Journal: Materials Today Bio

Article Title: Bipolar CD4-targeted dual-DARPin-55/57 lipid nanoparticle enables efficient CRISPR/Cas-mediated HIV-1 DNA excision and reactivation blockade in latent CD4 T cell lines

doi: 10.1016/j.mtbio.2026.102939

Figure Lengend Snippet: Design and physicochemical characterization of CD4-targeted CRISPR/Cas9-loaded LNPs. (A) Schematic of LNP formulation encapsulating spCas9 mRNA (Sp9m) and HIV-1-targeting LTR1 and GagD sgRNAs (LGsg), followed by surface functionalization with CD4-binding DR to generate DR-Sp9m/LGsg LNP. (B) DR thiolation using Traut's reagent (2-iminothiolane) to introduce reactive sulfhydryls for site-directed conjugation. (C) Maleimide–thiol coupling of thiolated DR to maleimide-PEG–modified LNPs, yielding bipolar attachment while preserving CD4-binding activity. A representative structural model depicts CD4 (brown ribbon) bound to DR (green), illustrating an outward-facing presentation of the CD4-binding interface on DR-decorated LNPs after conjugation. (D) Particle size, concentration, polydispersity index (PDI), and ζ-potential of uncoated Sp9m/LGsg LNPs and DARPin-coated DR-Sp9m/LGsg LNPs. Data are mean ± SEM from six independent Sp9m/LGsg batches and five DR-Sp9m/LGsg batches. Statistics: two-way ANOVA with Šídák multiple comparisons (∗p < 0.05, ∗∗∗p < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Sp9m and LGsg were synthesized using the HiScribe T7 ARCA mRNA Kit (NEB; E2060) with N1-methyl-pseudouridine (TriLink BioTechnologies; N-1081).

Techniques: CRISPR, Formulation, Binding Assay, Introduce, Conjugation Assay, Modification, Preserving, Activity Assay, Concentration Assay

Journal: Materials Today Bio

Article Title: Bipolar CD4-targeted dual-DARPin-55/57 lipid nanoparticle enables efficient CRISPR/Cas-mediated HIV-1 DNA excision and reactivation blockade in latent CD4 T cell lines

doi: 10.1016/j.mtbio.2026.102939

Figure Lengend Snippet: Formulation and cell-type targeting of DARPin-spCas9 mRNA LNPs in human PBMCs. (A, B) Physicochemical characterization of untargeted Sp9m LNPs and DARPin-decorated DR-Sp9m LNPs (size, PDI, ζ-potential, and particle concentration). Data are mean ± SEM from five independent Sp9m batches (n = 5). (C) Representative flow-cytometry gating strategy to quantify spCas9-GFP expression across PBMC subsets. (D) Time-course of spCas9-GFP expression in CD4 T cells, monocytes, B cells, and CD8 T cells after exposure to untargeted or DARPin-targeted LNPs (two independent LNP batches). (E) Uptake/expression kinetics in CD4 T cells and monocytes. Data are mean ± SE. Statistics: two-way ANOVA with Šídák multiple comparisons (∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant).

Article Snippet: Sp9m and LGsg were synthesized using the HiScribe T7 ARCA mRNA Kit (NEB; E2060) with N1-methyl-pseudouridine (TriLink BioTechnologies; N-1081).

Techniques: Formulation, Concentration Assay, Flow Cytometry, Expressing

Journal: Materials Today Bio

Article Title: Bipolar CD4-targeted dual-DARPin-55/57 lipid nanoparticle enables efficient CRISPR/Cas-mediated HIV-1 DNA excision and reactivation blockade in latent CD4 T cell lines

doi: 10.1016/j.mtbio.2026.102939

Figure Lengend Snippet: Elimination of HIV-1 Proviral DNA in J-Lat 10.6 Cell Line via DARPin-LNP Delivery of spCas9 mRNA (Sp9m) and LTR/GagD sgRNAs (LGsg). (A) Representative PCR gel images showing a significant reduction (80.6% ± 4.9% normalized to β-actin, n = 8) of the HIV-1 genomic region spanning from the 5′-LTR to Gag in J-Lat 10.6 cells treated with DR-Sp9m/LGsg LNPs. Data are from two independent experiments using two LNP batches (B5 and B7) at different dosages. (B) Complete excision of the full-length HIV-1-GFP genome integrated within intron 2 of the SEC16A gene. PCR was performed using primers (V250/V251) flanking the integration site at position 136,468,579 (GRCh38/hg38). (C–G) Digital PCR (dPCR) analysis quantifying HIV-1 proviral DNA, confirming a marked reduction in proviral copies following DR-Sp9m/LGsg LNP treatment. (C) Schematic of the HIV-1-GFP genome, dPCR probe locations, the excised region, and a representative 2D scatter plot distinguishing intact from excised genomes. (D, E) Representative 1D plots showing signal from individual probes and corresponding HIV-1 genome copy numbers. (F, G) Representative 2D plots from duplex probe assays, illustrating quantification of intact versus excised HIV-1 genomes.

Article Snippet: Sp9m and LGsg were synthesized using the HiScribe T7 ARCA mRNA Kit (NEB; E2060) with N1-methyl-pseudouridine (TriLink BioTechnologies; N-1081).

Techniques: Digital PCR

Journal: Materials Today Bio

Article Title: Bipolar CD4-targeted dual-DARPin-55/57 lipid nanoparticle enables efficient CRISPR/Cas-mediated HIV-1 DNA excision and reactivation blockade in latent CD4 T cell lines

doi: 10.1016/j.mtbio.2026.102939

Figure Lengend Snippet: Model for dual DARPin-mediated LNP delivery of spCas9 mRNA and duplex HIV-1 sgRNAs to human CD4 T cells to enforce functional proviral excision. A LNP incorporating spCas9 mRNA and duplex sgRNAs (targeting LTR1 and GagD) is functionalized with CD4-specific DARPin-55 and DARPin-57 molecules via bipolar thioether linkages. The dual-DARPin coating facilitates selective binding to CD4 T cells and receptor-mediated uptake. Following endosomal escape, Cas9/sgRNA complexes are assembled and execute targeted excision of integrated HIV-1 proviral DNA. Removal of the provirus abrogates HIV-1 reactivation upon latency-reversing stimulation, demonstrating a receptor-guided, non-viral strategy for HIV-1 proviral excision.

Article Snippet: Sp9m and LGsg were synthesized using the HiScribe T7 ARCA mRNA Kit (NEB; E2060) with N1-methyl-pseudouridine (TriLink BioTechnologies; N-1081).

Techniques: Functional Assay, Binding Assay

Journal: Molecular Therapy Oncology

Article Title: Suppression of PARP1 enhances PTEN mRNA therapy in castration-resistant prostate cancer by glycolysis disruption

doi: 10.1016/j.omton.2026.201133

Figure Lengend Snippet: The antitumor effects of mPsiP@miLAND on PC-3 subcutaneous tumor model (A) Brief description of the experimental process and grouping information. (B) Tumor growth curves and (C) bodyweight changes of the treated animals during the entire study period. (D) Tumor weights, (E) serum biochemical indicators, and (F) relative mRNA expression of PARP1, recorded or analyzed at the end of the study. Seven indicators including creatine kinase (CK, U/liter), aspartate aminotransferase (AST, U/liter), alkaline phosphatase (ALP, U/liter), total protein (TP, g/liter), urea nitrogen (UREA, mmol/L), serum creatinine (CREA, mmol/L), and triglyceride (TG, mmol/L) were recorded. (G) Analysis of PI3K-Akt signaling, glycolysis, apoptosis, transcription, and translation in tumors after treatment. (H) The impacts of the formulations on tumor proliferation and apoptosis, as measured by Ki67 (top) and TUNEL (bottom) staining. ∗ p < 0.05, considered as statistically significant; ∗∗∗∗ p < 0.0001, each bar chart represents the mean ± SD.

Article Snippet: BstZ17I-HF endonuclease (R3594L), rCutSmart Buffer (B6004S), and HiScribe T7 ARCA mRNA in vitro transcription kit (E2060S) were purchased from NEB (MA, USA).

Techniques: Expressing, TUNEL Assay, Staining