Pretreatment periodontitis is predictive of a poorer prognosis after esophagectomy for esophageal cancer.

BACKGROUND: Poor oral health is an independent risk factor for upper-aerodigestive tract cancers, including esophageal cancer. Several studies have investigated short-term outcomes after esophagectomy and the impact of periodontal disease, but few have examined the impact of periodontal disease on long-term outcomes. The purpose of this study was to investigate the rate of periodontitis among esophagectomy patients and the prognostic value of periodontitis and its effect on prognosis after esophagectomy. METHODS: A total of 508 patients who underwent esophagectomy received oral health care from a dentist before cancer treatment at Akita University Hospital between January 2009 and December 2021. We assessed the presence and severity of the patients' periodontitis and divided them into no-periodontitis, mild periodontitis, severe periodontitis and edentulous jaw groups. We then assessed 10-year overall survival (OS) and disease-specific survival (DSS) and determined whether periodontitis was an independent prognostic factor affecting OS and DSS. RESULTS: We found that 101 (19.9%) patients had no periodontitis, 207 (40.8%) had mild periodontitis, 176 (34.6%) had severe periodontitis requiring tooth extraction, and 24 (4.7%) had edentulous jaw. Both OS and DSS were significantly poorer in the periodontitis than no-periodontitis group (p < 0.001). In detail, the edentulous jaw group had the poorest prognosis (p < 0.001). Multivariate analysis showed that periodontitis was an independent risk factor affecting OS and DSS. CONCLUSION: Esophageal cancer patients had a high prevalence of periodontitis. Moreover, the presence of periodontitis and severity of periodontitis are independent risk factors contributing to a poorer prognosis after esophagectomy.

Retrograde Axonal Transport of Liposomes from Peripheral Tissue to Spinal Cord and DRGs by Optimized Phospholipid and CTB Modification.

Despite recent advancements in therapeutic options for disorders of the central nervous system (CNS), the lack of an efficient drug-delivery system (DDS) hampers their clinical application. We hypothesized that liposomes could be optimized for retrograde transport in axons as a DDS from peripheral tissues to the spinal cord and dorsal root ganglia (DRGs). Three types of liposomes consisting of DSPC, DSPC/POPC, or POPC in combination with cholesterol (Chol) and polyethylene glycol (PEG) lipid were administered to sciatic nerves or the tibialis anterior muscle of mature rats. Liposomes in cell bodies were detected with infrared fluorescence of DiD conjugated to liposomes. Three days later, all nerve-administered liposomes were retrogradely transported to the spinal cord and DRGs, whereas only muscle-administered liposomes consisting of DSPC reached the spinal cord and DRGs. Modification with Cholera toxin B subunit improved the transport efficiency of liposomes to the spinal cord and DRGs from 4.5% to 17.3% and from 3.9% to 14.3% via nerve administration, and from 2.6% to 4.8% and from 2.3% to 4.1% via muscle administration, respectively. Modification with octa-arginine (R8) improved the transport efficiency via nerve administration but abolished the transport capability via muscle administration. These findings provide the initial data for the development of a novel DDS targeting the spinal cord and DRGs via peripheral administration.

Development of Lipid Nanoparticles for the Delivery of Macromolecules Based on the Molecular Design of pH-Sensitive Cationic Lipids.

Considerable efforts have been made on the development of lipid nanoparticles (LNPs) for delivering of nucleic acids in LNP-based medicines, including a first-ever short interfering RNA (siRNA) medicine, Onpattro, and the mRNA vaccines against the coronavirus disease 2019 (COVID-19), which have been approved and are currently in use worldwide. The successful rational design of ionizable cationic lipids was a major breakthrough that dramatically increased delivery efficiency in this field. The LNPs would be expected to be useful as a platform technology for the delivery of various therapeutic modalities for genome editing and even for undiscovered therapeutic mechanisms. In this review, the current progress of my research, including the molecular design of pH-sensitive cationic lipids, their applications for various tissues and cell types, and for delivering various macromolecules, including siRNA, antisense oligonucleotide, mRNA, and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system will be described. Mechanistic studies regarding relationships between the physicochemical properties of LNPs, drug delivery, and biosafety are also summarized. Furthermore, current issues that need to be addressed for next generation drug delivery systems are discussed.

High TLR4 expression predicts a poor prognosis after esophagectomy for advanced thoracic esophageal squamous cell carcinoma.

BACKGROUND: Poor oral health is an independent risk factor for upper aerodigestive tract cancers, including esophageal squamous cell carcinoma (ESCC). The pattern recognition receptor Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide in the cell walls of Gram-negative periodontal pathogens associated with the development and progression of ESCC. It is, therefore, plausible that TLR4 plays a crucial role in the pathogenesis of ESCC. METHODS: We used an ESCC tissue microarray to confirm expression of TLR4 in patients with ESCC and to determine whether TLR4 expression status correlates with the clinicopathological features of these patients or their prognosis after esophagectomy. We also tested whether the combined expression statuses of TLR4 and TLR3 better correlate with prognosis in these patients than either parameter alone. RESULTS: Clinical ESCC samples from all 177 patients tested showed expression of TLR4. Moreover, high TLR4 expression (3 + and 2 +) correlated with poorer 5-year overall survival after esophagectomy than lower TLR4 expression (1 +) (p = 0.0491). Patients showing high TLR4 expression tended to have a poorer prognosis whether treated with surgery alone or with surgery and adjuvant chemotherapy. Univariate and multivariate analyses showed TLR4 expression status to be an independent prognostic factor affecting 5-year overall survival. Patients exhibiting high TLR4 expression with low TLR3 expression had a much poorer prognosis than other patients (p = < 0.0001). CONCLUSION: High TLR4 expression predicts a poor prognosis in advanced thoracic ESCC patients after esophagectomy.

An intervention study to test Locker's conceptual framework of oral health in edentulous elders.

OBJECTIVE: To test a previously described conceptual framework of oral health in edentulous elders using an intervention study that included complete denture replacement. Confirmatory factor analysis (CFA) was also conducted to substantiate construct validity. BACKGROUND DATA: To date, the model proposed by Locker has been tested on edentulous elders using structural equation model (SEM) analysis. However, cross-sectional designs and the Short-Form Oral Health Impact Profile (OHIP-14) cannot adequately express cause-effect relationships and distribution in edentulous patients. Accordingly, the authors investigated Locker's model using an interventional design that included complete denture replacement using the OHIP for edentulous subjects (OHIP-EDENT). METHODS: A total of 265 edentulous participants who visited the Dental Hospital of Tokyo Medical and Dental University (Tokyo, Japan) for new complete dentures were recruited. Locker's model was investigated, and CFA was performed using the change in subscale scores in the Japanese version of the OHIP-EDENT before and after complete denture replacement. RESULTS: CFA demonstrated an excellent model fit after adding several covariates. The Locker model also met the criteria of fit in all indices after 1 nonsignificant path was omitted. All path coefficients were significant. CONCLUSION: The findings of the present interventional study demonstrated an empirical fit to Locker's model in edentulous elders using SEM analysis, which included complete denture replacement. It is anticipated that clarification of causal mechanisms of oral health-related quality of life will lead to improvement of overall quality of life, thus maintaining or improving the activities of normal daily life for edentulous elders.

A structural equation model to test a conceptual framework of oral health in Japanese edentulous patients with an item weighting method using factor score weights: a cross-sectional study.

BACKGROUND: To investigate Locker's multidimensional model of oral health in Japanese edentulous patients with an item weighting method using factor score weights, which is more accurate than the sum scoring method. A previous study tested Locker's model in edentulous elders in the UK, using empirical evidence from the Short-Form Oral Health Impact Profile (OHIP-14). Investigating the model using the OHIP for edentulous subjects (OHIP-EDENT), which contains 19 items suitable for these patients, may complement that study. Testing Locker's model in Japanese patients may support generalization of the model. METHODS: A total of 394 patients who were edentulous in both arches and visited the Dental Hospital of Tokyo Medical and Dental University for new complete dentures were recruited. This cross-sectional study had a non-probabilistic sampling design and included the following: data collection; application of the new item weighting method that involves hierarchical confirmatory factor analysis (CFA) to derive factor score weights for each item, using the bootstrap method, to check the significance of the factor score weights; and empirical testing of Locker's conceptual model of oral health in Japanese edentulous patients, using structural equation modelling analysis with the bootstrap method for precise estimations and model generation. RESULTS: Factor score weights derived from CFA were significant. After item weighting, the initial model was analyzed and found to have an inconsistent direct path (functional limitation to disability). This path was eliminated from the model and the modified model was re-run. All effects were significant. The model showed acceptable fit on indices including the model chi-squared, standardized root-mean-square residual, root mean-square error of approximation, goodness-of-fit index, comparative fit index, and P-value. CONCLUSIONS: Our findings showed an empirical fit to Locker's model in Japanese edentulous patients when using the item weighting method, which was more accurate than the sum scoring method. These results could contribute to the generalization of Locker's model. TRIAL REGISTRATION: The experimental procedures were published in the University hospital Medical Information Network (UMIN) Center (UMINCTR Clinical Trial, Unique trial Number: UMIN000028711 ).

Size-Dependency of the Surface Ligand Density of Liposomes Prepared by Post-insertion.

In the active targeting of a drug delivery system (DDS), the density of the ligand on the functionalized liposome determines its affinity for binding to the target. To evaluate these densities on the surface of different sized liposomes, 4 liposomes with various diameters (188, 137, 70, 40 nm) were prepared and their surfaces were modified with fluorescently labeled ligand-lipid conjugates by the post-insertion method. Each liposomal mixture was fractionated into a series of fractions using size exclusion chromatography (SEC), and the resulting liposome fractions were precisely analyzed and the surface ligand densities calculated. The data collected using this methodology indicate that the density of the ligand on a particle is greatly dependent on the size of the liposome. This, in turn, indicates that smaller liposomes (75-40 nm) tend to possess higher densities. For developing active targeting systems, size and the density of the ligands are two important and independent factors that can affect the efficiency of a system as it relates to medical use.

Prognoses of new complete dentures from the patient's denture assessment of existing dentures.

OBJECTIVES: The aim was to determine prognostic factors affecting frequent post-delivery adjustments of new complete dentures using patients' assessments of existing complete dentures. MATERIALS AND METHODS: A total of 125 edentulous participants (56 men, 69 women; mean age, 76.4 years) who required new complete dentures evaluated existing dentures using the patient's denture assessment (PDA), a questionnaire regarding the self-assessment of dentures composed of 22 question items and containing six subscales: "function," "lower denture," "upper denture," "expectation," "esthetics and speech," and "importance." Moreover, the numbers of post-delivery adjustments of new dentures were recorded. A multiple logistic regression analysis was performed to identify significant factors for frequent adjustments of new dentures with five subscales of the PDA (excluding "importance"), level of mandibular ridge resorption, and age as independent variables. RESULTS: The analysis showed that "function," "esthetics and speech," and level of mandibular ridge resorption were significant variables for a frequent number of post-delivery adjustments of new complete dentures. CONCLUSIONS: The results suggested that patients' assessments of existing dentures using the PDA might allow a prediction of prognosis for complete denture treatments. Additionally, low "function" scores and high "esthetics and speech" scores for existing dentures and high levels of mandibular ridge resorption were significant prognostic factors affecting frequent post-delivery adjustments of new complete dentures. CLINICAL RELEVANCE: It may be difficult for edentulous persons to adapt to new complete dentures, especially those who have complaints about mastication and swallowing with existing dentures and poor mandibular ridges, but are satisfied with esthetics and speech.

A structural equation model to assess the influence of neuroticism on oral health-related quality of life in complete denture wearers.

OBJECTIVE: To investigate the influence of patient neuroticism on oral health-related quality of life (OHRQoL) in complete denture wearers. BACKGROUND: There are some indicators of complete denture prognosis. The relationship between personality traits, mainly neuroticism and OHRQoL in complete denture wearers remains unclear. MATERIALS AND METHODS: A total of 199 edentulous patients participated in this study. Neuroticism was investigated using the Japanese version of the modified short form of the Eysenck Personality Questionnaire (MS-EPQ). OHRQoL was measured according to the Japanese version of the Oral Health Impact Profile for edentulous subjects (OHIP-EDENT-J). Structural equation modelling was used to evaluate the relationship between neuroticism and subscales of the OHIP-EDENT-J. RESULTS: The High and Low neuroticism groups fit the same model for the 5 subscales of the OHIP-EDENT-J-functional limitation, pain, disability, discomfort and handicap-with different path coefficients. The path from functional limitation to handicap via pain, disability and discomfort was the same as previously reported. However, path coefficients for pain to disability differed significantly between the 2 neuroticism groups. The High neuroticism group had higher estimated path coefficients for Pain compared with those for other subscales than did the Low neuroticism group. This finding indicates that the participants who are high in neuroticism felt pain more strongly, and their disability increased. CONCLUSIONS: Neuroticism may influence OHRQoL in complete denture wearers because of patients' perception of pain.

Learning effects of different training models for border molding from the perspective of dental students.

BACKGROUND: The aim of this study was to investigate the effects of different practical training models on the comprehension and evaluation of practical training among dental students. METHODS: The study subjects were all sixth-year dental students at our institute, and the study took place over three consecutive years (n = 58, 63, and 65, respectively). In practical training, all students learned border molding, and practical models were modified each year from plaster models to silicone models and then to silicone models mounted in mannequins. Immediately after completing clinical training, all students were asked to complete questionnaires consisting of 21 items regarding their overall practical training and their clinical comprehension of border molding. All items were rated on a five-point Likert scale, and in order to reduce the large number of interrelated questions, exploratory factor analysis was carried out using maximum likelihood estimation with promax rotation (κ = 4) and Kaiser normalization. The number of factors was chosen using the Kaiser-Guttman rule, which states that the eigenvalue should be larger than 1, and the scree plot criteria. Items that scored less than 0.25 in communality and exhibited factor loading greater than 0.35 for more than one item were excluded. The defined factors were analyzed for the plaster models, the silicone models alone, and the silicone models with mannequins using the Kruskal-Wallis test and follow-up tests using Bonferroni-corrected Mann-Whitney U tests. The significance level was set at p < 0.05. RESULTS: Exploratory factor analysis identified the following three factors: "knowledge of border molding"; "contents of practical training"; and "personal learning attitude". The students who used silicone models and mannequins gave significantly better evaluations on the "knowledge of border molding" (p < 0.001, both) and "contents of practical training" (p = 0.046, p < 0.001, respectively) subscales than those who used plaster models. No significant differences were observed between those who used silicone models and those who used mannequins. Moreover, no significant differences were found on the "personal learning attitude" subscale among students for any model. CONCLUSIONS: The change in practical training models from plaster to silicone improved student evaluations of border molding training.

Novel pH-sensitive multifunctional envelope-type nanodevice for siRNA-based treatments for chronic HBV infection.

BACKGROUND & AIMS: Antiviral agents including entecavir (ETV) suppress the replication of the hepatitis B virus (HBV) genome in human hepatocytes, but they do not reduce the abundance of viral proteins. The present study focused on effectively reducing viral protein levels. METHODS: We designed siRNAs (HBV-siRNA) that target consensus sequences in HBV genomes. To prevent the emergence of escaped mutant virus, we mixed three HBV-siRNAs (HBV-siRNAmix); the mixture was encapsulated in a novel pH-sensitive multifunctional envelope-type nanodevice (MEND), a hepatocyte-specific drug delivery system. Coagulation factor 7 siRNA was used to assess delivery and knockdown efficiencies of MEND/siRNA treatments in mice. The potency of MEND/HBV-siRNAmix was evaluated in primary human hepatocytes and in chimeric mice with humanized liver persistently infected with HBV. RESULTS: Effective knockdown of targets, efficient delivery of siRNA, and liver-specific delivery were each observed with MEND. MEND/HBV-siRNA caused efficient reduction of HBsAg and HBeAg in vitro and in vivo. However, ETV treatment did not efficiently reduce HBsAg or HBeAg when compared with a single MEND/HBV-siRNAmix treatment. Furthermore, the suppressive effects of a single dose of MEND/HBV-siRNAmix persisted for 14days in vitro and in vivo. CONCLUSION: We demonstrated that MEND/HBV-siRNA controlled HBV more efficiently than did ETV. Furthermore, the effect of a single dose of MEND/HBV-siRNA persisted for a long time. These results indicated that MEND/HBV-siRNA may be a promising novel HBV treatment that is more effective than reverse transcriptase inhibitors.

Topology of Surface Ligands on Liposomes: Characterization Based on the Terms, Incorporation Ratio, Surface Anchor Density, and Reaction Yield.

The surface topology of ligands on liposomes is an important factor in active targeting in drug delivery systems. Accurately evaluating the density of anchors and bioactive functional ligands on a liposomal surface is critical for ensuring the efficient delivery of liposomes. For evaluating surface ligand density, it is necessary to clarify that on the ligand-modified liposomal surfaces, some anchors are attached to ligands but some are not. To distinguish between these situations, a key parameter, surface anchor density, was introduced to specify amount of total anchors on the liposomal surface. Second, the parameter reaction yield was introduced to identify the amount of ligand-attached anchors among total anchors, since the conjugation efficiency is not always the same nor 100%. Combining these independent parameters, we derived: incorporation ratio=surface anchor density×reaction yield. The term incorporation ratio defines the surface ligand density. Since the surface anchor density represents the density of polyethylene glycol (PEG) on the surfaces in most cases, it also determines liposomal function. It is possible to accurately characterize various PEG and ligand densities and to define the surface topologies. In conclusion, this quantitative methodology can standardize the liposome preparation process and qualify the modified liposomal surfaces.

Esophageal Cancer Patients Have a High Incidence of Severe Periodontitis and Preoperative Dental Care Reduces the Likelihood of Severe Pneumonia after Esophagectomy.

BACKGROUND: Poor oral health is a risk factor for causing upper aerodigestive tract tumors, including esophageal cancer. Our aim was to determine the periodontitis rate in our cohort of esophageal cancer patients. We also analyzed whether preoperative dental examination and care reduces the likelihood of severe pneumonia after esophagectomy. STUDY DESIGN: Between 2003 and 2014, 529 esophageal cancer patients received esophagectomy at Akita University Hospital. We studied 232 patients who had preoperative dental examinations and care (dental care group) retrospectively and assessed the severity of their periodontitis. The dental care group was compared to 297 patients who did not have preoperative dental care (control group) with respect to the incidence of severe pneumonia after esophagectomy. RESULTS: Ninety-one patients (39.2%) in the dental care group were diagnosed with slight periodontitis and 69 (29.7%) were diagnosed with severe periodontitis. Among all the patients, 69 patients (13.0%) were diagnosed with grade 3B postoperative severe pneumonia. The dental care group had a significantly lower incidence of severe pneumonia than the control group. Moreover, multivariable logistic regression analysis revealed that anastomotic leakage, preoperative dental care, gender and %VC were correlated significantly with the occurrence of postoperative severe pneumonia. CONCLUSION: Preoperative dental examination and care by a dentist are essential to reduce the likelihood of postoperative severe pneumonia in esophageal cancer patients.

In vitro optimization of 2'-OMe-4'-thioribonucleoside-modified anti-microRNA oligonucleotides and its targeting delivery to mouse liver using a liposomal nanoparticle.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally. Previous studies, which characterized miRNA function, revealed their involvement in fundamental biological processes. Importantly, miRNA expression is deregulated in many human diseases. Specific inhibition of miRNAs using chemically modified anti-miRNA oligonucleotides (AMOs) can be a potential therapeutic strategy for diseases in which a specific miRNA is overexpressed. 2'-O-Methyl (2'-OMe)-4'-thioRNA is a hybrid type of chemically modified oligonucleotide, exhibiting high binding affinity to complementary RNAs and high resistance to nuclease degradation. Here, we evaluate 2'-OMe-4'-thioribonucleosides for chemical modification on AMOs. Optimization of the modification pattern using a variety of chemically modified AMOs that are perfectly complementary to mature miR-21 revealed that the uniformly 2'-OMe-4'-thioribonucleoside-modified AMO was most potent. Further investigation showed that phosphorothioate modification contributed to long-term miR-122 inhibition by the 2'-OMe-4'-thioribonucleoside-modified AMO. Moreover, systemically administrated AMOs to mouse using a liposomal delivery system, YSK05-MEND, showed delivery to the liver and efficient inhibition of miR-122 activity at a low dose in vivo.

Gene silencing via RNAi and siRNA quantification in tumor tissue using MEND, a liposomal siRNA delivery system.

Small interfering RNA (siRNA) would be predicted to function as a cancer drug, but an efficient siRNA delivery system is required for clinical development. To address this issue, we developed a liposomal siRNA carrier, a multifunctional envelope-type nanodevice (MEND). We previously reported that a MEND composed of a pH-sensitive cationic lipid, YSK05, showed significant knockdown in both in vitro and in tumor tissue by intratumoral injection. Here, we report on the development of an in vivo siRNA delivery system that is delivered by systemic injection and an analysis of the pharmacokinetics of an intravenously administered siRNA molecule in tumor tissue. Tumor delivery of siRNA was quantified by means of stem-loop primer quantitative reverse transcriptase PCR (qRT-PCR) method. PEGylation of the YSK-MEND results in the increase in the accumulation of siRNA in tumor tissue from 0.0079% ID/g tumor to 1.9% ID/g tumor. The Administration of the MEND (3 mg siRNA/kg body weight) showed about a 50% reduction in the target gene mRNA and protein. Moreover, we verified the induction of RNA interference by 5' RACE-PCR method. The collective results reported here indicate that an siRNA carrier was developed that can deliver siRNA to a target cell in tumor tissue through an improved siRNA bioavailability.

Reliability and validity of a questionnaire for self-assessment of complete dentures.

BACKGROUND: Demand for complete denture treatment is expected to rise over several decades. However, to date, no questionnaire on complete dentures, as evaluated by edentulous patients, has been shown to be reliable and valid. This study sought to assess the reliability and validity of Patient's Denture Assessment (PDA), which provides a multidimensional evaluation of dentures among edentulous patients. METHODS: Patients, who had new complete dentures fabricated at the University Hospital of Dentistry, Tokyo Medical and Dental University through 2009 to 2010, were enrolled. The reliability of the PDA was determined by examining internal consistency and test-retest reliability. Internal consistency for all of the question items and the six subscales was measured using Cronbach's α and average inter-item correlation coefficients among 93 participants. For 33 of these participants, test-retest reliability was determined at a 2 month-interval using the interclass correlation coefficients (ICCs) and 95% confidence interval for the summary scores and the six subscale scores. The PDA was validated in 93 participants by examining the difference in the summary score and the six subscale scores of the PDA before and after replacement with new dentures by the paired t-test. Ability to detect change was also tested in 93 patients using effect size. RESULTS: The Cronbach's α for the PDA ranged from 0.56 to 0.93. The average inter-item correlation coefficients ranged from 0.28 to 0.83. ICCs for the PDA ranged from 0.37 to 0.83. The paired t-test showed a significant difference between the summary score and the six subscale scores before and after replacement with new dentures (p < 0.05) and the effect size was 0.97. CONCLUSIONS: The PDA demonstrated good reliability by assessing internal consistency and test-retest reliability. In addition, the PDA demonstrated good validity by assessing discriminant validity. Thus, the PDA could help dentists obtain a detailed understanding of the patients' perceptions in using their dentures.

The impact of, and expectations for, lipid nanoparticle technology: From cellular targeting to organelle targeting.

The success of mRNA vaccines against COVID-19 has enhanced the potential of lipid nanoparticles (LNPs) as a system for the delivery of mRNA. In this review, we describe our progress using a lipid library to engineer ionizable lipids and promote LNP technology from the viewpoints of safety, controlled biodistribution, and mRNA vaccines. These advancements in LNP technology are applied to cancer immunology, and a potential nano-DDS is constructed to evaluate immune status that is associated with a cancer-immunity cycle that includes the sub-cycles in tumor microenvironments. We also discuss the importance of the delivery of antigens and adjuvants in enhancing the cancer-immunity cycle. Recent progress in NK cell targeting in cancer immunotherapy is also introduced. Finally, the impact of next-generation DDS technology is explained using the MITO-Porter membrane fusion-based delivery system for the organelle targeting of the mitochondria. We introduce a successful example of the MITO-Porter used in a cell therapeutic strategy to treat cardiomyopathy.

Impact of in vivo fate of STING agonist-loaded lipid nanoparticles on antitumor immunity.

Therapeutically manipulating the stimulator of interferon genes (STING) pathway has promising potential for enhancing antitumor immunity. Agonists of this pathway (STING agonists) are being evaluated in clinical trials. Loading the STING agonists into lipid nanoparticles (LNPs) increases their safety and efficacy. We previously developed STING agonists loaded LNPs consisting of the ionizable lipid YSK12-C4 (YSK12-LNPs), which showed significant antitumor effects. However, it is largely unclear how the in vivo fate of STING agonists loaded LNPs affects the antitumor immune responses. In this study, we compared the YSK12-LNPs with LNPs composed of DLin-MC3-DMA (MC3-LNPs) showing different in vivo fates. Biodistribution and flow cytometry analyses of mouse tissues revealed that the MC3-LNPs delivered higher amounts of STING agonists to the liver than the YSK12-LNPs. Additionally, significantly more liver leukocytes internalized the MC3-LNPs than the YSK12-LNPs. In contrast, the YSK12-LNPs delivered higher amounts of STING agonists to the liver leukocytes than the MC3-LNPs, leading to the effective induction of innate immunity and inflammation in the tumors. However, the antitumor effects in the B16-F10 lung metastasis and CT26 tumor models were comparable. Interestingly, flow cytometry analyses suggested that the YSK12-LNPs were more likely to activate natural killer cells and M1 macrophages, while the MC3-LNPs were more likely to activate CD8+ T cells. Our data suggest that different antitumor immune response mechanisms may operate depending on the characteristics and distribution of the LNPs.

Overcoming thermostability challenges in mRNA-lipid nanoparticle systems with piperidine-based ionizable lipids.

Lipid nanoparticles (LNPs) have emerged as promising platforms for efficient in vivo mRNA delivery owing to advancements in ionizable lipids. However, maintaining the thermostability of mRNA/LNP systems remains challenging. While the importance of only a small amount of lipid impurities on mRNA inactivation is clear, a fundamental solution has not yet been proposed. In this study, we investigate an approach to limit the generation of aldehyde impurities that react with mRNA nucleosides through the chemical engineering of lipids. We demonstrated that piperidine-based lipids improve the long-term storage stability of mRNA/LNPs at refrigeration temperature as a liquid formulation. High-performance liquid chromatography analysis and additional lipid synthesis revealed that amine moieties of ionizable lipids play a vital role in limiting reactive aldehyde generation, mRNA-lipid adduct formation, and loss of mRNA function during mRNA/LNP storage. These findings highlight the importance of lipid design and help enhance the shelf-life of mRNA/LNP systems.

Development of Polymer-Lipid Hybrid Nanoparticles for Large-Sized Plasmid DNA Transfection.

RNA and DNA delivery technologies using lipid nanoparticles (LNPs) have advanced significantly, as demonstrated by their successful application in mRNA vaccines. To date, commercially available RNA therapeutics include Onpattro, a 21 bp siRNA, and mRNA vaccines comprising 4300 nucleotides for COVID-19. However, a significant challenge remains in achieving efficient transfection, as the size of the delivered RNA and DNA increases. In contrast to RNA transfection, plasmid DNA (pDNA) transfection requires multiple steps, including cellular uptake, endosomal escape, nuclear translocation, transcription, and translation. The low transfection efficiency of large pDNA is a critical limitation in the development of artificial cells and their cellular functionalization. Here, we introduce polymer-lipid hybrid nanoparticles designed for efficient, large-sized pDNA transfection. We demonstrated that LNPs loaded with positively charged pDNA-polycation core nanoparticles exhibited a 4-fold increase in transfection efficiency for 15 kbp pDNA compared with conventional LNPs, which encapsulate a negatively charged pDNA-polycation core. Based on assessments of the size and internal structure of the polymer-lipid nanoparticles as well as hemolysis and cellular uptake analysis, we propose a strategy to enhance large-sized pDNA transfection using LNPs. This approach holds promise for accelerating the in vivo delivery of large-sized pDNA and advancing the development of artificial cells.