Biomimetic mineralization mediated by proteins and peptides is a promising strategy for enamel remineralization, and its specific application model needs more research. However, the presence of a variety of cells, bacteria, proteases and inflammatory substances in the oral environment might become adverse factors to destroy the structure or inhibit the activity of peptides, which largely limited their oral clinical application. Peptide drugs encapsulated in the carrier, could be isolated from the external environment, effectively maintaining its structure and activity for targeted delivery. Liposomes are a kind of lipid vesicles mainly composed of phospholipids. This special structure is mainly formed by hydrophobic interaction between phospholipids. The amphiphilicity of phospholipids can encapsulate hydrophilic and hydrophobic drugs in the water nucleus and lipid bilayer membrane of liposomes, respectively. Therefore, as drug delivery carriers, liposomes have been widely used in the field of biomedicine[37]. Among different methods for preparing liposomal delivery system, active loading method, also known as remote loading method, is the direct loading of drug molecules into the prepared liposomes[38]. The pH gradient and potential difference are usually the main driving forces for drug entry into the liposomes. Active loading method has high efficiency and large loading capacity, and can prevent the degradation of bioactive compounds in the preparation process[29, 39, 40]. Among them, calcium acetate gradient method can be used for the preparation of weakly acidic drug liposomes, which takes advantage of the difference in permeability coefficient between lipid bilayer of cations and acetic acid molecules[41]. In this study, DK5-Lips was prepared by calcium acetate gradient method according to the weak acidity of salivary biomimetic anti-caries peptide DK5.
The in vitro release study showed that as compared with the rapid release of DK5 bulk, the DK5-Lips was released in a biphasic pattern, with a rapid initial rate followed by a slow and persistent release. The main reason probably was that at the initial stage of drug release, DK5 attached to the liposomes surface leached out and quickly dissolved in the release medium, making the drug concentration in the medium rise quickly. Subsequently, drugs encapsulated in liposomes were driven by diffusion control mechanism and released slowly through lipid bilayer to achieve sustained drug release[42].
The pH-cycling model could measure the net effect of inhibiting demineralization and enhancing remineralization by repeatedly alternating demineralization and remineralization solutions on demineralized samples, mimicking the periodic pH changes during caries in vivo and the kinetics of mineral loss and gain in dental hard tissue[43]. Hence, a classical pH-cycling model was applied to quantify whether DK5-Lips showed significant remineralization activity in vitro. After 7 and 14 days of pH-cycling, SMH analysis showed a higher SMHR% in the DK5-Lips group than in the negative groups, which indicated a significant increase in surface hardness of enamel lesions after treatment with DK5-Lips. The results were also confirmed by PLM, exhibiting a brighter remineralization layer and shallower caries lesions. Lippert et al. confirmed the strong correlation between TMR and surface microhardness in the study of initial enamel caries formation[44]. And TMR could provide supplementary information about the structural changes and mineral content of initial enamel caries. Similarly, TMR analysis showed that DK5-Lips reduced the mineral loss and lesion depth, and increased the mineral content of the surface layer and lesion body after pH-cycling, indicating that the demineralized subsurface of enamel was remineralized by attracting mineral crystal deposition under the treatment of DK5-Lips. The above results of DK5-Lips were preferable compared with DK5, which confirmed that DK5-Lips could promote the mineralization-inducing effect of peptide DK5 on initial enamel caries.
The reasons for promoting mineralization-inducing effect of peptide DK5 might be as follows: DK5-Lips could promote remineralization over a long period by slowly releasing peptide DK5 on the surface of demineralized enamel and maintaining DK5 concentration levels[45]. In addition, liposomes might isolate calcium and phosphate ions from saliva by simulating the role of matrix vesicles in human hard tissue formation, provide special compartments for biological mineralization, control the deposition of mineral particles, and promote the occurrence of mineralization reactions[46]. Therefore, the application of DK5-Lips in the remineralization of artificial initial enamel caries might be more advantageous than direct utilization of DK5. However, the mechanism of DK5-Lips promoting remineralization need to be further explored.
The rodent caries model is of great significance for the study of the etiology, pathology and prevention of caries, and makes a clearer understanding of the occurrence and development of caries in the complex oral environment[47]. Therefore, the Sprague-Dawley rat caries model was used for in vivo experiments in this study. The results of body weight, HE staining, blood routine and biochemistry all showed good biocompatibility of DK5-Lips. Only the level of alkaline phosphatase (ALP) in all groups was 1.5-2 times of the normal range, possibly due to the fact that ALP is considered to regulate fatty acid absorption, which is related to diet-induced obesity[48]. Keyes caries scoring standard is a classical method for evaluating the development and severity of rat caries in animal studies. It is a fast and intuitive method for caries evaluation by classifying and scoring the caries location and depth[36]. In the present study, compared with negative groups, caries scores for smooth surfaces and fissures were significantly lower after treatment with DK5-Lips and NaF, followed by DK5 treatment, which indicated that DK5-Lips promoted remineralization and reversed progression of carious lesions in vivo. PLM results were consistent with the caries scoring results. Micro-CT is a non-destructive and high-resolution imaging technology, which can carry out three-dimensional analysis of the structural characteristics of samples, and has good application value in the evaluation of dental hard tissue volume and mineral density[49]. The higher mineral density of residual molar enamel indicated stronger mineralization, and the larger volume of residual molars enamel suggested the smaller caries range and the stronger anti-caries capability[34]. The rats treated with DK5-Lips and NaF showed higher volume and density values of residual molar enamel than the negative groups, followed by DK5 treatment, demonstrating a relatively better remineralization effect of DK5-Lips. Besides, as compared with negative groups, the results after three-dimensional reconstruction also showed increased enamel density in the DK5-Lips group and no significant decrease in dentin density was observed. Taken together, the above findings proved that DK5-Lips could hold potentials of bioactive therapeutics for initial enamel caries through significantly inducing the increase of enamel mineral content and reducing the degree of caries.
We successfully developed a liposomal delivery system for a novel peptide derived from histatin-1 (DK5-Lips) as a new biomimetic mineralization strategy against initial enamel caries. The liposomes showed slow-release effect of peptide DK5 with good stability in saliva. Furthermore, the system could exert significant anti-caries effect both in vitro and in vivo, evidenced by the increased level of remineralization and the reduced degree of caries decay (Fig. 8). Given those, DK5-Lips provides a promising and efficient remineralization method for initial caries management and has translation potential for the clinical application of peptides.