Preparation and In Vitro Characterization of Lactococcus lactis-Loaded Alginate Particles as a Promising Delivery Tool for Periodontal Probiotic Therapy

Abstract

Probiotic microorganisms are used in a variety of food supplements and medical formulations to promote human health. In periodontal therapy, probiotics are mainly used in the form of gels, tablets or rinses that often tend to leak from the periodontal pocket, resulting in a strongly reduced therapeutic effect. In this pilot in vitro study, we present biodegradable alginate-based particles as an alternative, highly efficient system for a periodontal delivery of probiotic bacteria to the inflammation site. For this purpose, Lactococcus (L.) lactis was encapsulated using a standardized pump-controlled extrusion-dripping method. Time-dependent bacterial release in artificial saliva was investigated over 9 days. The effect of freeze drying was explored to ensure long-term storage of L. lactis-loaded particles. Additionally, the particles were bound to dentin surface using approved bioadhesives and subjected to shear stress in a hydrodynamic flow chamber that mimics the oral cavity in vitro. Thus, round particles within the range of 0.80-1.75 mm in radius could be produced, whereby the diameter of the dripping tip had the most significant impact on the size. Although both small and large particles demonstrated a similar release trend of L. lactis, the release rate was significantly higher in the former. Following lyophilization, particles could restore their original shape within 4 h in artificial saliva; thereby, the bacterial viability was not affected. The attachment strength to dentin intensified by an adhesive could resist forces between 10 and 25 N/m². Full degradation of the particles was observed after 20 days in artificial saliva. Therefore, alginate particles display a valuable probiotic carrier for periodontal applications that have several crucial advantages over existing preparations: a highly stable form, prolonged continuous release of therapeutic bacteria, precise manufacturing according to required dimensions at the application site, strong attachment to the tooth with low risk of dislocation, high biocompatibility and biodegradability.

Keywords: alginate particles; periodontal health; probiotic therapy.

Figure 1

Illustration of methods used to produce and characterize alginate particles. (a) Schematic workflow highlights the main technical requirements for production. A dosing pump controlled the pressure piston, which advanced the alginate solution mixed with L. lactis through the dripping tip into a rotating CaCl₂-enriched gelation bath placed on a magnetic stirrer. (b) Selected microscopic images exemplify the measurement of size (left) and roundness (right) of the particles in terms of radius (in mm) and aspect ratio (d_max/d_min), respectively. Magnification: 100×. (c) Schematic set-up of the spinning disk device illustrates the laminar flow chamber filled with artificial saliva that was used to define the bond strength of different adhesives. An alginate particle was adhered to a tooth specimen, which was attached to the circular disk. Rotating shaft translated the increasing angular velocity (ω) until particle detachment. All dimensions in mm. (d) Schema represents the calculation formula of the shear stress (τ_RES), which acts tangentially on the alginate particle.

Figure 2

Effect of different production settings on size (radius) and roundness (aspect ratio) of alginate particles. The impact of (a) a small (27 G) vs. large (20 G) diameter of the dripping tip, (b) dosing pump rate (60 mL/h vs. 80 mL/h), (c) alginate concentration (1% vs. 2% vs. 3%), (d) calcium chloride concentration (1% vs. 3% vs. 5%) and (e) stirring speed (100 vs. 400 vs. 800 rpm) on radius (top) and aspect ratio (bottom) was determined for each sample produced using either a 27 G tip (left) or 20 G tip (right). Data represent mean ± SD of (a) n = 72 analyzed particles per condition from 24 independent experiments with three technical replicates per experiment or (b–e) n = 9 particles per condition from three independent experiments with three technical replicates per experiment (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 analyzed with unpaired t-test (a,b) or one-way ANOVA with Tukey post hoc test (c–e)).

Figure 3

Morphological appearance and release kinetics of L. lactis or latex beads enclosed in alginate particle. (a) SEM images demonstrate general morphology of freshly prepared alginate particle (magnification 1000×) and (b,c) L. lactis under the surface of a particle after 3 h incubation in artificial saliva (magnification 5000× and 24,000×, respectively). (d) The LBC of L. lactis released over 216 h from one small vs. one large particle was determined. Data represent mean ± SD of n = 15 (particles) from three independent experiments with five technical replicates (** p < 0.01, *** p < 0.001 analyzed by two-way ANOVA). (e–g) The release of L. lactis was additionally analyzed by SEM after 24 h incubation in artificial saliva. Magnification: 755× (left), 2350× (middle), 5000× (right). (h) Viability of released L. lactis in artificial saliva was analyzed over 216 h. Data represent mean ± SD of n = 15 (particles) from three independent experiments with five technical replicates. (i–k) Surface morphology of freshly prepared alginate particle carrying latex beads was visualized by SEM. Magnification: 1000× (left), 5000× (middle, right). (l) Representation of latex bead release from large alginate particle into artificial saliva analyzed over 360 h. Data represent mean ± SD of n = 3 (particles).

Figure 4

The effect of freeze-drying on particle morphology, weight, size, roundness and bacterial release. (a) SEM image displays a freeze-dried alginate particle. Magnification: 41×. (b) Enlarged SEM image of the particle surface after freeze-drying. Magnification: 10,000×. Following rehydration in artificial saliva or water for 24 h, the particle (c) weight, (d) radius and aspect ratio (e) were evaluated and compared to a non-dried reference particle. Data represent mean ± SD of n = 5 (particles) from three independent experiments with two or three technical replicates. (f) Comparison of LBC of L. lactis released from a non-lyophilized vs. lyophilized particle after 24 h incubation in saliva. Data represent mean ± SD of (c–e) n = 9 (particles) or (f) n = 15 (particles) as three or five technical replicates from three independent experiments, respectively.

Figure 5

Analysis of the detachment of alginate particles fixed with fibrin vs. skin adhesive from the dentin surface. (a) The maximum rotation speed (rpm) leading to the detachment of particles from the dentin was determined. (b) Calculated shear stress (τ_RES) that led to a particle detachment. The values represent mean ± SD for n = 9 (particles) analyzed using an unpaired t-test (*** p < 0.001).