +28 1.05PLLA-CTACl-40 40 90/23 +24 1.12PLLA-PVA-80 80 220/23 +2 0.94PLLA-PVA-60 60 230/16 +2 1.03PLLA-PVA-40 40 230/11 +1 1.11PLLA-HES-80 80 240/27 +15 0.67PLLA-HES-60 60 240/27 +14 0.79PLLA-HES-40 40 225/29 +10 0.98PLLA-HSA-80 80 220/26 −2 0.95PLLA-HSA-60 60 240/24 −3 0.99PLLA-HSA-40 40 240/26 −5 1.05

The amount of PLLA (300 mg) and chloroform (10 mL) was kept constant in all experiments.

133G. Baier et al / Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 131–139

The obtained values of average size and size distributionwere determined by dynamic light scattering (DLS) (seeTable 1). The electro-kinetic potential (zeta potential) and theamount of carboxylic groups that are present on the PLLA-NPs

surface (determined by the particle charge detector, PCD)measurements are also shown in Table 1. The molecularweights of PLLA after ultrasonication, determined by gelpermission chromatography (GPC), were in the range between

Figure 2. SEM images of the polymeric PLLA nanoparticles synthesized via solvent evaporation method combined with the miniemulsion technique. (A)PLLA-SDS-60-NPs. (B) PLLA-CTACl-60-NPs. (C) PLLA-PVA-60-NPs. (D) PLLA-HES-60-NPs. (E) PLLA-HSA-60-NPs.


102,000 and 137,000 g · mol−1.The influence of the ultrasoni-cation duration on the PLLA molecular weight was studied andit was obtained that a longer duration of ultrasonication reducesthe molecular weight of the polymer, and as a result theviscosity decreases, leading to the formation of smaller dropletsand subsequently composite particles.35

From the obtained data in Table 1 it can be seen that the sizeof PLLA nanoparticles, stabilized with an ionic surfactant, is inthe diameter range between 90 and 160 nm, whereas thediameter of nanoparticles increases up to 240 nm when a non-ionic stabilizer was employed. Interestingly, PLLA-NPs withoutthe antimicrobial agent octenidine and stabilized with HES orHSA (data not shown) were unstable after the crosslinkingreaction. Due to the low water solubility, the hydrophobicoctenidine molecules (low molecular weight) act as an osmoticpressure agent, thus enhancing the stability of the nanodroplets.Therefore, as expected, using octenidine leads to smallernanoparticle sizes compared to nanoparticles without octenidine.The values of the zeta potential are slightly more positive whenusing higher amounts of octenidine. Since octenidine is acationic surface active agent and able to decrease the interfacialtension at the water/chloroform interface from≈30 mN ⋅m−1 tominimum 7 mN ⋅ m−1 the octenidine chains could either coverthe nanoparticles surface or/and they can protrude into theaqueous phase. For the PVA, HES and HSA-stabilized PLLA-NPs, the amount of carboxylic groups per nanoparticle wascalculated from the results of the particle charge titration.36 Thecarboxylic groups on the surface of the PLLA particles aredetectable because PLLA degrades during the ultrasonicationprocess which leads to lactic acid and therefore COOHisrising.31 The lower density of carboxylic groups for thesamples with 80 mg octenidine independent of the usedstabilizing molecules in comparison to the samples with loweroctenidine amount (40 mg) could be due to the “shielding” of thenegative carboxylic charges by the cationic character ofoctenidine. The obtained nanoparticles as studied by SEM(Figure 2) are spherical nanoparticles with a monodisperse sizedistribution in all cases.

Enzymatic degradation of the PLLA-NPs and releaseof octenidine

The enzymatic-triggered degradation of the PLLA-NPs with60 mg octenidine (PLLA-surfactant-60-NPs) stabilized withSDS, PVA, HES and HSA molecules followed by the release

of octenidine was monitored by HPLC. Additionally the changesin the pH values, in size and size distribution and in themorphology were studied after the enzyme exposure of the NPs.For the HPLC experiments, the NP dispersion and thesupernatant (after centrifugation of the PLLA-NPs) weremeasured and the total amount of octenidine found inside theNPs was compared with the amount of octenidine found in thesupernatant (see Figure 4). As an example, the single curves(proteinase K 10 mg · mL−1, day 40) obtained from themeasurements using an ELSD (evaporative light scatteringdetector, curves above) and an UV detector (curves below) areshown in Figure 3.

The ELSD detector allows the detection of the release of theantimicrobial agent octenidine and the monitoring of the PLLAfragments (after degradation) within a single experiment. Theoctenidine peak can be observed in the NP dispersions and in thesupernatants with one exception. The peak is not visible in theSDS-stabilized PLLA-NPs. This is due to the fact that using SDSas stabilizing molecules the surfactant influences and inhibits theactivity of the enzyme. From the curves of the NP dispersion(Figure 3, graph above left), the PLLA fragments at an elutiontime of about 15 min can be detected as well with the exceptionof SDS-stabilized PLLA-NPs. For these NPs only a fairly smallpeak was observed which could be due to the same reason. Thesame result was found using an UV detector (280 nm). Again, nooctenidine peak was visible in the supernatant of the PLLA-NPsloaded with 60 mg octenidine and stabilized with SDS (PLLA-SDS-60-NPs). For the PVA, HES and HSA-stabilized PLLA-60-NPs a sharp peak for octenidine can be observed in thesupernatant. The results of the comparison between the totalamount of octenidine found inside the NPs and the amount ofoctenidine found in the supernatant for the proteinase K (1 and10 mg · mL−1, days 23 and 40)-treated SDS, PVA, HES andHSA-treated PLLA-NPs are depicted in Figure 4. The experi-ments using esterase (1 and 10 mg · mL−1 treatment) under thesame conditions did not show any release even after longerincubation times. From Figure 4 it can be seen that with the usageof 10 mg · mL−1 (yellow columns) proteinase K with asignificant increase in the amount of octenidine found in thesupernatant was detected compared to the usage of 1 mg · mL−1

(gray columns). Furthermore, only a small increase of theamount of octenidine found in the supernatant could be observedbetween day 23 and day 40. No octenidine was found for theSDS-stabilized PLLA-NPs because of the reason mentionedabove. About 90% of the octenidine was found in the HES and

image of Figure�2

Figure 4. Results obtained from HPLC measurements of the comparisonbetween the total amount of octenidine found inside theNPs and the amount ofoctenidine found in the supernatant for the proteinase K (1 and 10 mg · mL−1,days 23 and 40)-treated SDS, PVA, HES, and HSA-treated PLLA-NPs.

Figure 3. HPLC curves of PLLA-NPs obtained from the measurements using an ELSD (see above) and a UV detector (280 nm; see below).


HSA-stabilized PLLA-NPs which means that neither thepolysaccharide HES nor the protein HSA as a stabilizer has aninfluence on the degradation behavior of proteinase K-treated

PLLA-NPs. For the PVA-stabilized PLLA-NPs about 70%octenidine was found in the supernatant. This is about 20% lessthan for the HES and HSA-stabilized PLLA-NPSs and could bedue to a shielding of the particles because of the PVA as astabilizing molecule.

The pH changes were monitored as an indication of thePLLA-NPs degradation obtained from the proteinase K andesterase experiments are depicted in Figure 5. A remarkable dropin pH of about 1.5 when using proteinase K (1 mg · mL−1) orabout 2 when using (10 mg · mL−1) can be seen for the PLLA-PVA-60-NPs, PLLA-HES-60-NPs and PLLA-HSA-60-NPsduring the 6-week test. This is explained by producing largequantities of lactic acid or its oligomers leading to a decrease inpH values. For the PLLA-SDS-60 and PLLA-CTACl-60-stabilized NPs there was only a small reduction in pH for thePLLA-NPs with and without octenidine and at both proteinase Kconcentrations which is due to the fact that ionic surfactants areknown to eventually inhibit the enzyme activity completely andonly very limited hydrolysis takes place. Using esterase at bothconcentrations (1 or 10 mg · mL−1) did not lead to a drop in pHvalues (see Figure 5). From the pH studies it can be concludedthat proteinase K accelerates the degradation of PLLA-PVA-60,PLLA-HES-60 and PLLA-HSA-60 nanoparticles whereas

image of Figure�4

image of Figure�3

Figure 5. Monitoring of the pH values for degradation studies of PLLA using proteinase K and esterase (1 and 10 mg · mL−1).

Table 2Influence on diameter and standard deviation after enzyme treatment byproteinase K (10 mg · mL−1, 40 days) of PLLA-NPs.

PLLA samples Diameter (nm)/standarddeviation (%) beforeenzyme treatment

Diameter (nm)/sizedistribution (%) afterenzyme treatment

PLLA-SDS-60-NPs 125/28 120/31PLLA-CTACl-60-NPs 115/25 115/27PLLA-PVA-60-NPs 230/16 550, 44 (Peak1)

280, 37 (Peak 2)PLLA-HES-60-NPs 240/27 520, 42 (Peak1)

300, 37 (Peak 2)PLLA-HSA-60-NPs 240/24 500, 41 (Peak1)

340, 40 (Peak 2)


esterase did not influence the degradation of PLLA-NPs.Furthermore, for the PLLA-PVA-60-NPs (please note that thecapsules PLLA-HES-60-NPs and PLLA-HSA-60-NPs are notstable without octenidine), it can be concluded that theencapsulated octenidine did not influence the degradationbehavior of PLLA.

In addition to the HPLC and pH degradation studies, theaverage sizes and size distributions of the PLLA-NPs (for thePLLA-NPs with 60 mg octenidine) before and after enzymetreatment (proteinase K, 10 mg · mL−1, after 40 days) weremeasured by DLS (see Table 2). Once again, no influence on sizeand size distribution was observed for the NPs stabilized by theionic surfactants; a slight increase of the size distribution mightbe due to the residuals from the enzyme which is attached to thenanoparticles surface. For the PLLA-PVA, PLLA-HES andPLLA-HSA-stabilized NPs, the influence on the size and sizedistribution by showing a second peak and a broader sizedistribution can be seen. This indicates the destruction of thenanoparticle system.

Morphology studies by SEM performed for the PLLA-PVA-60, PLLA-HES-60 and PLLA-HSA-60-stabilized NPs after40 days of proteinase K (10 mg · mL−1) exposure (Figure 6),show a clear disintegration of the nanoparticle system. After

40 days, only few nanoparticles remained intact and themorphology was remarkably altered due to the presence of theproteinase K.

Immobilization of fluorescent-labeled HES-stabilizedPLLA-NPs by electrospinning

In order to immobilize HES-stabilized fluorescent-labeledPLLA-NPs in fibers, the dispersion of PLLA-HES with

image of Figure�5

Figure 6. SEM images of the polymeric PLLA nanoparticles after enzymatic degradation by proteinase K (10 mg · mL−1, 40 days). (A) PLLA-PVA-NPs. (B)PLLA-HES-NPs. (C) PLLA-HSA-NPs.

Table 3MIC of PLLA nanoparticles against S. aureus (ATCC 29213), S. aureus(ATCC 43300), and E. coli (ATCC 25922) (μg · mL−1 nanoparticles).

Sample S. aureusATCC 29213

S. aureusATCC 43300

E. coliATCC 25922

PLLA-CTACl-NPs 6.25 12.5 N200PLLA-CTACl-60-NPs 3.13 12.5 100PLLA-PVA-60-NPs 12.5 25.0 100PLLA-HES-60-NPs 25.0 25.0 50.0PLLA-HSA-60-NPs 12.5 50.0 50.0


octenidine was electrospun using a 10% wt/wt aqueous solutionof PVA as a fiber template. To check the incorporation of thePLLA-HES-NPs inside the electrospun network, the fibers weredeposited directly on silicon or glass slides, to make themsuitable for SEM and cLSM measurements. The schematicsetup of the electrospinning device and a SEM micrograph andcLSM micrograph is shown in Figure 1. In the SEM, smoothfibers are detected; the cLSM image clearly shows thesuccessful incorporation of the fluorescently labeled PLLA-NPs inside the fibers.

Minimum inhibitory concentration

The antibacterial activities of nanoparticles loaded with60 mg octenidine were evaluated for their antibacterial proper-ties in terms of minimum inhibitory concentration (MIC) againstgram-positive S. aureus (ATCC 29213 and ATCC 43300) andgram-negative E. coli (ATCC 25922) (see Table 3). The resultsindicate that the E. coli strain ATCC 25922 was more resistant toall of the nanoparticles tested compared to the S. aureus strainssuggesting that the E. coli has a greater ability to resist the effectof the octenidine.

Discussion

In the present study stable nano-sized distributed PLLAnanoparticles stabilized with different molecules were formed bythe combination of the miniemulsion/solvent evaporationprocess. The hydrophobic antimicrobial agent octenidine (lowmolecular weight) acts as an osmotic pressure agent, thusenhancing the stability of the nanodroplets and leads to smallernanoparticle sizes compared to nanoparticles without octenidine.

In measuring the zeta potential, slightly more positive valueswere observed by using higher amounts of octenidine sinceoctenidine is a cationic surface active agent. The octenidinechains could be on the nanoparticles surface and they canprotrude into the aqueous phase. From the particle chargedetector titration experiments a lower density of carboxylicgroups for the samples with 80 mg octenidine independent of theused stabilizing molecules in comparison to the samples withlower octenidine amount (40 mg) was obtained. This could bedue to the “shielding” of the negative carboxylic charges by thecationic character of octenidine. The observed data from HPLCmeasurements show that the antimicrobial agent octenidine isreleased out of PLLA-NPs which are stabilized with PVA, HESor HSA and from the pH studies which can be concluded thatproteinase K accelerates the degradation of these nanoparticles,whereas esterase did not influence the degradation of PLLA-NPs. No degradation was observed for the SDS und CTACl-stabilized NPs. This is due to the fact that by using ionicsurfactant as stabilizing molecules the surfactant influences andinhibits the activity of the enzyme. Furthermore, morphologystudies and an increase in size and size distribution show a cleardisintegration of the nanoparticle system.

The antibacterial activity of nanoparticles loaded with60 mg octenidine was evaluated for their antibacterialproperties in terms of minimum inhibitory concentration(MIC) against S. aureus and E. coli. The PLLA-CTACl-NPs(without octenidine) showed the ability to inhibit growth ofboth strains of S. aureus tested which is only due to thequaternary ammonium group of the CTACl.37 However,PLLA-CTACl-NPs seemed to show no apparent antibacterialeffect on the E. coli. The addition of octenidine to the CTACl-stabilized PLLA (PLLA-CTACl-60-NPs) showed an increasein activity against S. aureus ATCC 29213 and E. coli ATCC25922. The MIC decreased from 6.25 to 3.13 μg mL−1 andfrom N200 to 100 μg·mL−1 for the S. aureus ATCC 29213and E. coli, respectively. The MIC against S. aureus ATCC43300 did not change with the addition of octenidine to theCTACl-stabilized particles. It appears that S. aureus ATCC43300 might be more resistant to octenidine. This is alsosuggested by the MIC values determined for the otheroctenidine-loaded nanoparticles that were tested (Table 3). Itis also possible that in samples PLLA-CTACl-NPs (withoutoctenidine) and PLLA-CTACl-60-NPs (with octenidine)a combination of effects contribute to their antimicrobial

image of Figure�6


activity, for example, the quaternary ammonium group of theCTACl and the presence of octenidine.

PLLA-HES-60-NPs and PLLA-HSA-60-NPs were observedto have a lower ability to inhibit the growth of both strains of S.aureus tested compared to PLLA-CTACl-NPs and PLLA-CTACl-60-NPs. However, they were more efficient against E.coli. Interestingly, PLLA-HSA-60-NPs showed the sameinhibition potential against S. aureus ATCC 29213 as PLLA-PVA-60-NPs but lower against S. aureus ATCC 43300 andhigher against E. coli ATCC 25922. Regardless of surfactantused, all particles showed antibacterial activity against bothgram-positive and gram-negative bacteria. Overall, the particlesshowed a greater ability to inhibit the growth of S. aureuscompared to E. coli.

In the present publication, stable narrowly size-distributedPLLA-NPs stabilized with different molecules were formed bythe combination of the miniemulsion/solvent evaporationprocess. The observed data from HPLC measurements showthat the antimicrobial agent octenidine is released out of PLLA-NPs which are stabilized with PVA, HES or HSA and from thepH studies it can be concluded that proteinase K accelerates thedegradation of these nanoparticles, whereas esterase did notinfluence the degradation of PLLA-NPs. No degradation wasobserved for the SDS and CTACl-stabilized NPs. Furthermore,morphology studies and an increase in size and size distributionshow a clear disintegration of the nanoparticle system. In bacteriatests the PLLA nanoparticles showed a greater ability to inhibitthe growth of S. aureus compared to E. coli. All aspectsconsidered, the PLLA nanoparticles containing octenidine havethe potential to be used in the biomedical field as they possessantimicrobial properties. Prospective studies using this new typeof PLLA nanoparticles are needed to clarify the biocompatibility.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.nano.2013.07.002.

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enzymatic degradation of poly(l-lactide) nanoparticles followed by the release of octenidine and...

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