Rapid in situ mutation detection in extracellular vesicle-DNA.

A PCR- and sequencing-free mutation detection assay facilitates cancer diagnosis and reduces over-reliance on specialized equipment. This benefit was highlighted during the pandemic when high demand for viral nucleic acid testing often sidelined mutation analysis. This shift led to substantial challenges for patients on targeted therapy in tracking mutations. Here, we report a 30-minute DNA mutation detection technique using Cas12a-loaded liposomes in a microplate reader, a fundamental laboratory tool. CRISPR-Cas12a complex and fluorescence-quenching (FQ) probes are introduced into tumor-derived extracellular vesicles (EV) through membrane fusion. When CRISPR-RNA hybridizes with the DNA target, activated Cas12a can trans-cleave FQ probes, resulting in fluorescence signals for the quantification of DNA mutation. Future advancements in multiplex and high-throughput mutation detection using this assay will streamline self-diagnosis and treatment monitoring at home.

Chimeric nanobody-decorated liposomes by self-assembly.

Liposomes as drug vehicles have advantages, such as payload protection, tunable carrying capacity and improved biodistribution. However, due to the dysfunction of targeting moieties and payload loss during preparation, immunoliposomes have yet to be favoured in commercial manufacturing. Here we report a chemical modification-free biophysical approach for producing immunoliposomes in one step through the self-assembly of a chimeric nanobody (cNB) into liposome bilayers. cNB consists of a nanobody against human epidermal growth factor receptor 2 (HER2), a flexible peptide linker and a hydrophobic single transmembrane domain. We determined that 64% of therapeutic compounds can be encapsulated into 100-nm liposomes, and up to 2,500 cNBs can be anchored on liposomal membranes without steric hindrance under facile conditions. Subsequently, we demonstrate that drug-loaded immunoliposomes increase cytotoxicity on HER2-overexpressing cancer cell lines by 10- to 20-fold, inhibit the growth of xenograft tumours by 3.4-fold and improve survival by more than twofold.

Anthropometry in predicting Semitendinosus and Gracilis graft diameter for arthroscopic ACL reconstruction among the Bengali population.

Semitendinosus and Gracilis autografts are commonly used for anterior cruciate ligament (ACL) reconstruction. However, its main drawback is individual variation in the tendon diameter. This study aimed to evaluate the usefulness of the anthropometric measurements for the prediction of Semitendinosus and Gracilis combined (quadrupled) strands graft diameter in arthroscopic ACL reconstruction among the Bengali population. Sixty consecutive patients who underwent ACL reconstruction between July 2019 to June 2020 were observed in this cross-sectional study. In all cases, the doubled (2 strands) Gracilis and Semitendinosus tendons were combined to get the final quadrupled (combined 4 strands) graft for use. Anthropometries such as body mass index (BMI), body weight, and height were recorded preoperatively and quadrupled combined tendon diameter was measured at the operating theater. The relationship between these parameters was statistically determined using the Pearson correlation coefficient, and scatter diagrams were plotted. Among the study subjects, most (54) were male, and the mean age of all subjects was 24 ±â€…6.92 years. The average graft diameter was 7.20 ±â€…0.76 mm. Correlations between the mean graft diameter with BMI (r = 0.018, and P = .891), body height (r = 0.011 and P = .933), and weight (r = 0.028 and P = .832) were not significant. Scatter diagrams also showed that the variables were not correlated. Anthropometries like BMI, body weight, or body height cannot be used in predicting Semitendinosus and Gracilis tendon graft diameter for arthroscopic ACL reconstruction among the Bengali population.

Bioinspired engineering of fusogen and targeting moiety equipped nanovesicles.

Cell-derived small extracellular vesicles have been exploited as potent drug vehicles. However, significant challenges hamper their clinical translation, including inefficient cytosolic delivery, poor target-specificity, low yield, and inconsistency in production. Here, we report a bioinspired material, engineered fusogen and targeting moiety co-functionalized cell-derived nanovesicle (CNV) called eFT-CNV, as a drug vehicle. We show that universal eFT-CNVs can be produced by extrusion of genetically modified donor cells with high yield and consistency. We demonstrate that bioinspired eFT-CNVs can efficiently and selectively bind to targets and trigger membrane fusion, fulfilling endo-lysosomal escape and cytosolic drug delivery. We find that, compared to counterparts, eFT-CNVs significantly improve the treatment efficacy of drugs acting on cytosolic targets. We believe that our bioinspired eFT-CNVs will be promising and powerful tools for nanomedicine and precision medicine.

DNA-induced fusion between lipid domains of peptide-lipid hybrid vesicles.

Peptide-lipid hybrid vesicles were prepared with complementary DNA strands in their lipid domains. Hybridization of the complementary DNA strands induced the controlled fusion of the vesicles during repeated heating and cooling cycles. Vesicle fusion was indicated by a decrease in the efficiency of Förster resonance energy transfer between lipid-localized probes (from 72 to 42%) and transmission electron microscopy analysis. We suggest that this approach is a general strategy for the creation of polymersomes with membrane-fusion functionality.

Tubular Network Formation by Mixing Amphiphilic Polypeptides with Differing Hydrophilic Blocks.

Artificial tubular networks are promising structures for biomaterial applications because of their large surface areas. A tubular network was formed by co-assembling two different amphiphilic polypeptides, poly(ethylene glycol)-b-(l-Leu-Aib)6 (PL12) and polysarcosine-b-(l-Leu-Aib)6 (SL12). They both have the same hydrophobic 12-mer helical block (l-Leu-Aib)6 but different hydrophilic chains, poly(ethylene glycol) and polysarcosine. In water, both polypeptides self-assembled into a tubular structure having a uniform 80 nm diameter that was formed by packing among the hydrophobic L12 blocks. The SL12 nanotubes were short (200 nm), straight, and robust. PL12 formed long (>1 µm), bendable, and fusogenic nanotubes. The amphiphiles were then co-assembled with various mixing ratios to form tubular networks. Higher concentrations of PL12 made the nanotubes more bendable and fusogenic between open tube ends, which produced branching junctions under heat treatment.

Spontaneous Formation of Gating Lipid Domain in Uniform-Size Peptide Vesicles for Controlled Release.

Hybrid assemblies composed of phospholipids and amphiphilic polymers have been investigated previously as a biomimetic model of biological cells. However, these studies focused on the functions of polymers in a sea of membrane lipids. Here, we prepared a highly stable peptide-lipid hybrid vesicle from a combination of an amphiphilic polypeptide and the phospholipid, 1,2-dimyristoyl- sn-glycero-3-phosphocholine, with a mixing molar ratio of 1:1. The phase-separated structure of the hybrid vesicle was demonstrated by fluorescence resonance energy transfer analysis. The lipid domain of the hybrid vesicle had a phase-transition temperature of 38 °C and allowed the permeation of a hydrophilic molecule, fluorescein isothiocyanate-labeled polyethylene glycol ( Mw: 2000), above 38 °C. The designed peptide-lipid hybrid vesicle and a "lipidic gate" are a promising tool for smart drug delivery.