Bioactive Polymeric Nanoparticles for Periodontal Therapy

Abstract

Aims: to design calcium and zinc-loaded bioactive and cytocompatible nanoparticles for the treatment of periodontal disease.

Methods: PolymP-nActive nanoparticles were zinc or calcium loaded. Biomimetic calcium phosphate precipitation on polymeric particles was assessed after 7 days immersion in simulated body fluid, by scanning electron microscopy attached to an energy dispersive analysis system. Amorphous mineral deposition was probed by X-ray diffraction. Cell viability analysis was performed using oral mucosa fibroblasts by: 1) quantifying the liberated deoxyribonucleic acid from dead cells, 2) detecting the amount of lactate dehydrogenase enzyme released by cells with damaged membranes, and 3) by examining the cytoplasmic esterase function and cell membranes integrity with a fluorescence-based method using the Live/Dead commercial kit. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests.

Results: Precipitation of calcium and phosphate on the nanoparticles surfaces was observed in calcium-loaded nanoparticles. Non-loaded nanoparticles were found to be non-toxic in all the assays, calcium and zinc-loaded particles presented a dose dependent but very low cytotoxic effect.

Conclusions: The ability of calcium-loaded nanoparticles to promote precipitation of calcium phosphate deposits, together with their observed non-toxicity may offer new strategies for periodontal disease treatment.

Fig 1. Zinc and calcium chelation ability of NPs.

a. μg Ca²⁺/mg of NPs and b. μg Zn²⁺/ mg of NPs. Values were determined at two different pH values, measured by means of an inductively coupled plasma optical emission spectrometer. All tests were performed in triplicate. After Kruskal-Wallis and Mann-Whitney tests, all groups were significantly different (p<0.05), except when NPs were immersed in CaCl₂ aqueous solutions containing 40 ppm at 6.5 or 8.5 pH values (p>0.05).

Fig 2. TEM images of NPs.

a. Non-loaded NPs, zinc and calcium are absent in the EDX spectrum (Ep1). b. Calcium loaded NPs. Calcium is detected in the EDX spectrum analysis (Ep2) c. Zn loaded NPs. Zinc and calcium were detected in the EDX spectrum analysis (Ep3). Light circular objects inside of the NPs were artifacts that developed during electron beam transmission. Ni, Si and Ti in the EDX spectra were contaminant elements from the sample holder. Presented EDX spectra correspond to qualitative analysis and to determine the identification of the elements present and is not a normalized quantitative analysis.

Fig 3. XRD spectra of NPs, Zn-NPs or Ca-NPs before and after 7 days of SBFS immersion.

An amorphous crystallization pattern of calcium phosphate deposits is shown. Sodium chloride crystals formation (vertical bars) was also detected.

Fig 4. FESEM micrographs of NPs.

a. Non-loaded NPs, before immersion in SBFS were spherical in shape. b. Ca-NPs, before immersion in SBFS. c. Zn-NPs, before immersion in SBFS. d. NPs after immersion in SBFS for 7 d. A negligible amount of calcium was encountered in EDX spectra (Ep1). e. Ca-NPs after immersion in SBFS for 7 d. Spotty calcium deposits were uniformly distributed throughout particles surfaces. Particles lost their regular morphology. Calcium and phosphorous were identified after EDX analysis (Ep2). f. Zn-NPs after 7 days immersion in SBFS. Calcium was identified after elemental analysis (Ep3). At the EDX spectra, chloride and sodium were detected after SBFS immersion; aluminum and magnesium were contaminant elements from the sample holder. Presented EDX spectra correspond to qualitative analysis and to determine the identification of the elements present and is not a normalized quantitative analysis.

Fig 5

a. Normalized average percentage of DNA liberation of human fibroblasts to the culture medium for the different experimental groups. Mean values and standard deviations of 3 independent experiments for each experimental group are presented. C+ and C–are positive and negative control for cytotoxicity respectively. All the experimental groups were significantly different from controls (p<0.05). The rest of the groups were equal except for NPs 1% group that was different from Zn-NPs 1% (p<0.05), and NPs 100% group that was different from Zn-NPs 100% (p<0.05), being Zn-NPs less toxic to cells. b. Normalized average percentage of LDH liberation of human fibroblasts to the culture medium for the different experimental groups. Mean values and standard deviations of 5 independent experiments for each experimental group are presented. C+ and C–are positive and negative control for cytotoxicity respectively. All the experimental groups were significantly different from controls (p<0.05). The rest of the groups were similar (p>0.1). c. Average cell viability according to the Live/Dead assay in human fibroblasts cells incubated with different NPs dilutions and controls. Mean values and standard deviations of 5 independent experiments for each experimental group are presented. C+ and C–are positive and negative control for cytotoxicity respectively. All the experimental groups were significantly different from controls (p<0.05). The rest of the groups were equal except for NPs 100% group that was less cytotoxic than Zn-NPs 100% and Ca-NPs 100% groups (p<0.01). 1, 10, 50 and 100% dilutions correspond to 30, 15, 3 and 0.3 mg of NPs/ml respectively. Ca-NPs contain 0.99 μg of Ca²⁺/mg of NP and Zn-NPs contain 2.18 μg of Zn²⁺/mg of NP.

Fig 6. Fluorescence microscopy images corresponding to the analysis of cell viability according to the LIVE/DEAD assay, in human fibroblast cells, incubated with different NPs dilution for 24 h.

Green cells correspond to live cells whereas dead cells are stained in red. a, b, c and d are cells exposed to 1,10,50 and 100% NPs dilutions. e, f, g and h correspond to cells exposed to calcium loaded NPs at 1, 10, 50 and 100% dilutions. i, j, k, and l are cells incubated with 1, 10, 50, and 100% dilutions of zinc loaded NPs. 1, 10, 50 and 100% dilutions correspond to 30, 15, 3 and 0.3 mg of NPs/ml respectively. Ca-NPs contain 0.99 μg of Ca²⁺/mg of NP and Zn-NPs contain 2.18 μg of Zn²⁺/mg of NP.