Bioengineering & Translational Medicine最新文献

Lentiviral vectors (LVVs) are emerging as an enabling tool in gene and cell therapies, yet the toolkit for purifying them at scale is still immature. A pivoting moment in LVV isolation technology was marked by the introduction of affinity ligands for LVVs pseudo-typed with Vesicular Stomatitis Virus G (VSV-G) protein. Camelid antibody ligands were initially discovered and utilized to functionalize a resin with a capacity of 10¹⁴ LVV particles per liter (vp/L). Shortly thereafter, our team introduced VSV-G-targeting peptides and assessed their application as ligands for purifying LVVs from HEK293 cell harvests. In this study, we utilized these peptides to develop novel affinity resins and—first in this field—affinity membranes with optimal binding capacity, productivity, and removal of host cell contaminants. To that end, we evaluated resins of different material, particle and pore size, and functional density, as well as membranes with different fiber morphology, porosity, and ligand distribution. The lead peptide-functionalized resin and membrane featured high capacity (5 × 10⁹ and 1.2 × 10⁹ transducing LVV units per mL of adsorbent, TU/mL) and productivity (2.9 × 10⁹ and 1.7 × 10⁹ TU/mL min) and afforded a substantial enrichment of cell-transducing LVVs and reduction of contaminants (110–170-fold) in the eluates. Finally, we demonstrated an LVV purification process in four steps: clarification and nuclease treatment, affinity capture in bind-and-elute mode, polishing in flow-through mode, and ultra/dia-filtration and sterile filtration. The processes afforded yields of 33%–46%, a residual HCP level below 5 ng/mL, and productivity of 1.25–1.5 × 10¹⁴ active LVV particles per hour and liter of adsorbent.

The house dust mite Dermatophagoides pteronyssinus produces major allergens (Der p 1, Der p 2, and Der p 23) that require precise IgE detection for clinical diagnosis. We developed a multiplex digital ELISA using fluorescence-encoded micromagnetic beads (532 nm/638 nm dual-wavelength system) coupled with microfluidics to simultaneously quantify serum IgE against these components, with comprehensive evaluation against the clinical standard UniCAP system. The 532 nm channel measured allergen-specific signals via average brightness increase (ABMB) of enzymatically amplified fluorescence, while 638 nm enabled spectral bead differentiation. Comparative evaluation with UniCAP showed the improved digital ELISA achieved uniform 75.0% sensitivity but variable specificity (42.9%–54.5%) across allergens at the 15.8% ABMB threshold. Sample classification results (Der p 1: 9 positive/6 negative; Der p 2: 7/8; Der p 23: 7/8) demonstrated suboptimal positive predictive values (33.3%–60.0%) versus more favorable negative predictive values (60.0%–85.7%), with likelihood ratios (LR+: 1.31–1.65) and Cohen's κ (0.12–0.25) suggesting limited diagnostic reliability. The automated platform offered 60% reduced sample volume (20 μL vs. 50 μL), multiplex capability, and maintained sensitivity for low-titer samples, representing an efficient screening solution pending specificity enhancement.

The homeostasis of branched-chain amino acids (BCAAs) plays a crucial role in maintaining health, and the accumulation of BCAAs can lead to various diseases. Therefore, exogenous degradation or conversion of excessive BCAAs may help alleviate diseases caused by BCAA accumulation, such as maple syrup urine disease. This study utilized synthetic biology approaches to engineer two strains for efficient BCAA catabolism successfully—ECN-Deg and ECN-Tra—by integrating specific metabolic pathways into the chassis strain, Escherichia coli Nissle 1917 (ECN). ECN-Deg integrates a metabolic module for BCAA degradation, while ECN-Tra integrates a metabolic module for BCAA transformation. Both engineered strains demonstrate efficient BCAA catabolism in vitro and in vivo. In a high-BCAA mouse model, ECN-Deg and ECN-Tra alleviated liver and ileal damage caused by excessive BCAAs and reduced systemic inflammation levels. Furthermore, ECN-Deg and ECN-Tra were able to modulate the gut microbiota, increasing the richness of Akkermansia muciniphila and Mucispirillum schaedleri, which are associated with health benefits. Additionally, they reduced the richness of the pathogenic bacterium Streptococcus pasteurianus. Thus, this study lays the foundation for the development of probiotics for the treatment of BCAAs metabolic disorders and BCAAs-related chronic diseases.

A major concern of conventional photodynamic therapy is its non-specific toxicity due to off-site drug accumulation. Micelles tend to localize the drug to the tumor site. However, rapid drug release at high concentrations from the micelles to kill the cancer cells remains a formidable task. In this manuscript, we have introduced the 2-nitrobenzyl (2NB)-moiety as the linker between mPEG and the photosensitizer, chlorin e6 (Ce6), to prepare the conjugate, mPEG(2-nitrobenzyl)Ce6. We envision that 2NB as a linker between hydrophobic, Ce6, and hydrophilic mPEG would be more effective in releasing Ce6 by disassembling PEGylated 2-nitrobenzyl chlorin e6 (mPNCe6) Ms. Characterization through Fourier transform infrared spectroscopy and ¹H, ¹³C nuclear magnetic resonance spectra validated the successful synthesis of the conjugate. By conjugating Ce6 into the hydrophobic core of the micelles, exposure to near-infrared light significantly hastened the dissociation of the micelles, facilitating a controlled and rapid release of Ce6's hydrophobic components within the micelles. A cellular uptake study was performed, showing that Ce6 conjugation has improved the uptake of Ce6. The cell viability assay revealed that the formulation had shown concentration-dependent cytotoxicity upon laser irradiation. mPNCe6 group with laser irradiation has generated abundant reactive oxygen species (ROS) inside cells and exhibited green solid fluorescence, indicating the efficient delivery of Ce6 by mPNCe6 micelles and its excellent ROS generation ability inside cells upon laser irradiation. Further, in vivo studies on MOC2 tumor-bearing mice demonstrate reduced tumor growth, lung metastasis, and drug accumulation in the tumor region. The developed nanomedicine could be a potential treatment strategy for oral cancer, minimizing the occurrence of lung metastasis.