Sustained Release of Antibacterial Lipopeptides from Biodegradable Polymers against Oral Pathogens.

The development of antibacterial drugs to overcome various pathogenic species, which inhabit the oral cavity, faces several challenges, such as salivary flow and enzymatic activity that restrict dosage retention. Owing to their amphipathic nature, antimicrobial peptides (AMPs) serve as the first line of defense of the innate immune system. The ability to synthesize different types of AMPs enables exploitation of their advantages as alternatives to antibiotics. Sustained release of AMPs incorporated in biodegradable polymers can be advantageous in maintaining high levels of the peptides. In this study, four potent ultra-short lipopeptides, conjugated to an aliphatic acid chain (16C) were incorporated in two different biodegradable polymers: poly (lactic acid co castor oil) (PLACO) and ricinoleic acid-based poly (ester-anhydride) (P(SA-RA)) for sustained release. The lipopeptide and polymer formulations were tested for antibacterial activity during one week, by turbidometric measurements of bacterial outgrowth, anti-biofilm activity by live/dead staining, biocompatibility by hemolysis and XTT colorimetric assays, mode of action by fluorescence-activated cell sorting (FACS) and release profile by a fluorometric assay. The results show that an antibacterial and anti-biofilm effect, as well as membrane disruption, can be achieved by the use of a formulation of lipopeptide incorporated in biodegradable polymer

PLACO formulation biocompatibility by the XTT assay.

<p>Mice macrophages RAW-246 were cultivated in wells of a 96 well microtiter plate. Each polymer was tested. PLACO exhibited the highest cell survivability (A). Each formulation containing lipopeptide and PLACO polymer was tested. All the formulations showed high cell survivability. C16-KL<u>L</u>K exhibited the highest cell survival vs the C16-K<u>K</u>K, C16-KGGK and C16-KA<u>A</u>K formulations (B).</p

The bacterial membrane is disrupted by lipopeptide after a 1 hr exposure.

<p>A representative experiment showing lipopeptide C16-KGGK and its interaction with the <i>E</i>. <i>faecalis</i> bacterial membrane. Fluorescence-activated cell sorting was used to measure cytoplasmic membrane depolarization and to determine membrane disruption. At high cytoplasmic concentrations the DiOC₂ (3) self-associates and the green fluorescence emission (FL1-A axis) shifts to red (FL3-A axis). The bacteria were stained with DiOC₂ (3), exhibiting green fluorescence (FL1-A) with a shift to red emission shift as the dye molecules self-associate at the higher cytosolic concentrations caused by the larger membrane potential (FL3-A). Left shift of the bacteria exposed to KGGK is shown in the dot plot (A). The red/green ratio is lower for the bacteria exposed to C16-KGGK, indicating that the bacterial membrane was disrupted and thus revealing the lipopeptide antibacterial mechanism (B).</p

Biofilm growth inhibition by lipopeptide incorporated in biodegradable polymers.

<p>The antibiofilm effect was evaluated with the use of a dead/live dying kit against a 72 hr formed biofilm. The live bacteria were stained with a green dye, the dead bacteria were stained with a red dye. Results are shown for <i>E</i>. <i>faecalis</i>, <i>F</i>. <i>nucleatum</i> and <i>S</i>. <i>mutans</i>. The control group included 0.025% CHX.</p

Lipopeptide hemolysis assay.

<p>All four lipopeptides: C16-KGGK, C16-K<u>K</u>K, C16-KA<u>A</u>K and C16-KL<u>L</u>K were tested for hemolysis in sheep RBC, at concentrations of 5, 10, 20, 50 and 100 μg/ml. Insignificant hemolysis was detected at the MICs. The control group was considered complete hemolysis.</p

Lipopeptide release profile from biodegradable polymer.

<p>A representative release profile of C16-KGGK lipopeptide from P(SA:RA) and PLACO polymers evaluated at 1, 8, 24, 48, 72, 96 and 168 hrs. C16-KGGK release from PLACO peaked after about 72 hrs, whereas the release from P(SA:RA) was continuous throughout the week. The amount of accumulated peptide released from the polymer was calculated according to calibration curves made before the experiment.</p

Growth inhibition of bacteria by lipopeptides released from P(SA:RA) or from PLACO.

<p>The tested formulation 100 μg peptide + 100 mg polymer, ratio 1:1000 was evaluated for its antibacterial effect every 24 hrs during 1 week. (A, B) KGGK formulations against <i>E</i>. <i>faecalis</i>. (C, D) C16-K<u>K</u>K formulations against <i>F</i>. <i>nucleatum</i> and (E, F) KL<u>L</u>K formulations against <i>S</i>. <i>mutans</i>.</p