Supramolecular assembly of polycation/mRNA nanoparticles and in vivo monocyte programming.

Size-dependent phagocytosis is a well-characterized phenomenon in monocytes and macrophages. However, this size effect for preferential gene delivery to these important cell targets has not been fully exploited because commonly adopted stabilization methods for electrostatically complexed nucleic acid nanoparticles, such as PEGylation and charge repulsion, typically arrest the vehicle size below 200 nm. Here, we bridge the technical gap in scalable synthesis of larger submicron gene delivery vehicles by electrostatic self-assembly of charged nanoparticles, facilitated by a polymer structurally designed to modulate internanoparticle Coulombic and van der Waals forces. Specifically, our strategy permits controlled assembly of small poly(ß-amino ester)/messenger ribonucleic acid (mRNA) nanoparticles into particles with a size that is kinetically tunable between 200 and 1,000 nm with high colloidal stability in physiological media. We found that assembled particles with an average size of 400 nm safely and most efficiently transfect monocytes following intravenous administration and mediate their differentiation into macrophages in the periphery. When a CpG adjuvant is co-loaded into the particles with an antigen mRNA, the monocytes differentiate into inflammatory dendritic cells and prime adaptive anticancer immunity in the tumor-draining lymph node. This platform technology offers a unique ligand-independent, particle-size-mediated strategy for preferential mRNA delivery and enables therapeutic paradigms via monocyte programming.

Iron-Catalyzed Multicomponent C-H Alkylation of in Situ Generated Imines via Photoinduced Ligand-to-Metal Charge Transfer.

Herein, we describe a novel photoinduced iron-catalyzed strategy for multicomponent C-H alkylation of in situ generated imines. By utilizing the alkyl radicals generated through iron-mediated photocatalytic C-H activation, the imines formed in situ are further subjected to addition reactions, resulting in the synthesis of various secondary and tertiary amine products. This method is simple to operate and does not require additional oxidants. It is applicable to inert alkane substrates such as cyclic alkanes, cyclic ethers, toluene, and ketones. The reaction is also compatible with various aromatic amines, alkyl amines, halogenated aromatic amines, as well as aromatic aldehydes, alkyl aldehydes, and cinnamaldehyde, among other different types of aldehydes.

Feasibility of thoracoscopic monosubsegmentectomy for small ground-glass opacity dominant lung cancer.

BACKGROUND: Monosubsegmentectomy (MSS) involves removal of less lung parenchyma than monosegmentectomy (MS) does. However, the clinical efficacy of MSS in lung cancer treatment remains unclear, with concerns regarding insufficient surgical margins and increased complications. METHODS: Between February 2015 and December 2019, patients who underwent thoracoscopic MSS (n = 126) or MS (n = 678) for small ground-glass opacity (GGO) dominant lung cancer were examined. The primary endpoints were the procedure success rate (defined as resection with a surgical margin ≥2 cm or tumor size) and surgical margin. RESULTS: There were no significant differences in age, sex, smoking history, or comorbidities between the groups. Both groups achieved a success rate of 100%. No significant group differences were observed in the number of lymph nodes removed (p = 0.060), overall complications (p = 0.147), or major complications (p = 0.450). The MSS group had a smaller surgical margin (median, 17 vs. 21mm, p < 0.001) and longer operative time (median, 138 vs. 130 min, p = 0.005) than the MS group did. Propensity score-matched analysis of 122 pairs of patients revealed consistent results. Surgical margins were further compared based on the number of resected subsegments. The 1 subsegment group had the smallest median surgical margin, followed by the 2 and 3 subsegments groups (17, 20, and 23 mm, p < 0.001). CONCLUSIONS: Thoracoscopic MSS is an acceptable option for treating patients with small-sized GGO-dominant lung cancer. However, the close surgical margins and prolonged operative time should be considered.

Liter-scale manufacturing of shelf-stable plasmid DNA/PEI transfection particles for viral vector production.

The transfection efficiency and stability of the delivery vehicles of plasmid DNA (pDNA) are critical metrics to ensure high-quality and high-yield production of viral vectors. We previously identified that the optimal size of pDNA/poly(ethylenimine) (PEI) transfection particles is 400-500 nm and developed a bottom-up assembly method to construct stable 400-nm pDNA/PEI particles and benchmarked their transfection efficiency in producing lentiviral vectors (LVVs). Here, we report scale-up production protocols for such transfection particles. Using a two-inlet confined impinging jet (CIJ) mixer with a dual syringe pump set-up, we produced a 1-L batch at a flow rate of 100 mL/min, and further scaled up this process with a larger CIJ mixer and a dual peristaltic pump array, allowing for continuous production at a flow rate of 1 L/min without a lot size limit. We demonstrated the scalability of this process with a 5-L lot and validated the quality of these 400-nm transfection particles against the target product profile, including physical properties, shelf and on-bench stability, transfection efficiency, and LVV production yield in both 15-mL bench culture and 2-L bioreactor runs. These results confirm the potential of this particle assembly process as a scalable manufacturing platform for viral vector production.

Path towards mRNA delivery for cancer immunotherapy from bench to bedside.

Messenger RNA (mRNA) has emerged as a promising therapeutic agent for the prevention and treatment of various diseases. mRNA vaccines, in particular, offer an alternative approach to conventional vaccines, boasting high potency, rapid development capabilities, cost-effectiveness, and safe administration. However, the clinical application of mRNA vaccines is hindered by the challenges of mRNA instability and inefficient in vivo delivery. In recent times, remarkable technological advancements have emerged to address these challenges, utilizing two main approaches: ex vivo transfection of dendritic cells (DCs) with mRNA and direct injection of mRNA-based therapeutics, either with or without a carrier. This review offers a comprehensive overview of major non-viral vectors employed for mRNA vaccine delivery. It showcases notable preclinical and clinical studies in the field of cancer immunotherapy and discusses important considerations for advancing these promising vaccine platforms for broader therapeutic applications. Additionally, we provide insights into future possibilities and the remaining challenges in mRNA delivery technology, emphasizing the significance of ongoing research in mRNA-based therapeutics.

Screening for lipid nanoparticles that modulate the immune activity of helper T cells towards enhanced antitumour activity.

Lipid nanoparticles (LNPs) can be designed to potentiate cancer immunotherapy by promoting their uptake by antigen-presenting cells, stimulating the maturation of these cells and modulating the activity of adjuvants. Here we report an LNP-screening method for the optimization of the type of helper lipid and of lipid-component ratios to enhance the delivery of tumour-antigen-encoding mRNA to dendritic cells and their immune-activation profile towards enhanced antitumour activity. The method involves screening for LNPs that enhance the maturation of bone-marrow-derived dendritic cells and antigen presentation in vitro, followed by assessing immune activation and tumour-growth suppression in a mouse model of melanoma after subcutaneous or intramuscular delivery of the LNPs. We found that the most potent antitumour activity, especially when combined with immune checkpoint inhibitors, resulted from a coordinated attack by T cells and NK cells, triggered by LNPs that elicited strong immune activity in both type-1 and type-2 T helper cells. Our findings highlight the importance of optimizing the LNP composition of mRNA-based cancer vaccines to tailor antigen-specific immune-activation profiles.

Difficulty of Video-Assisted Thoracoscopic Surgery Segmentectomy: Proposal for a New Classification.

BACKGROUND: Segmentectomy has classically been distinguished as "simple" or "complex" based on the number of intersegmental planes (ISPs) dissected. However, with the increasing variety and complexity of segmentectomies, it is clear that a classification based on the number of ISPs alone is inadequate. This study aimed to develop a new classification to predict the surgical difficulty of video-assisted thoracoscopic surgery (VATS) segmentectomy. METHODS: The study retrospectively reviewed 1868 patients who underwent VATS segmentectomy between January 2014 and December 2019. Uni- and multivariate analyses were performed to identify predictors associated with prolonged operative time (>140 min), and a scoring system was constructed to classify the surgical difficulty of VATS segmentectomy. RESULTS: Altogether, 1868 VATS segmentectomies were divided into three groups: group 1 (low difficulty, including segmentectomy with only one intersegmental plane [ISP] dissection), group 2 (intermediate difficulty, including a single segmentectomy with more than one ISP dissection and a single subsegmentectomy), group 3 (high difficulty level, including combined resection with more than one ISP dissection). This classification effectively differentiated the three groups in terms of operative time, estimated blood loss, major complications, and overall complications (all p < 0.001). For receiver operating characteristic analysis, the new classification showed significantly better differentiation performance in terms of operative time (p < 0.001), estimated blood loss (p = 0.004), major complications (p = 0.002), and overall complications (p = 0.012) than the simple/complex classification. CONCLUSIONS: This new three-level classification accurately predicted the surgical difficulty of VATS segmentectomy.

Cloaking Mesoporous Polydopamine with Bacterial Membrane Vesicles to Amplify Local and Systemic Antitumor Immunity.

As adjuvants or antigens, bacterial membranes have been widely used in recent antibacterial and antitumor research, but they are often injected multiple times to achieve therapeutic outcomes, with limitations in biosafety and clinical application. Herein, we leverage the biocompatibility and immune activation capacity of Salmonella strain VNP20009 to produce double-layered membrane vesicles (DMVs) for enhanced systemic safety and antitumor immunity. Considering the photothermal effect of polydopamine upon irradiation, VNP20009-derived DMVs are prepared to coat the surface of mesoporous polydopamine (MPD) nanoparticles, leading to the potential synergies between photothermal therapy mediated by MPD and immunotherapy magnified by DMVs. The single dose of MPD@DMV can passively target tumors and activate the immune system with upregulated T cell infiltration and secretion levels of pro-inflammatory factors as well as antitumor related cytokines. All of these promoted immune responses result in malignant melanoma tumor regression and extended survival time on local or distant tumor-bearing mouse models. Importantly, we further explore the advantages of intravenous injection of the MPD@DMV agent compared with its intratumoral injection, and the former demonstrates better long-term immune effects on animal bodies. Overall, this formulation design brings broader prospects for the autologous vaccine adjuvant by bacterial membrane vesicles in cancer therapy.

Visible-light-induced dehydrogenative amidation of aldehydes enabled by iron salts.

A direct dehydrogenative amidation reaction of aldehydes and amines under a visible light mediated ligand-to-metal charge transfer (LMCT) process was described. In this protocol, aldehyde substrates were activated by photoinduced hydrogen atom abstraction (HAA), generating acyl chloride intermediates followed by nucleophilic addition of amines. The synthetic method furnishes good functional group tolerance and broad substrate scope toward both aliphatic and aromatic components.

Totally mechanical Collard versus circular stapled cervical esophagogastric anastomosis for minimally invasive esophagectomy.

BACKGROUND: Previous studies have proposed that the totally mechanical Collard (TMC) method may reduce anastomotic leakage and stricture. This study aimed to compare the TMC method and the circular stapled (CS) method for cervical anastomosis after minimally invasive esophagectomy (MIE) for esophageal cancer. METHODS: From May 2017 to September 2020, 308 patients (165 in the CS group and 143 in the TMC group) were included in this study. The primary endpoints were anastomotic leakage and anastomotic stricture within 12 months. Propensity score matching was used to control potential selection bias. RESULTS: Anastomotic leak, anastomotic stricture, and refractory stricture (≥ 3 dilations) occurred in 30 (9.7%), 28 (9.1%), and 18 (5.8%) patients, respectively. The rate of anastomotic leak was similar in the CS and TMC methods (9.7 vs. 9.8%; P = 0.978), but anastomotic stricture (3.5 vs. 13.9%; P = 0.001) and refractory stricture (2.8 vs. 9.1%, P = 0.022) occurred less frequently in the TMC method. Propensity score matching yielded 128 patient pairs and confirmed these results. Multivariable analyses found that CS method, anastomotic leakage, and diabetes were independent predictors for both anastomotic stricture and refractory stricture. Subgroup analysis revealed that for patients with anastomotic leakage, the postoperative hospital stay in the TMC group was significantly longer than that in the CS group. CONCLUSION: In cervical anastomosis after MIE, the TMC method is superior to the CS method regarding anastomotic stricture and refractory stricture formation. However, compared to the CS method, the TMC method cannot lower the probability of anastomotic leakage, and anastomotic leakage with the TMC method requires a longer healing time.

Biostimulatory Micro-Fragmented Nanofiber-Hydrogel Composite Improves Mesenchymal Stem Cell Delivery and Soft Tissue Remodeling.

Functional microgels are preferred stem cell carriers due to the ease of delivery through minimally invasive injection and seamless integration with the surrounding host tissue. A biostimulatory nanofiber-hydrogel composite (NHC) has been previously developed through covalently crosslinking a hyaluronic acid hydrogel network with surface-functionalized poly (ε-caprolactone) nanofiber fragments. The NHC mimics the microarchitecture of native soft tissue matrix, showing enhanced cell infiltration, immunomodulation, and proangiogenic properties. Here, injectability of the pre-formed NHC is improved by mechanical fragmentation, making it into micro-fragmented NHC (mfNHC) in a granular gel form as a stem cell carrier to deliver mesenchymal stem cells (MSCs) for soft tissue remodeling. The mfNHC shows a similar storage modulus but a significantly reduced injection force, as compared with the corresponding bulk NHC. When injected subcutaneously in a rat model, mfNHC-MSC constructs initiate an elevated level of host macrophage infiltration, more pro-regenerative polarization, and subsequently, improved angiogenesis and adipogenesis response when compared to mfNHC alone. A similar trend of host cell infiltration and pro-angiogenic response is detected in a swine model with a larger volume injection. These results suggest a strong potential for use of the mfNHC as an injectable carrier for cell delivery and soft tissue remodeling.

Exploring Potential Causal Genes for Uterine Leiomyomas: A Summary Data-Based Mendelian Randomization and FUMA Analysis.

Objective: To explore potential causal genetic variants and genes underlying the pathogenesis of uterine leiomyomas (ULs). Methods: We conducted the summary data-based Mendelian randomization (SMR) analyses and performed functional mapping and annotation using FUMA to examine genetic variants and genes that are potentially involved in the pathogenies of ULs. Both analyses used summarized data of a recent genome-wide association study (GWAS) on ULs, which has a total sample size of 244,324 (20,406 cases and 223,918 controls). We performed separate SMR analysis using CAGE and GTEx eQTL data. Results: Using the CAGE eQTL data, our SMR analysis identified 13 probes tagging 10 unique genes that were pleiotropically/potentially causally associated with ULs, with the top three probes being ILMN_1675156 (tagging CDC42, PSMR = 8.03 × 10-9), ILMN_1705330 (tagging CDC42, PSMR = 1.02 × 10-7) and ILMN_2343048 (tagging ABCB9, PSMR = 9.37 × 10-7). Using GTEx eQTL data, our SMR analysis did not identify any significant genes after correction for multiple testing. FUMA analysis identified 106 independent SNPs, 24 genomic loci and 137 genes that are potentially involved in the pathogenesis of ULs, seven of which were also identified by the SMR analysis. Conclusions: We identified many genetic variants, genes, and genomic loci that are potentially involved in the pathogenesis of ULs. More studies are needed to explore the exact underlying mechanisms in the etiology of ULs.

Nanoparticles with rough surface improve the therapeutic effect of photothermal immunotherapy against melanoma.

Photothermal therapy has been intensively investigated for treating cancer in recent years. However, the long-term therapeutic outcome remains unsatisfying due to the frequently occurred metastasis and recurrence. To address this challenge, immunotherapy has been combined with photothermal therapy to activate anti-tumor immunity and relieve the immunosuppressive microenvironment within tumor sites. Here, we engineered silica-based core‒shell nanoparticles (JQ-1@PSNs-R), in which silica cores were coated with the photothermal agent polydopamine, and a bromodomain-containing protein 4 (BRD4) inhibitor JQ-1 was loaded in the polydopamine layer to combine photothermal and immune therapy for tumor elimination. Importantly, to improve the therapeutic effect, we increased the surface roughness of the nanoparticles by hydrofluoric acid (HF) etching during the fabrication process, and found that the internalization of JQ-1@PSNs-R was significantly improved, leading to a strengthened photothermal killing effect as well as the increased intracellular delivery of JQ-1. In the animal studies, the multifunctional nanoparticles with rough surfaces effectively eradicated melanoma via photothermal therapy, successfully activated tumor-specific immune responses against residual tumor cells, and further prevented tumor metastasis and recurrence. Our results indicated that JQ-1@PSNs-R could serve as an innovative and effective strategy for combined cancer therapy.

Advances in Salmonella Typhimurium-based drug delivery system for cancer therapy.

The clinical application of bacteria-mediated immune therapy dates back over a century ago. In recent years, these strategies have advanced greatly with the rapid development of synthetic biology and nanotechnology. Several bacterial therapies have been developed allowing for more effective treatments for cancers, and Salmonella is one of the most studied bacterial species. Here, we review the advances in the bioengineered and functionalized Salmonella Typhimurium strains as drug delivery carries, including the various genetic circuits for programing these bacteria, the surface modification strategies using nanoparticles or other therapeutic agents for richer and broader features, and the bacterial component-based vehicles for cancer immunotherapy. This review will include the promises and challenges of these optimized Salmonella-based delivery systems and their related clinical trials. Ultimately, we hope to provide a spark of thought in the field of drug delivery and find important crosstalk between bacteria-mediated therapy and other different forms of treatments.

Aluminum nanoparticles deliver a dual-epitope peptide for enhanced anti-tumor immunotherapy.

Tumor peptide vaccines contain only key amino acid sequences of tumor neoantigens, and therefore can provide precise activation of immune responses. Recent research has found that short peptide vaccines restricted to MHC-I epitopes are insufficient to activate effective CD8+ T cell responses for tumor elimination, and assistance from CD4+ T cell immunity could significantly improve the therapeutic outcome. Herein, we proposed an innovative peptide vaccine strategy to simultaneously activate CD8+ and CD4+ T cell responses by combining MHC-I and MHC-II epitopes into one long peptide antigen. To further strengthen the anti-tumor immune response induced by this dual-epitope peptide, we engineered a PEG derivative (PpASE) stabilized aluminum nanoparticle for delivering the synthetic long peptides (ANLs). The synthesized nanovaccine with a diameter of ~100 nm showed good stability and enhanced antigen uptake by antigen-presenting cells (APCs). As a result, ANLs promoted the presentation of MHC-I epitope in APCs and induced stronger activation and proliferation of CD8+ T cells as compared to aluminum nanoparticle loaded with MHC-I epitope restricted peptides (ANSs). After subcutaneous vaccination, the developed nanovaccine significantly inhibited tumor growth and prolonged mouse survival in both B16-OVA and B16F10 tumor models. Finally, ANLs were also able to elevate the maturation level of human dendritic cells (DCs), showing a great possibility of clinical translation.

Long Noncoding RNA PCAT6 Regulates Cell Proliferation and Migration in Human Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the sixth most common cancer type in East Asian countries. Mounting evidences illustrated that long noncoding RNAs (lncRNAs) play important roles in a variety of human cancers, including ESCC. LncRNA PCAT6 has been identified as a tumor promoter in multiple cancers. However, the roles and underlying mechanism of PCAT6 in ESCC remain largely unclear. In the current study, we discovered that lncRNA PCAT6, which was aberrantly upregulated in ESCC tumor tissues, significantly promoted cell proliferation and migration in ESCC cell lines Eca-109 and Kyse-30 cells. Flow cytometry assays showed that PCAT6 knockdown promoted the apoptosis of ESCC cells. Mechanistically, RNA-seq and Gene Ontology analyses indicated that PCAT6 knockdown influenced the expression of genes that participated in cell proliferation and migration. Furthermore, real-time PCR and western blot assays validated that knockdown of PCAT6 could increase the levels of GDF15 and DUSP4 in Eca-109 and Kyse-30 cells. In brief, our findings reveal that lncRNA PCAT6 plays an oncogenic role in the progression of ESCC by inhibiting the expression of genes related to cell proliferation and migration.

Recent Advances in Delivery Systems for Genetic and Other Novel Vaccines.

Vaccination is one of the most successful and cost-effective prophylactic measures against diseases, especially infectious diseases including smallpox and polio. However, the development of effective prophylactic or therapeutic vaccines for other diseases such as cancer remains challenging. This is often due to the imprecise control of vaccine activity in vivo which leads to insufficient/inappropriate immune responses or short immune memory. The development of new vaccine types in recent decades has created the potential for improving the protective potency against these diseases. Genetic and subunit vaccines are two major categories of these emerging vaccines. Owing to their nature, they rely heavily on delivery systems with various functions, such as effective cargo protection, immunogenicity enhancement, targeted delivery, sustained release of antigens, selective activation of humoral and/or cellular immune responses against specific antigens, and reduced adverse effects. Therefore, vaccine delivery systems may significantly affect the final outcome of genetic and other novel vaccines and are vital for their development. This review introduces these studies based on their research emphasis on functional design or administration route optimization, presents recent progress, and discusses features of new vaccine delivery systems, providing an overview of this field.

Surface-Functionalized PEGylated Nanoparticles Deliver Messenger RNA to Pulmonary Immune Cells.

Nanoparticles designed as messenger RNA (mRNA) carriers to deliver gene medicine have shown great potential to change the way lung disease states are managed. Controlling their delivery to the lung and the transgene expression in a specific population of cells remains a challenge. Here, we developed a series of nanoparticles with polyethylene glycol (PEG) corona prepared by condensing mRNA with PEG-grafted-polyethyleneimine (PEI-g-PEG) with different PEG terminal functional groups and grafting ratios. PEGylated nanoparticles (PEG grafting ratio was 0.5%) with amino or amino acid terminal groups showed the highest transgene expression levels in the lung following systemic administration, and cell profiling analysis indicated that pulmonary immune cells contributed to the majority of expression. We also showed that these nanoparticles can be prepared by the flash nanocomplexation method, which is a scalable and reproducible process, yielding lyophilizable nanoparticles that were stable for at least 4 months at -20 °C. These results suggest that these surface-functionalized PEGylated nanoparticles may serve as desirable carriers to deliver mRNA to the lung for pulmonary immunomodulation.

[Assessment of the Accuracy of Modified Inflation-deflation Methods for Distinguishing the Intersegmental Border].

BACKGROUND: For early-stage lung cancer, segmentectomy can get the same oncological benefits as lobectomy. Accurate identification of the intersegmental border is the key to segmentectomy. This study used extended segmentectomy and extended subsegmentectomy to treat lung intersegmental and intersubsegmental ground-glass nodules (GGN) by utilizing modified inflation-deflation methods to distinguish the intersegmental and intersubsegmental borders. The accuracy of modified inflation-deflation methods and the effectiveness of extended resection to guarantee a safe surgical margin were evaluated. METHODS: A retrospective analysis of 83 cases of extended segmentectomy and extended subsegmentectomy was conducted. Preoperative three-dimensional computed tomography bronchography and angiography (3D-CTBA) revealed that nodules were involved in intersegmental or intersubsegmental veins. Based on preoperative three-dimensional reconstruction, the surgery was designed to extendedly remove the dominant lung segment or subsegment with nodules involved. When the dominant lung segment or subsegment could not be identified, the simpler lung segment or subsegment was selected for the resection. After the target vessel and bronchus were cut off during the operation, modified inflation-deflation method was used to determine the border, and a stapler was used to resect the adjacent lung segment or subsegment tissue by 2 cm-3 cm around the inflation-deflation boundary line. Then, the relationship between the inflation-deflation boundary line and the nodule and the width of the surgical margin were measured. Clinical data were collected during the perioperative period. RESULTS: 56 extended segmentectomies and 27 extended subsegmentectomies were performed. The average diameter of pulmonary nodules was (0.9±0.3) cm. There were 79 cases with clearly inflation-deflation boundary lines. The average time needed for the appearance of the lines was (13.6±6.5) min. In 55 cases, the nodules were involved with the inflation-deflation boundary lines. Meanwhile, the remaining 24 cases revealed an average minimum distance of (0.6±0.3) cm between nodules and the boundary lines. The average width of surgical margin was (2.1±0.3) cm in these 79 cases. No deaths or major complications appeared during 30 d after operation. CONCLUSIONS: The modified inflation-deflation method can effectively define the intersegmental and intersubsegmental borders, and guarantee the safe surgical margins of extended segmentectomy and extended subsegmentectomy to treat intersegmental and intersubsegmental small lung tumors.