Pharmacodynamics of siRNA RANKL and Oestrogen Loaded MSNs-CADY on Human Periodontal Ligament Stem Cells with Porphyromonas gingivalis infection

Background: Periodontitis irreversibly invades and destroys periodontal supporting tissues, loses the ability of periodontal regeneration and restoration, and eventually leads to tooth loosening and loss. periodontal ligament stem cells (PDLSCs) hold great promises for periodontal tissue regeneration which was the potential target of periodontitis treatment, siRNA RANKL and oestrogen can help PDLSCs maintain normal function, however, it was very difficult for siRNA RANKL and oestrogen to get into PDLSCs. Here, Cell penetrating peptide CADY was modified on the surface of siRNA RANKL and oestrogen loaded mesoporous silica nanoparticles (MSNs) to carry them into Porphyromonas gingivalis infected PDLSCs, Then further affect the proliferation of PDLSCs. Methods: 120-150 nm Mesoporous silica nanoparticles (MSNs) was prepared, and the biocompatibility, loading capacity and drug release properity were tested; MSNs was modified by penetrating peptide CADY and the prepared MSNs/CADY was loaded with siRNA RANKL and oestrogen; In vitro drug release of siRNA RANKL /MSNs-CADY and oestrogen/MSNs-CADY was tested by using semi-permeable dialysis bag diffusion; Cellular uptake and internalization of FITC-Labeled MSNs and FITC-Labeled MSNs-CADY was observed by use of Laser confocal microscopy; Finally, the effect of siRNA RANKL and oestrogen loaded MSNs-CADY on cell proliferation of Porphyromonas gingivalis infected human periodontal ligament stem cells was tested by MTT assay. Results: according to the results, MSNs-CADY with a concentration of 6.25-200 ug/mL have no toxic to PDLSCs; 24.6 mg oestrogen and 0.5 mM siRNA RANKL can be loaded into 1mg of MSNs-CADY; and drug loaded MSNs-CADY nanodrug carriers can release siRNA RANKL and oestrogen stably for at least 48 h; After modification with cell penetrating peptide CADY, more MSNs-CADY can be taken by PDLSCs. siRNA RANKL /oestrogen/MSNs-CADY can increase the proliferation of PDLSCs significantly. Conclusion: siRNA RANKL /oestrogen/MSNs-CADY constructed can significantly improve the cell proliferation of P-gingivalis infected PDLSCs, this nano drug carrier has the potential to be used in PDLSCs -based periodontitis treatment, this work provided a useful theoretical basis and therapeutic ideas for the treatment of periodontitis.

periodontal regeneration and restoration, and eventually leads to tooth loosening and loss, it is an inflammatory disease negatively affecting up to 15% of adults worldwide [1]. Plaque bacteria and their metabolites are the initiating factors of periodontal disease. Porphyromonas gingivalis (P.gingivalis) is one of the most virulent periodontal pathogens. It is closely related to the severity and recurrence of periodontal lesions [2]. It is generally agreed that P.gingivalis infection effect bone formation and bone resorption and then effect the reconstruction of the periodontal ligament (PDL) [3]. Recent studies reported that periodontal ligament stem cells (PDLSCs) hold great promises for periodontal tissue regeneration, PDLSCs possess the capacity for self-renewal and multipotent differentiation, and are able to form cementum/PDL-like tissue in vivo [4,5].
As we know, the Receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) system plays a very important role in the regulation of osteoclastogenesis in bone systems [6], Lipopolysaccharide (LPS) is the most important pathogenic factor of P.gingivalis, the main pathogen of periodontitis. It can promote the up-regulation of lymphocyte inflammatory factor secretion, activate osteoclast expression, and lead to alveolar bone destruction and absorption [7]. It has been reported that LPS of P.gingivalis can up-regulate the expression of RANKL in periodontal ligament cells and osteoblasts [8]. Oestrogen (E2) is a hormone that affecting biological processes that include bone formation, angiogenesis and autoimmunity [9][10][11]. It was also reported that exogenous E2 enhanced the proliferation and osteogenic differentiation of hPDLSCs during in vitro culture [12]. Theoretically, the expression of RANKL should be decreased in hPDLSCs which infected by P.gingivalis, and exogenous E2 at an appropriate concentration can also help hPDLSCs maintain normal function. Therefore, drug carrier with high drug loading capacity and good release performance was needed.
The main purpose of this study was to construct a mesoporous nano drug carrier which can improve the internalization efficiency of siRNA RANKL and oestrogen, and then to evaluate the pharmacodynamics of the siRNA RANKL and oestrogen loaded drug carrier on proliferation of P-gingivalis infected PDLSCs.our study provided a useful theoretical basis and therapeutic ideas for the treatment of periodontitis.

Construction of CADY modified MSNs
Firstly, Mesoporous silica nanoparticles with an diameter of about 120 nm were prepared according to Liu's method with some modification [13].
In brief, 5 mL NH 3 · H 2 O, 5 mL ddH 2 O and 50 mL ethanol were mixed in and flasked，then the mixed solution was heated to 60 ℃. and then 2 mL

Cellular Internalization
The PDLSCs cells were seeded on 60 mm dishes (Corning Incorporated).

Statistical Analysis
One-way ANOVA was used for statistical analysis of data to confirm the presence of significant differences in measured parameters between test groups; Individual points were compared using a Student's t test, and differences were considered significant at p<0.05. All the experimental data were expressed as mean ± SD. All the experiments were repeated at least three times (n = 3).

MSNs-CADY
The MSNs with the diameter about 120nm were first synthesized, then the black MSNs were modified with carboxyl, finally, cell penetrating peptide CADY was attached on the MSNs by use of EDC and NHS reaction. TEM images of each nanoparticle were shown in figure 1, and zeta potential of each nanoparticle indicated that the CADY has been modified on MSNs and 120 nm of MSNs-CADY was prepared successfully.

FITC-Labeled MSNs-CADY
Suit nano-drug carriers should effectively carry drugs into cells, membrane-penetrating peptides such as CADY has been reported can improve the cellular internalization efficiency of drug-carriers [14][15][16]. Here

Effect of siRNA RANKL /oestrogen/MSNs-CADY on the cell proliferation of P-gingivalis infected PDLSCs
The direct effect of bacterial infection will cause to greatly reduction of cell proliferation. Here, we tested the effect of nano-drug carriers on the proliferation of p-gingivalis infected PDLSCs, as the result shown in

Discussion
Periodontitis is a common complex disease with relatively late onset, which is characterized by inflammatory destruction of periodontal attachment and bone loss. PDLCs) are able to participate in connective tissue remodeling while suffering mechanical stress from orthodontic appliances. If the disease is not treated in time, repeated attacks over a long period of time can cause dental defects, and then affect oral function.
In clinic, systemic or oral local therapy is often used [2,17,18]. The P-gingivalis is the main pathogen of periodontal disease [19].  [24]. Here, we studied the effect of siRNA RANKL and oestrogen loaded MSNs-CADY on the cell proliferation of P-gingivalis infected PDLSCs, and results shown that siRNA RANKL /oestrogen/MSNs-CADY can increase the proliferation of P-gingivalis infected PDLSCs significantly.

Conclusion
In short, siRNA RANKL /oestrogen/MSNs-CADY we constructed here can significantly improve the cell proliferation of P-gingivalis infected PDLSCs, this nano drug carrier has the potential to be used in PDLSCs -based periodontitis treatment, this work provided a useful theoretical basis and therapeutic ideas for the treatment of periodontitis. In addition, we will continue to evaluate the efficacy of the nano drug carrier in periodontitis treatment in vivo in the future and to provide preclinical theoretical basis for the treatment of periodontitis.