Preparation And Study Of Anti-Porphyromonas Gingivalis Liposome-Rh2 In Periodontitis

Periodontitis is associated with the interaction between oral microorganisms and host immunity.P.gingivalis is the keystone pathogen of periodontitis.It can affect the interaction between symbiotic microbial communities and trigger an inflammatory response,which induces the blooming of inflammophilic pathogens.Small populations of P.gingivalis can alter the relative abundance of specific taxa and thus contribute to the progression of the periodontitis.In addition,the resulting dysbiosis in the oral microbiota further manipulates the host immune response by entering the host cell.It can disrupt the immune system,evade the phagocytosis of macrophages,and invade other tissues.Due to the properties of P.gingivalis,multiple factors interventions are required for periodontitis,which is more intractable than infectious disorders caused by typical species.Ginsenoside Rh2,as a phytochemical,has broad-spectrum bactericidal efficiency,anti-inflammatory functions,and a prominent role in modulating the immune system.It is an ideal drug for the treatment of bacterial infections.However,the application of such phytochemicals is often limited due to their high insolubility,and poor permeability.Although it has been widely used in clinical practice,it has not been used in the treatment of periodontitis.Liposome-based carriers have outstanding stability and the ability to increase the bioavailability of phytochemicals.Treatment with liposomes can overcome the permeability limitation of the cellular barrier,increasing the intracellular antimicrobial and anti-inflammatory activities of the drug.Moreover,in order to both target bacteria and enter cells,antibody targeted liposome was selected as the nanocarrier.Targeted drugs can not only reduce bacterial resistance and cytotoxic effects on host cells but also maintain the bacterial homeostasis.Based on the above,we developed a multifunctional nanomaterial that could target P.gingivalis,enter cells to remove intracellular bacteria,and regulate immune response: A-L-R.A-L-R could effectively target P.gingivalis,maintain the bacterial homeostasis,promote intracellular release,and inhibit bacterial intracellular survival.In addition,A-L-R could reduce the inflammatory response by inhibiting the expression of inflammatory cytokines.Accordingly,this multifunctional nanomedicine could offer multiple strategies for periodontitis therapy.A-L-R could provide a foundation for other oral bacterial infections.Part I Design,synthesis,and characterization of L-R and A-L-RObjective: P.gingivalis can affect the occurrence and progression of periodontitis.The mechanism involved in the actions of P.gingivalis comprises two parts: 1.P.gingivalis can indirectly affect the progression of the disease by affecting the interspecies interactions of the symbiotic microbial community.It induces the blooming of inflammophilic pathogens.The resulting dysbiosis in the oral microbiota forms an inflammatory microenvironment.2.P.gingivalis can cause inflammatory diseases directly by interfering with host immunity.P.gingivalis can disrupt the immune system,evade phagocytosis,and further manipulates the host immune.In order to reduce inflammation and kill intracellular bacteria,Rh2 was coated with liposome,and the antibody was conjugated with L-R.With the cell affinity of liposomes and the antimicrobial,anti-inflammatory properties of Rh2,A-L-R could reduce cell inflammation and kill intracellular bacteria.A-L-R could also target P.gingivalis and maintained the stability of the bacterial community.Moreover,A-L-R could achieve the effect of low toxicity and high efficacy.Methods: By HPLC,the content of free Rh2 was determined to determine the drug loading and encapsulation rate.ELISA was used to detect the specificity and sensitivity of MIg G1(Serum Type B specific cell surface antigen antibody)and MIg G2a(anti-gingival protease antibody)to P.gingivalis.FTIR was used to detect the chemical structure changes of liposomes and L-R.Subsequently,particle size and zeta potential were determined using a nanoparticle size analyzer.TEM was used to observe the particle morphology.A-L-R containing free Rh2 was stored at 60℃,37℃,4℃,-80℃,or under 6500 K light for at least 10 h every day.Or 10 times the volume of 0.9% Na Cl(p H6.5),1 m M HCl(p H3.0),1 m M Na OH(p H11.4),or H2O2 were added to the remaining samples for 4,10,20,and 28 days.Observed its appearance at each point in time and measured its encapsulation rate to test its stability.Results: Liposomes were mainly composed of cholesterol and phospholipid.Liposomes had the structure of cell membranes.In this study,DPPC and mal-PEG-DSPE were selected as pegylated phospholipids.The prepared liposome was incubated with Rh2 to obtain L-R.The proportions of cholesterol,DPPC,and mal-PEG-DSPE were adjusted according to drug loading and encapsulation rate by orthogonal experiment.When the molar ratio of cholesterol,DPPC,mal-PEG-DSPE,and Rh2 was 2:2:10:1,the optimal loading rate of L-R was 78.86% and the encapsulation rate was 20.69%.MIg G1 and MIg G2 a could both effectively target P.gingivalis.The sensitivity of MIg G1 was slightly higher than MIg G2 a.Then A-L-R with MIg G1 was prepared.The FTIR assay confirmed that Rh2 and the binding antibody were successfully encapsulated in the liposome without damaging the structure of the liposome.In the FTIR spectra of pure liposomes and A-L-R,antisymmetric and symmetric vibrations of phosphatidylcholine adipose chains were found.The particle size of A-L-R in aqueous solution was less than 100 nm and the zeta potential is-3.78 m V.It was observed by TEM that the diameter of A-L-R compressed by 100 nm filter was about 52.99 nm and the surface was slightly rough,which had similar characteristics to liposome(the diameter of liposome was about44.94 nm and the surface was slightly rough).A-L-R had good stability(except precipitation in an alkaline environment),which maintained a stable encapsulation rate in high temperature,low temperature,strong light,strong acid,and other environments.Conclusion: In this study,A-L-R with good drug loading and encapsulation rate was successfully prepared.A-L-R retained the basic structure of liposome,successfully encapsulated Rh2,and coupled antibody.A-L-R was an antibody liposome nano-drug with a uniform particle size of less than 100 nm and had good stability.Part Ⅱ Anti-Porphyromonas gingivalis A-L-R for maintaining bacterial homeostasis in periodontitisObjective: In this study,the drug toxicity of A-L-R and Rh2 was evaluated with macrophage RAW264.7 cells and HGF cells.Hemolysis assay of A-L-R and Rh2 was evaluated.Then,the antibacterial effect and mechanism of A-L-R and Rh2 on phytoplankton and biofilm cultured by P.gingivalis(as the keystone pathogenic bacteria of periodontitis),E.faecalis and S.sanguis(as symbiotic bacteria) respectively were studied.Subsequently,the protective effects of A-L-R and Rh2 on bactericidal and flora balance of plankton and biofilm in a mixed culture of the three bacteria were further studied.Methods: CCK-8 was used to calculate the drug toxicity of A-L-R and Rh2 on RAW264.7 cells and HGF cells.The biocompatibility of A-L-R and L-R was evaluated by hemolysis assay.ELISA was used to detect the specificity and sensitivity of A-L-R to P.gingivalis,E.faecalis,and S.sanguis.Subsequently,the efficacy of A-L-R against P.gingivalis,E.faecalis,or S.sanguis was tested: CLSM using live/dead staining assay was performed for the bactericidal effect of biofilm;Absorbance was recorded at 600 nm in a microplate reader for bacteriostatic effect;The MTS assay was performed for bacterial inhibition;The CFU were measured for bactericidal effect.The targeting of A-L-R to P.gingivalis,E.faecalis,and S.sanguis in mixed culture was tested: FISH was used to detect the inhibitory effect of A-L-R in monospecies biofilms;PMA-q PCR was used to detect the inhibitory effect of A-L-R in monospecies plankton.Results: The IC50 of Rh2 and A-L-R for the reduction of RAW264.7 cells was 61.50 and 128.90 μg/m L.The IC50 of Rh2 and A-L-R for HGF cells was 67.94 and 77.68μg/m L.Rh2 and L-R showed high hemolysis activity,while A-L-R showed good biocompatibility.The hemolysis rate of A-L-R was lower than 5%,even after prolonged incubation time.MIg G1 and A-L-R prepared with MIg G1 could selectively and specifically bind to P.gingivalis,but not to symbiotic bacteria.Live/dead cell staining results showed that A-L-R only had a bactericidal effect on P.gingivalis,and had no inhibitory effect on the growth of E.faecalis and S.sanguis,while Rh2 could effectively remove both P.gingivalis,E.faecalis,and S.sanguis.At 32 μg/m L,A-L-R could only inhibit the growth of P.gingivalis,while Rh2 significantly inhibited the growth of those all.125 μg/m L A-L-R had no significant inhibitory effect on the growth of E.faecalis and S.sanguis,while Rh2 had a significant inhibitory effect on the growth of all three bacteria.L-R had a similar effect to Rh2,while A-L did not inhibit any bacterial growth.However,in the mixed bacteria,Rh2 showed a less inhibitory effect,while with only 32 μg/m L A-L-R could reduce the total bacterial load and effectively reduce the proportion of P.gingivalis.A-L-R was effective against only P.gingivalis,but not symbiotic bacteria.Conclusion: MIg G1 antibody was an ideal antibody in the anti-P.gingivalis nanomaterials.A-L-R prepared with MIg G1 antibody could effectively target P.gingivalis,inhibit the growth of P.gingivalis,and kill P.gingivalis.At the same time,it had no effect on other bacteria.In monospecies,A-L-R could effectively reduce the proportion of P.gingivalis,and had a better bactericidal effect than Rh2.A-L-R was effective against only P.gingivalis,but not against E.faecalis and S.sanguis,.Part Ⅲ A-L-R and L-R for anti-inflammatory and intracellular bactericidal effect in vitroObjective: In this study,the drug toxicity of L-R and Rh2 was further evaluated with macrophage RAW264.7 cells and HGF cells.Then,the anti-inflammatory effect of A-L-R,L-R,and Rh2 on LPS-induced RAW264.7 cells and HGF cells,at a non-bacterial toxic dose,was studied.The mechanism and target of drug action were studied.The intracellular bactericidal effect of the drugs on P.gingivalis was evaluated.Methods: The drug toxicity of L-R and Rh2 on RAW264.7 cells and HGF cells was evaluated using CCK-8.Subsequently,LPS was used to induce the production of inflammatory cytokines TNF-α,IL-6,and IL-18 in RAW264.7 cells,and the production of inflammatory cytokines TNF-α,IL-6,and IL-8 in HGF cells.q RT-PCR and ELISA were used to evaluate the anti-inflammatory effect of A-L-R,L-R,and Rh2.Bioinformatics analysis of PPI,KEGG,and GO databases was used to obtain the high-pathway pathway by network pharmacological analysis,and its anti-inflammatory mechanism was further studied.In addition,RAW264.7 cells were co-cultured with P.gingivalis to obtain intracellular bacteria,and the CFU counting method was used to evaluate the intracellular bactericidal effect of A-L-R,L-R,and Rh2.In addition,the bactericidal effect of A-L-R on intracellular bacteria and its targeting of P.gingivalis were observed under CLSM using IF staining.The microstructure of the effect of A-L-R on intracellular bacteria was observed by I-TEM.Results: A-L and low concentration A-L-R had little effect on the growth of HGF and RAW264.7 cells.A high concentration of A-L-R promoted cell growth and had a similar growth-promoting effect as low concentration of Rh2.High concentration of Rh2 was toxic to HGF and RAW264.7 cells.Over time,the effects of L-R on cells remained relatively stable.Both A-L-R and L-R could significantly decrease the levels of TNF-α,IL-6,and IL-18 in LPS-induced RAW264.7 cells and TNF-α,IL-6,and IL-8 in LPS-induced HGF cells.The anti-inflammatory effects of 8 μg/m L A-L-R and L-R were comparable to or better than those of 16 μg/m L Rh2.A-L-R co-target and kill P.gingivalis in RAW264.7 cells more effectively than Rh2.A-L had no effect on bacterial growth and even promoted the entry of P.gingivalis into host cells.Conclusions: A-L-R showed low cytotoxicity and high stability in both concentration-dependent and time-dependent cytotoxicity studies.A-L-R showed significant anti-inflammatory effects on RAW 264.7 and HGF cells.Even at lower doses,they had better anti-inflammatory effects.With low doses but high effects,it gained low toxicity.Meanwhile,L-R and A-L-R had a better intracellular bactericidal effect than Rh2.Part Ⅳ The effect of A-L-R and L-R on periodontitis in vivoObjective: In this study,we evaluated the biosafety of A-L-R in health and periodontitis disease models.The periodontitis model induced by gingival ligation and P.gingivalis was established in rats.The effect of A-L-R on periodontitis in vivo was evaluated.Furthermore,the bactericidal effect of A-L-R on P.gingivalis in vivo was further studied.The protective effect on the balance of microbial flora,the anti-inflammatory effect,and the effect of eliminating bacteria in tissues were studied.Methods: 6-week-old rats were used.Blood was collected from eyeballs on days 2ed,8th,and 15 th after a high dose of subgingival injection.ALP,ALT,BUN,and CREA were detected by an automatic blood biochemical analyzer.The rats were weighed every two days for testing the biological toxicity of the drug.Another group of6-week-old rats were treated with periodontal silk ligature and inoculated with P.gingivalis for 13 days to induce periodontitis.The subgingival injection was administered every 4 days(on days 2ed,6th,10 th,and 14th)and then untreated for one week.During the experiment,the rats were weighed every three days.And at the end of the experiment(day 21st),blood,heart,liver,spleen,lung,kidney,and tongue were collected for detecting the biological toxicity of the drug.On days 6th,14 th,and 21 st,GCF samples were collected,and the amount of P.gingivalis and total bacteria in GCF were measured by CFU counting.The proportion of P.gingivalis in GCF was detected by q RT-PCR.Next next-generation sequencing technology was used for detecting bacterial genomic DNA and the changes in oral microbiomes.The rats were sacrificed on the day 21 st,and the teeth and periodontal tissues were collected.The periodontal histological changes were observed after H-E staining,and the degree of tooth bone defect was evaluated by Micro-CT and SM.At the same time,the periodontal tissue was IF stained to observe the changes in periodontal inflammatory factors.In addition,the gingival tissues were collected.The total amount of P.gingivalis and bacteria in gingival tissues were measured by CFU counting with defibrinated sheep blood medium.Results: In the toxicity test with normal rats,the body weight and blood between groups were not statistically different.In the experimental periodontitis model,the body weight,blood,and visceral tissues of groups were not significantly different or within the normal range.The experimental periodontitis rat model was successfully prepared.In the control group,there were more alveolar bone loss,bifurcation defect,and horizontal penetrating alveolar bone defect.A-L group showed the same result as control group.There was,but not much,therapeutic effect in Rh2 group.By contrast,the gingiva of L-R and A-L-R groups showed a healthier appearance.H-E staining showed that the A-L-R group had the least collagen structure damage.After administration,P.gingivalis in L-R and Rh2 groups significantly increased.The total number of bacteria in both L-R and Rh2 groups was also increased.The q RT-PCR detection and next next-generation sequencing technology showed that the amount of P.gingivalis in the A-L-R group remained at a low level and effectively protected E.faecalis and related symbiotic species.A-L-R maintained the stability of the flora.IF histological analysis showed that the expressions of inflammatory factors(IL-6 and TNF-α)in Rh2 and L-R groups were significantly lower than those in control group but significantly higher than those in A-L-R group.Conclusion: L-R and A-L-R could be used in normal rats and experimental periodontitis model rats with good biological safety.The gingiva of L-R and A-L-R groups showed better periodontal condition and less bone defect than those of control,A-L,and Rh2 groups.L-R and A-L-R groups effectively reduced inflammatory factors and bacteria in and outside tissues.A-L-R group could effectively and continuously reduce the number of P.gingivalis and maintain the stability of microflora in vivo.