Surface-engineered extracellular vesicles for targeted delivery of therapeutic RNAs and peptides for cancer therapy.

The advent of novel therapeutics in recent years has urged the need for a safe, non-immunogenic drug delivery vector capable of delivering therapeutic payloads specifically to diseased cells, thereby increasing therapeutic efficacy and reducing side effects. Extracellular vesicles (EVs) have garnered attention in recent years as a potentially ideal vector for drug delivery, taking into account their intrinsic ability to transfer bioactive cargo to recipient cells and their biocompatible nature. However, natural EVs are limited in their therapeutic potential and many challenges need to be overcome before engineered EVs satisfy the levels of efficiency, stability, safety and biocompatibility required for therapeutic use. Here, we demonstrate that an enzyme-mediated surface functionalization method in combination with streptavidin-mediated conjugation results in efficient surface functionalization of EVs. Surface functionalization using the above methods permits the stable and biocompatible conjugation of peptides, single domain antibodies and monoclonal antibodies at high copy number on the EV surface. Functionalized EVs demonstrated increased accumulation in target cells expressing common cancer associated markers such as CXCR4, EGFR and EpCAM both in vitro and in vivo. The functionality of this approach was further highlighted by the ability of targeting EVs to specifically deliver therapeutic antisense oligonucleotides to a metastatic breast tumor model, resulting in increased knockdown of a targeted oncogenic microRNA and improved metastasis suppression. The method was also used to equip EVs with a bifunctional peptide that targets EVs to leukemia cells and induces apoptosis, leading to leukemia suppression. Moreover, we conducted extensive testing to verify the biocompatibility, and safety of engineered EVs for therapeutic use, suggesting that surface modified EVs can be used for repeated dose treatment with no detectable adverse effects. This modular, biocompatible method of EV engineering offers a promising avenue for the targeted delivery of a range of therapeutics while addressing some of the safety concerns associated with EV-based drug delivery.

The Role of in silico Research in Developing Nanoparticle-Based Therapeutics.

Nanoparticles (NPs) hold great potential as therapeutics, particularly in the realm of drug delivery. They are effective at functional cargo delivery and offer a great degree of amenability that can be used to offset toxic side effects or to target drugs to specific regions in the body. However, there are many challenges associated with the development of NP-based drug formulations that hamper their successful clinical translation. Arguably, the most significant barrier in the way of efficacious NP-based drug delivery systems is the tedious and time-consuming nature of NP formulation-a process that needs to account for downstream effects, such as the onset of potential toxicity or immunogenicity, in vivo biodistribution and overall pharmacokinetic profiles, all while maintaining desirable therapeutic outcomes. Computational and AI-based approaches have shown promise in alleviating some of these restrictions. Via predictive modeling and deep learning, in silico approaches have shown the ability to accurately model NP-membrane interactions and cellular uptake based on minimal data, such as the physicochemical characteristics of a given NP. More importantly, machine learning allows computational models to predict how specific changes could be made to the physicochemical characteristics of a NP to improve functional aspects, such as drug retention or endocytosis. On a larger scale, they are also able to predict the in vivo pharmacokinetics of NP-encapsulated drugs, predicting aspects such as circulatory half-life, toxicity, and biodistribution. However, the convergence of nanomedicine and computational approaches is still in its infancy and limited in its applicability. The interactions between NPs, the encapsulated drug and the body form an intricate network of interactions that cannot be modeled with absolute certainty. Despite this, rapid advancements in the area promise to deliver increasingly powerful tools capable of accelerating the development of advanced nanoscale therapeutics. Here, we describe computational approaches that have been utilized in the field of nanomedicine, focusing on approaches for NP design and engineering.

Effects of Periodontal Treatment in Patients with Periodontitis and Kidney Failure: A Pilot Study.

Periodontitis and chronic kidney disease are both chronic inflammatory diseases and share some common risk factors. This 3-month pilot study aimed to clarify whether non-surgical periodontal therapy is beneficial in clinical, biochemical, and microbiological conditions in patients with periodontitis and kidney failure. Kidney failure patients with moderate to severe periodontitis were recruited from two hospitals. Treatment group received non-surgical periodontal therapy, and control group received oral hygiene instruction only. Outcome assessments were conducted 1 and 3 months after treatment. Non-parametric tests were used to analyze the patient-level data. Periodontal site-level assessments were analyzed by Student t-test and paired t-test. Statistical significance was set at p-value < 0.05. A total of 11 subjects completed the study. There was no significant difference between groups in all-cause mortality, cardiovascular events, infection events, systemic parameters, and serum biomarkers. Comparing to control group, clinical periodontal parameters, gingival crevicular fluid interleukin-1ß (IL-1ß) level and periodontal pathogens showed significant improvement in the treatment group. Non-surgical periodontal treatment did not change systemic outcomes in kidney failure patients, but changed the local micro-environment.

Salivary Pro-Inflammatory Markers and Smoking Status Influences the Treatment Effectiveness of Periodontal Disease Patients with Hypertension.

BACKGROUND: Hypertension and periodontal diseases share several risk factors. Inflammation biomarkers in saliva are related to hypertension and periodontal disease. The aim of this study was to explore the role of the salivary inflammatory biomarkers in the treatment effectiveness of patients with hypertension and periodontal disease. METHODS: This observational study enrolled 160 subjects diagnosed with periodontitis, 40 of which had a history of hypertension. All subjects had completed scaling and root planning therapeutic procedures within four weeks. The clinical periodontal parameters (i.e., bleeding on probing, plaque control record (PCR), and probing depth (PD)) were evaluated before and after the treatment. Pro-inflammatory markers were determined using a commercial kit. RESULTS: The recovery rate (PD 4-9 mm) in non-hypertensive subjects was significantly higher than in hypertensive subjects (60.47% vs. 52.60%, respectively; p = 0.04). All clinical parameters, excluding PCR, positively correlated with salivary IL-1ß at baseline and after completing treatment. Our results showed that increased salivary IL-1ß levels were positively associated with decreased PCR (ß = -27.65 and p = 0.05) and PD recovery rate (ß = -17.05 and p = 0.02) in hypertensive subjects. CONCLUSIONS: The present study sheds important light on the clinical use of salivary pro-inflammatory cytokines as valuable biomarkers for predicting the treatment effectiveness of patients suffering from hypertension and periodontitis.

IL-8 as a Potential Therapeutic Target for Periodontitis and Its Inhibition by Caffeic Acid Phenethyl Ester In Vitro.

Salivary levels of interleukin-8 (IL-8) are elevated in patients with periodontitis. Caffeic acid phenethyl ester (CAPE) improves the periodontal status in subjects. However, whether CAPE can reduce IL-8 expression is unclear. We collected saliva to determine proinflammatory cytokine levels and used subgingival calculus and surrounding tissues from patients with periodontitis for oral microbiota analysis via 16s ribosomal RNA gene sequencing. THP-1 cells were stimulated with sterile-filtered saliva from patients, and target gene/protein expression was assessed. IL-8 mRNA expression was analyzed in saliva-stimulated THP-1 cells treated with CAPE and the heme oxygenase-1 (HO-1) inhibitor tin-protoporphyrin (SnPP). In 72 symptomatic individuals, IL-8 was correlated with periodontal inflammation (bleeding on probing, r = 0.45; p < 0.001) and disease severity (bleeding on probing, r = 0.45; p < 0.001) but not with the four oral microbiota species tested. Reduced salivary IL-8 secretion was correlated with effective periodontitis treatment (r = 0.37, p = 0.0013). In THP-1 cells, saliva treatment induced high IL-8 expression and IKK2 and nuclear factor-κB (NF-κB) phosphorylation. However, the IKK inhibitor BMS-345541, NF-κB inhibitor BAY 11-7082, and CAPE attenuated saliva-induced IL-8 expression. CAPE induced HO-1 expression and inhibited IKK2, IκBα, and NF-κB phosphorylation. Blocking HO-1 decreased the anti-inflammatory activity of CAPE. The targeted suppression of IL-8 production using CAPE reduces inflammation and periodontitis.

Associations between the phenotype and genotype of MnSOD and catalase in periodontal disease.

BACKGROUND: Periodontal disease is an inflammatory disease in which pathogenic infections trigger a series of inflammatory responses and redox regulation. The hypothesis of this study was that a host's redox regulation, as modified by genetic polymorphisms, may affect periodontal disease activities (including the plaque index (PlI), bleeding on probing (BOP), and pocket depth (PD)) during periodontal therapy. METHODS: In total, 175 patients diagnosed with periodontitis were recruited from the Department of Periodontology, Taipei Medical University Hospital. Both saliva samples and clinical measurements (PlI, BOP, and PD) were taken at the baseline and at 1 month after completing treatment. Salivary manganese superoxide dismutase (MnSOD) and catalase, and corresponding genetic polymorphisms (MnSOD, T47C, rs4880 and Catalase, C-262 T, rs1001179) were determined. The extent of change (Δ) of MnSOD or catalase was calculated by subtracting the concentration after completing treatment from that at the baseline. RESULTS: Subjects who carried the Catalase CC genotype had significantly higher salivary MnSOD or catalase levels. The MnSOD genotype had a significant effect on the percentage of PDs of 4~9 mm (p = 0.02), and salivary ΔMnSOD had a significant effect on the PlI (p = 0.03). The Catalase genotype had a significant effect on the PlI (p = 0.01~0.04), but the effect was not found for the mean PlI or PD. There was a significant interaction between the MnSOD genotype and salivary ΔMnSOD on PDs of 4~9 mm. After adjusting for gender, years of schooling, smoking status, and alcohol consumption, subjects with ΔMnSOD of < 0 µg/ml or Δcatalase of < 0 µg/ml had significantly higher 5.58- or 5.17-fold responses to scaling and root planing treatment. CONCLUSIONS: The MnSOD T47C genotype interferes with the phenotype of salivary antioxidant level, alters MnSOD levels, and influences the PD recovery. MnSOD and catalase gene polymorphism associated with phenotype expression and susceptibility in periodontal root planing treatment responses.

A Cross-Sectional Study of Endogenous Antioxidants and Patterns of Dental Visits of Periodontitis Patients.

Periodontitis is an inflammatory disease, wherein endogenous antioxidants help to balance the inflammatory status. Oral health behaviors are related to the periodontal disease status. The aim of this study was to explore the associations between oral health behaviors and endogenous antioxidants in periodontitis patients. In total, 225 subjects diagnosed with periodontitis were enrolled in the study. Information obtained from the initial interview included socioeconomic and demographic characteristics, lifestyle factors, and oral health-related behaviors. The clinical periodontal parameters evaluated included bleeding on probing (BOP), the plaque index (PI), and probing depth (PD). Stimulated saliva was collected before periodontal therapy to determine five endogenous antioxidants (copper-zinc superoxide dismutase (Cu/Zn SOD), manganese SOD (MnSOD), thioredoxin 1 (Trx1), peroxiredoxin 2 (Prx2), and catalase (CAT)). When these five factors were adjusted for in patients whose last previous dental visit was >1 year, the patients' PI, BOP, and PD showed significant decreases because of an elevation in the Cu/Zn SOD level. Associations of endogenous antioxidants with levels of clinical periodontal parameters were much higher in subjects whose last previous dental visit was >1 year, compared to subjects whose last previous dental visit was <1 year. This study provides a better understanding of dental visit patterns and the salivary endogenous antioxidants that may underlie the symptomatic development of preclinical periodontitis.

Cigarette Smoking Aggravates the Activity of Periodontal Disease by Disrupting Redox Homeostasis- An Observational Study.

The aim of this study was to evaluate the associations between cigarette use and five salivary oxidative stress biomarkers, copper-zinc superoxide dismutase (Cu/Zn SOD), manganese superoxide dismutase (MnSOD), catalase, thioredoxin-1 (TRX1), and peroxiredoxin-2 (PRX2), to assess the effectiveness of non-surgical periodontal therapy. MATERIALS AND METHODS: This is an observational study,167 patients diagnosed with periodontitis were recruited. Both saliva samples and clinical measurements (plaque index (PI), bleeding on probing (BOP), and pocket depth (PD)) were taken at baseline and after completing non-surgical periodontal therapy. The Levels of salivary biomarkers were determined using a MILLIPLEX® MAP Human Oxidative Stress Magnetic Bead Panel kit. The overall reductions in PI and BOP were 31.56% and 42.16%, respectively. BOP reduction after treatment in female or male non-smokers was significantly higher than in male former smokers (p < 0.05). After completing non-surgical periodontal therapy, Cu/ZnSOD, MnSOD, catalase, and Prx2 significantly decreased. There was a significant interaction between smoking status and ΔCu/ZnSOD on PI and a significant interaction between smoking status and ΔCatalase on BOP. CONCLUSIONS: Cigarette smoking interferes with redox homeostasis in the body, alters antioxidants levels, and influences the periodontal disease activity.

Effects of Salivary Oxidative Markers on Edentulous Patients' Satisfaction with Prosthetic Denture Treatments: A Pilot Study.

OBJECTIVES: The purpose of this study was to assess relationships among periodontal conditions, salivary antioxidant levels, and patients' satisfaction with their prostheses. METHODS: This study was conducted at the Division of Prosthodontics, Department of Dentistry, Taipei Medical University Hospital. The periodontal condition of patients was based on an assessment of the plaque index (PI) and gingival index (GI). The pH value, flow rate, and buffer capacity of the saliva were estimated. The salivary total antioxidant status (TAS) and superoxide dismutase (SOD) level were also determined. Patients' satisfaction with prosthetic treatments was evaluated using the Chinese version of the short-form Oral Health Impact Profile (OHIP-14C). A multivariate regression model was used to determine whether patients' satisfaction with prosthetic treatment was affected by their oral health status. RESULTS: In total, 35 edentulous patients were recruited. In the Spearman correlation analysis, salivary pH (r = -0.36, p = 0.03) and the buffer ability (r = -0.48, p<0.01) were associated with OHIP-14C scores. In the multivariate analysis, patients who had a higher GI also had a higher score of physical disabilities (ß = 1.38, p = 0.04). Levels of SOD increased with the scores of psychological discomfort (ß = 0.33 U/g protein, p = 0.04). CONCLUSIONS: This study suggested that both the GI and SOD levels were associated with patients' satisfaction with prosthetic treatments. To the best of our knowledge, this is the first study to elucidate the relationship between OHIP scores and salivary oxidative markers in edentulous patients.

Scaling-stimulated salivary antioxidant changes and oral-health behavior in an evaluation of periodontal treatment outcomes.

AIM: Our goal was to investigate associations among scaling-stimulated changes in salivary antioxidants, oral-health-related behaviors and attitudes, and periodontal treatment outcomes. MATERIALS AND METHODS: Thirty periodontitis patients with at least 6 pockets with pocket depths of >5 mm and more than 16 functional teeth were enrolled in the study. Patients were divided into three groups: an abandoned group (AB group), a nonprogress outcome group (NP group), and an effective treatment group (ET group). Nonstimulated saliva was collected before and after scaling were received to determine superoxide dismutase (SOD) and the total antioxidant capacity (TAOC). RESULTS: Salivary SOD following scaling significantly increased from 83.09 to 194.30 U/g protein in patients who had irregular dental visit patterns (<1 visit per year). After scaling, the TAOC was significantly higher in patients who had regular dental visits than in patients who had irregular dental visits (3.52 versus 0.70 mmole/g protein, P < 0.01). The scaling-stimulated increase in SOD was related to a higher severity of periodontitis in the NP group, while the scaling-stimulated increase in the TAOC was inversely related to the severity of periodontitis in the AB group. CONCLUSIONS: These results demonstrate the importance of scaling-stimulated salivary antioxidants as prognostic biomarkers of periodontal treatment.

Human blood-derived fibrin releasates: composition and use for the culture of cell lines and human primary cells.

We have evaluated the capacity of two human blood fractions to substitute for FBS as growth medium supplement for human and animal cell cultures. Non-anticoagulated blood from volunteer donors (N = 13) was centrifuged to isolate a supernatant serum (SS) and a platelet-rich fibrin (PRF) clot which was squeezed to extract the releasate (PRFR). Both materials were characterized for the content in PDGF-AB, TGF-ß1, VEGF, bFGF, EGF, IGF, total protein, albumin, IgG, IgM IgA, fibrinogen, cholesterol, triglycerides, various chemistry analytes and hemoglobin. Cell growth promoting activity of pooled SS and PRFR at 1, 5, and 10% in growth medium was evaluated over 7 days using human (HEK293, MG-63) and animal (SIRC, 3T3) cell lines and two human primary cultures (gingival fibroblasts and periodontal ligaments). Viable cell count was compared to that in cultures in FBS free-medium and 10% FBS supplement. SS and PRFR at 1-10% stimulated cell growth significantly more than FBS-free medium and in a way similar to 10% FBS in all cultures apart from 3T3. These two human blood-derived fibrin releasates are equally efficient to substitute for FBS as supplement for cell cultures and could be useful for specialized applications in regenerative medicine, dentistry and oral implantology, or cell therapy.