Fight bacteria with bacteria: Bacterial membrane vesicles as vaccines and delivery nanocarriers against bacterial infections.

Bacterial membrane vesicles (MVs) are particles secreted by bacteria with diameter of 20-400 nm. The pathogen-associated molecular patterns (PAMPs) present on the surface of MVs are capable of activating human immune system, leading to non-specific immune response and specific immune response. Due to the immunostimulatory properties and proteoliposome nanostructures, MVs have been increasingly explored as vaccines or delivery systems for the prevention and treatment of bacterial infections. Herein, the recent progresses of MVs for antibacterial applications are reviewed to provide an overview of MVs vaccines and MVs-related delivery systems. In addition, the safety issues of bacterial MVs are discussed to demonstrate their potential for clinical translation. In the end of this review, the challenges of bacterial MVs as vaccines and delivery systems for clinical applications are highlighted with the purpose of predicting future research directions in this field.

In vitro dentin tubule occlusion by an arginine-containing dentifrice.

PURPOSE: To evaluate the ability of an arginine-containing dentifrice to occlude dentin tubules. METHODS: Dentin discs were divided equally into premolar and molar groups, which were then utilized in three treatment groups: a blank control group (distilled water treatment), a negative control group (common dentifrice with calcium carbonate) and an experimental group [dentifrice with 8% (w/w) arginine]. Each dentin disk was brushed with the dentifrice twice daily for 7 consecutive days. After this period, each disc was separated into two equal halves. One half was used for scanning electron microscopy (SEM) and energy-dispersive spectrometer (EDS) examinations, while the other half was brushed with distilled water twice daily for another 7 days prior to SEM observation. RESULTS: The plugging rate in the arginine dentifrice group was significantly higher and more sustainable than in the negative control group. The surface deposition of calcium and phosphorus on the dentin discs in the arginine dentifrice group was also significantly higher. CLINICAL SIGNIFICANCE: This study provided evidence that using arginine as an active ingredient in dentifrice can improve its ability to occlude dentin tubules, thus supporting future efforts to improve dentin hypersensitivity.

Mitochondria-Targeting, Intracellular Delivery of Native Proteins Using Biodegradable Silica Nanoparticles.

Mitochondria are key organelles in mammalian cells whose dysfunction is linked to various diseases. Drugs targeting mitochondrial proteins provide a highly promising strategy for potential therapeutics. Methods for the delivery of small-molecule drugs to the mitochondria are available, but these are not suitable for macromolecules, such as proteins. Herein, we report the delivery of native proteins and antibodies to the mitochondria using biodegradable silica nanoparticles (BS-NPs). The modification of the nanoparticle surface with triphenylphosphonium (TPP) and cell-penetrating poly(disulfide)s (CPD) facilitated their rapid intracellular uptake with minimal endolysosomal trapping, providing sufficient time for effective mitochondrial localization followed by glutathione-triggered biodegradation and of native, functional proteins into the mitochondria.

Healing of root and surrounding periodontium after root damage with miniscrew implants: a histomorphologic study in dogs.

OBJECTIVES: The main purpose of this study was to investigate the detailed healing process of the roots and surrounding periodontium [cementum, periodontal ligament (PDL), and bone] at different time points after intentional root damage with miniscrew implants (MSIs). MATERIALS AND METHODS: After cone-beam computed tomography examination and measurement, a total of 78 premolar and molar roots from five beagle dogs were intentionally damaged by implanting miniscrews in the interradicular region. MSIs were immediately removed, and the histological morphology was observed at days 0 and 3 and at weeks 1, 2, 3, 4, 6, 8, and 12 after root injury using haematoxylin and eosin and fluorescence stainings (fluorescence staining was performed at days 28 and 56). RESULTS: An early new attachment of PDL adhering on to the damaged root surface was found at week 2 after root injury. Tissue differentiation of newly formed bone tissue, PDL, and cementum began at week 3. Moreover, the newly formed cementum and bone were constantly forming and mineralising at weeks 4, 6, 8, and 12, and the width of PDL gradually narrowed until close to the normal width at week 12. CONCLUSIONS: This study demonstrated the complete healing process of the roots and surrounding periodontium after root damage with MSIs in dogs when the damage was limited to the cementum or dentin. CLINICAL RELEVANCE: The findings of this study may help provide a better understanding of the detailed healing process in roots and PDLs damaged by MSIs.

Conjugated-polymer-based red-emitting nanoparticles for two-photon excitation cell imaging with high contrast.

Two-photon fluorescence microscopy is a widely used noninvasive bioimaging technique because of unique advantages such as a large penetration depth and 3D mapping capability. Ideal two-photon fluorophores require large two-photon absorption cross sections and red emission with high quantum yields. Here we report red-emitting-dye-doped conjugated polymer nanoparticles that display high two-photon excitation brightness. In these nanoparticles, conjugated polymer (PFV) was chosen as a two-photon light-harvesting material, and red-emitting dyes (MgPc and Nile red) were chosen as the energy acceptors and red-emitting materials. Two-photon excitation fluorescence of MgPc and Nile red was enhanced by up to ∼53 and ∼240 times, respectively. We have successfully demonstrated the application of these conjugated polymer-based nanoparticles in two-photon excitation cancer cell imaging with an excellent contrast ratio. This concept could become a general approach to the preparation of two-photon excitation red-emitting materials for deep-tissue live-cell imaging with high contrast.

Biofunctional lipid nanoparticles for precision treatment and prophylaxis of bacterial infections.

The lack of bacterial-targeting function in antibiotics and their prophylactic usage have caused overuse of antibiotics, which lead to antibiotic resistance and inevitable long-term toxicity. To overcome these issues, we develop neutrophil-bacterial hybrid cell membrane vesicle (HMV)-coated biofunctional lipid nanoparticles (LNP@HMVs), which are designed to transport antibiotics specifically to bacterial cells at the infection site for the effective treatment and prophylaxis of bacterial infection. The dual targeting ability of HMVs to inflammatory vascular endothelial cells and homologous Gram-negative bacterial cells results in targeted accumulation of LNP@HMVs in the site of infections. LNP@HMVs loaded with the antibiotic norfloxacin not only exhibit enhanced activity against planktonic bacteria and bacterial biofilms in vitro but also achieve potent therapeutic efficacy in treating both systemic infection and lung infection. Furthermore, LNP@HMVs trigger the activation of specific humoral and cellular immunity to prevent bacterial infection. Together, LNP@HMVs provide a promising strategy to effectively treat and prevent bacterial infection.

High concentrations of NaF aggravate periodontitis by promoting M1 polarization in macrophages.

High-concentration fluoride treatment is commonly used to prevent dental caries in the oral cavity, and fluorine-containing protective paint is used to alleviate common root sensitivity symptoms in patients with periodontitis after periodontal treatment. Recent studies have confirmed its safe use in normal oral environments. However, whether fluoride treatment affects the progression of periodontitis in an inflammatory microenvironment remains unclear. Immunometabolism is crucial for maintaining bone regeneration and repair in periodontitis, and the precise regulation of macrophage polarisation is crucial to this process. Fluoride can influence the immune microenvironment of bone tissue by regulating immune metabolic processes. Herein, we investigated the effects of high concentrations of sodium fluoride (NaF) on periodontal tissues. We examined the expression of osteogenic and M1/M2 macrophage polarisation markers and glucose metabolism in macrophages. RNA sequencing was used to study differentially expressed genes related to M1 polarisation and glucose metabolism in treated macrophages. The results showed that NaF indirectly affects human periodontal ligament cells (hPDLCs), aggravating bone loss, tissue destruction, and submandibular lymph node drainage. Furthermore, NaF promoted glycolysis in macrophages and M1 polarisation while inhibiting osteogenic differentiation. These findings suggest that NaF has a direct effect on hPDLCs. Moreover, we found that high concentrations of NaF stimulated M1 polarisation in macrophages by promoting glycolysis. Overall, these results suggest that M1 macrophages promote the osteoclastic ability of hPDLCs and inhibit their osteogenic ability, eventually aggravating periodontitis. These findings provide important insights into the mechanism of action of NaF in periodontal tissue regeneration and reconstruction, which is critical for providing appropriate recommendations for the use of fluoride in patients with periodontitis.

Polymeric micellar nanoparticles for effective CRISPR/Cas9 genome editing in cancer.

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) gene editing has attracted extensive attentions in various fields, however, its clinical application is hindered by the lack of effective and safe delivery system. Herein, we reported a cationic micelle nanoparticle composed of cholesterol-modified branched small molecular PEI (PEI-CHO) and biodegradable PEG-b-polycarbonate block copolymer (PEG-PC), denoted as PEG-PC/PEI-CHO/pCas9, for the CRISPR/Cas9 delivery to realize genomic editing in cancer. Specifically, PEI-CHO condensed pCas9 into nanocomplexes, which were further encapsulated into PEG-PC nanoparticles (PEG-PC/PEI-CHO/pCas9). PEG-PC/PEI-CHO/pCas9 had a PEG shell, protecting DNA from degradation by nucleases. Enhanced cellular uptake of PEG-PC/PEI-CHO/pCas9 nanoparticles was observed as compared to that mediated by Lipo2k/pCas9 nanoparticles, thus leading to significantly elevated transfection efficiency after escaping from endosomes via the proton sponge effect of PEI. In addition, the presence of PEG shell greatly improved biocompatibility, and significantly enhanced the in vivo tumor retention of pCas9 compared to PEI-CHO/pCas9. Notably, apparent downregulation of GFP expression could be achieved both in vitro and in vivo by using PEG-PC/PEI-CHO/pCas9-sgGFP nanoparticles. Furthermore, PEG-PC/PEI-CHO/pCas9-sgMcl1 induced effective apoptosis and tumor suppression in a HeLa tumor xenograft mouse model by downregulating Mcl1 expression. This work may provide an alternative paradigm for the efficient and safe genome editing in cancer.

Liposomes Co-Delivering Curcumin and Colistin to Overcome Colistin Resistance in Bacterial Infections.

Antibiotic colistin is the last line of defense against multidrug-resistant (MDR) Gram-negative bacterial infections. Emergence of colistin resistance in microbes is a critical challenge. Herein, curcumin is discovered, for the first time, to reverse the resistance phenotype of colistin-resistant bacteria via a checkerboard assay. For the co-delivery of curcumin and colistin, negatively charged poly(ethylene glycol)-functionalized liposomes encapsulating both drugs (Lipo-cc) are prepared. Killing kinetics and live/dead assays confirm the antibacterial activity of Lipo-cc against colistin-resistant bacteria, which is more potent than that of the free curcumin and colistin combination. Mechanistical studies reveal that Lipo-cc restores the affinity of colistin for the bacterial membrane and improves the uptake of curcumin, which leads to reduced efflux pump activity, achieving a synergistic effect of colistin and curcumin. At the effective antibacterial dose, Lipo-cc does not exhibit any toxicity. The therapeutic efficacy of Lipo-cc is further demonstrated in an intestinal bacterial infection model induced with colistin-resistant Escherichia coli. Lipo-cc reduces the bacterial burden with over 6-log reduction and alleviated inflammation caused by infection. Importantly, unlike colistin, Lipo-cc does not affect the homeostasis of the intestinal flora. Taken together, Lipo-cc successfully overcame colistin resistance, indicating its potential for the treatment of colistin-resistant bacterial infections.

Antibiotic-Polymer Self-Assembled Nanocomplex to Reverse Phenotypic Resistance of Bacteria toward Last-Resort Antibiotic Colistin.

Colistin is the last-resort antibiotic to treat multidrug-resistant (MDR) Gram-negative bacterial infections that are untreatable by other clinically available antibiotics. However, the recently merged plasmid-borne gene mobilized colistin resistance (mcr) leads to modification of the colistin target (i.e., bacterial membrane), greatly compromising the therapy outcome of colistin. To address this unmet clinical need, a nanocomplex (CMS-pEt_20 NP) of anionic prodrug colistin methanesulfonate (CMS) and guanidinium-functionalized cationic polymer pEt_20 is developed through facile self-assembly for co-delivering an antibiotic and antimicrobial polymer with membrane affinity to reverse colistin resistance. The CMS-pEt_20 NP formation enables reversal of colistin resistance and complete killing of clinically isolated mcr-positive colistin-resistant bacteria including MDR E. coli and K. pneumoniae, while monotreatment of polymer or antibiotic at equivalent doses exhibits no antibacterial activity. Mechanistic studies reveal that the CMS-pEt_20 NP enhanced the affinity of delivered CMS to the modified membrane of colistin-resistant bacteria, reviving the membrane lytic property of colistin. The increased membrane permeability caused by colistin in turn promotes an influx of pEt_20 to generate intracellular ROS stress, resulting in elimination of colistin-resistant bacteria. More importantly, a colistin-resistant mouse peritonitis-sepsis infection model demonstrates the excellent therapeutic efficacy of CMS-pEt_20 NP with 100% survival of the infected mouse. In addition, the nanocomplex is proven not toxic both in vitro and in vivo. Taken together, the self-assembled antibiotic-polymer nanocomplex with two complementary antibacterial mechanisms successfully reverses the colistin resistance phenotype in bacteria, and it can be a potential strategy to treat untreatable colistin-resistant MDR bacterial infections.

Interpenetrating Polymer Network Hydrogels Formed Using Antibiotics as a Dynamic Crosslinker for Treatment of Infected Wounds.

Antibacterial hydrogels, particularly antibiotic-loaded hydrogels, are promising wound dressing materials for treatment of bacteria-infected wound. However, it is challenging to achieve sustained release of antibiotics from hydrogels through physical encapsulation of the antibiotics. Herein, an interpenetrating polymer network P(AA-co-HEMA)Gen hydrogel is reported with double crosslinking formed by free radical polymerization of 2-hydroxyethyl methacrylate (HEMA) and acrylic acid (AA), while using the antibiotic gentamicin (Gen) as the dynamic physical crosslinker. Gentamicin is incorporated into the hydrogel networks via electrostatic interaction between the carboxyl groups of poly(acrylic acid) and the amino groups of gentamicin, which leads to pH-responsive drug release and a significant increase in mechanical strength (i.e., elastic modulus, viscous modulus, and compressive modulus). More importantly, the hydrogels with optimal compositions demonstrate long-lasting antibacterial activity against both Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli) over 28 d. The in vivo studies that are conducted in an S. aureus-infected full-thickness skin wound model demonstrate that the double crosslinking hydrogels loaded with gentamicin eliminate bacteria in the wounds more effectively and significantly accelerate wound healing as compared to 3M dressing and the control without any treatment. Taken together, this antibiotic-loaded interpenetrating polymer network hydrogel is potentially a promising wound dressing material for the treatment of bacteria-infected wound.

[Effect of nanohydroxyapatite on surface mineralization in acid-etched dentinal tubules and adsorption of lead ions].

OBJECTIVE: To study the effect of nanohydroxyapatite (nHA) for promoting surface mineralization of demineralized dentin discs and adsorption of lead ions in simulated sewage water. METHODS: Sixty dentin disks were prepared from freshly extracted teeth with intact crown (including 30 premolars and 30 molars) and treated with 10% citric acid for 2 min to simulate dentinal tubules with dentin hypersensitivity. The etched dentin discs were brushed with distilled water, 0.2 g HA or 0.2 g nHA for 2 min twice a day for 7 consecutive days, after which scanning electron microscopy (SEM) was performed and calcium and phosphorus contents in the dentin discs were detected using EDS. Lead ion adsorption capacities of HA and nHA were tested by mixing 1 mL serial concentrations of HA and nHA suspensions with 50 mL lead ion solutions (1.0 mg/L). After 24 h, the residual lead ion concentration in the supernatant was measured using ICP to calculate lead ion adsorption rate and adsorption capacity of the materials. RESULTS: SEM showed a smooth surface and empty dentin tubules in the acid- etched dentin dics. The dentin dics treated with HA were covered with masses of particles that loosely attached to the surface, and the diameter of the dentin tubules was reduced. In nHA group, the dentin discs showed a fine and homogeneous surface clogging with a tight attachment, and the dentin tubule diameter was obviously reduced. Daily brushing with HA and nHA, especially the latter, significantly increased calcium and phosphorus contents on the surface of the dentin dics (P < 0.000). In lead ion adsorption experiment, the lead ion adsorption rate of HA decreased as its concentration increased with the highest adsorption rate of 83.01%; the adsorption rate of nHA increased with its concentration until the adsorption equilibrium was reached, and its highest adsorption rate was 98.79%. A good liner relationship was found between the adsorption ability and concentration of HA. CONCLUSIONS: Compared with HA, nHA has a better capacity for surface mineralization of acid-etched dentin discs and also a better ability of lead ion adsorption.

Effect of a dentifrice containing different particle sizes of hydroxyapatite on dentin tubule occlusion and aqueous Cr (VI) sorption.

BACKGROUND: Dentin hypersensitivity is a common negative oral condition that can be treated with dentifrice containing hydroxyapatite (HA). The study evaluated the effect of nano-HA dentifrice on plugging the dentinal tubules for an anti-sensitivity reaction compared to a dentifrice containing common-sized particles. Also, the adsorption capacity of different particle sizes of HA mixed in a dentifrice and which is the optimal particle size was considered. METHODS: Fourty premolar dentine discs and fourty molar dentine discs were randomly divided into 4 groups: distilled water group, ordinary dentifrice group and 80, 300 nm HA dentifrice group. Each dentin disc was brushed with a dentifrice twice daily at 7600 rpm under 100 g force for 2 mins for 7 consecutive days and divided into two parts, half of the dentin disc was detected by the scanning electron microscope (SEM) and energy dispersive spectrometer (EDS), the other half was brushed with distilled water and observed by SEM. One milliliter dentifrice solution (80 nm HA dentifrice, 300 nm HA dentifrice, ordinary dentifrice) was added to 50 ml potassium dichromate solution for 1, 14, and 28 d. The residual Chromium (Cr6+) concentration in the supernatant was measured by the diphenylcarbon phthalocyanine hydrazine method. The elemental constitution in the precipitate was detected by EDS. The Kruskal-Wallis test was used to analyze surface mineralization and different plugging rates of dentinal tubules. The absorption capacity of dentifrices were also evaluated by the Kruskal-Wallis test. RESULTS: The plugging rate in the HA dentifrice group was higher than that in the ordinary dentifrice group, and the 80 nm HA dentifrice group showed the best result. The atomic percentages of Ca and P of 80 nm dentifrice group on the surface of dentinal tubules were the highest. The 80 nm HA dentifrice group showed the best adsorption and stable effect of Cr6+, followed by the 300 nm HA dentifrice group. The 300 nm HA dentifrice and the ordinary dentifrice showed desorption phenomenon. CONCLUSIONS: The dentifrice containing HA, especially the 80 nm HA dentifrice, exerts good dentinal tubule occlusion and surface mineralization effect. This dentifrice was also a good adsorbent of Cr6+.

Effects of dentifrice containing hydroxyapatite on dentinal tubule occlusion and aqueous hexavalent chromium cations sorption: a preliminary study.

In order to endow environmental protection features to dentifrice, hydroxyapatite (HA) was added to ordinary dentifrice. The effects on dentinal tubule occlusion and surface mineralization were compared after brushing dentine discs with dentifrice with or without HA. The two types of dentifrice were then added to 100 µg/ml of hexavalent chromium cation (Cr(6+)) solution in order to evaluate their capacities of adsorbing Cr(6+) from water. Our results showed that the dentifrice containing HA was significantly better than the ordinary dentifrice in occluding the dentinal tubules with a plugging rate greater than 90%. Moreover, the effect of the HA dentifrice was persistent and energy-dispersive spectrometer (EDS) revealed that the atomic percentages of calcium and phosphorus on the surface of dentine discs increased significantly. Adding HA to ordinary dentifrice significantly enhanced the ability of dentifrice to adsorb Cr(6+) from water with the removal rate up to 52.36%. In addition, the sorption was stable. Our study suggests that HA can be added to ordinary dentifrice to obtain dentifrice that has both relieving dentin hypersensitivity benefits and also helps to control environmental pollution.

Gold nanorods as dual photo-sensitizing and imaging agents for two-photon photodynamic therapy.

Gold nanorods with three different aspect ratios were prepared and their dual capabilities for two-photon imaging and two-photon photodynamic therapy have been demonstrated. These gold nanorods exhibit large two-photon absorption action cross-sections, about two orders of magnitude larger than small organic molecules, which makes them suitable for two-photon imaging. They can also effectively generate singlet oxygen under two-photon excitation, significantly higher than traditional photosensitizers such as Rose Bengal and Indocyanine Green. Such high singlet oxygen generation capability under two-photon excitation was ascribed to their large two-photon absorption cross-sections. Polyvinylpyrrolidone (PVP) coated gold nanorods displayed excellent biocompatibility and high cellular uptake efficiency. The two-photon photodynamic therapy effect and two-photon fluorescence imaging properties of PVP coated gold nanorods have been successfully demonstrated on HeLa cells in vitro using fluorescence microscopy and indirect XTT assay method. These gold nanorods thus hold great promise for imaging guided two-photon photodynamic therapy for the treatment of various malignant tumors.

[Research on anatomy of posterior tooth in the Beagle dog and mini-screw implant implantation].

OBJECTIVE: The morphological characteristics of posterior tooth in the Beagle dog were investigated in order to provide reference to mini-screw implant (MSI) implantation in the root furcation site. METHODS: Seventy posterior teeth of five Beagle dogs were collected and measured as follows: First, the greatest mesio-distal diameter AB of the crown was measured, which was perpendicular to the tooth long axis. Second, mesial cusp of maxillary posterior tooth or central cusp of mandibular was marked as the piont C, and CD which was equal and perpendicular to AB was drawn. The piont D was planned to be the reference point for MSI implantation. Finally, Dm, Dd and Df were measured, which were the distance from the point D to the mesial and distal root surface and root furcation. RESULTS: The difference between the left and right sides teeth with the same name had no statistical significant (P > 0.05). The measurement results of all indices assumed a Gaussian distribution. The mean Dd was basically equal to Dm. All the average Df were more than 4 mm. CONCLUSION: The point D could be used as a reference point for MSI implantation in the root furcation site of posterior tooth in the Beagle dog. The data of this study can provide certain reference for MSI implantation in the study of the biomechanics of anchorage stability, and provide certain reference for MSI implantation in the study of root repair after injury.