PEGylated Fmoc–Amino Acid Conjugates as Effective Nanocarriers for Improved Drug Delivery

A structure–activity relationship (SAR) study was conducted using a series of PEGylated Fmoc-amino acid conjugates (PFA) as a simple model to gain more insight into carrier–drug interaction. Among the eight PEG₂₀₀₀-Fmoc conjugates with different neighboring structures of Fmoc motif, PEG₂₀₀₀-Fmoc-Lys (Cbz) (PFA₂)-based nanomicelles exhibited the smallest particle size distribution, lowest critical micelle concentration (CMC) value, and highest loading capacity with paclitaxel (PTX). These biophysical properties were largely attributed to the strengthened carrier–carrier and carrier–drug interaction, including π–π stacking, hydrophobic, and hydrogen bonding interaction, as confirmed by fluorescence quenching and ¹³C NMR study. In vitro and in vivo evaluation further demonstrated the effectiveness of PFA₂ as a nanocarrier for efficient delivery of PTX to achieve improved antitumor activity. Importantly, PFA₂ was also effective in formulating eight other model drugs of diverse structures, indicating a broad application potential. This work may shed insights into the molecular basis for the structural optimization of nanocarriers for improved delivery efficacy

Synthesis of the Trisaccharide Repeating Unit of the Atypical <i>O</i>-Antigen Polysaccharide from Danish <i>Helicobacter p</i><i>ylori</i> Strains Employing the 2‘-Carboxybenzyl Glycoside

Synthesis of the unique trisaccharide repeating unit of the O-polysaccharide of the lipopolysaccharide from Danish Helicobacter pylori strains has been accomplished. Key steps include the coupling of three monosaccharide moieties by glycosylations employing the 2‘-carboxybenzyl glycoside method. Also presented is a method for the synthesis of the novel branched sugar, 3-C-methyl-d-mannose, which is one of three monosaccharide components

Supplementary Figure 1 from Secretory TRAIL-Armed Natural Killer Cell–Based Therapy: <i>In Vitro</i> and <i>In Vivo</i> Colorectal Peritoneal Carcinomatosis Xenograft

Apoptotic activity of secretory TRAIL-armed NK cells in a contacting culture system</p

UBR4-loss induces the depletion of EGFR and PDGFR from the plasma membrane.

(A) Immunoblotting analysis of EGFR and PDGFR-β in +/+ and UBR4-/- MEFs. (B) Immunoblotting analysis of EGFR and PDGFR-β in siControl (siCtrl) and siUBR4-treated MEFs. (C) Immunostaining analysis of EGFR using +/+ and UBR4-/- MEFs. Solid white lines delineate the cell boundary (scale bar = 40 μm). Enlarged views show the areas indicated by yellow rectangles (scale bar = 3 μm). (D) Immunoblotting analysis of PDGFR-β using primary cultures of +/+ and UBR4-/- MEFs at passages 3. GAPDH was probed for loading control. (E) Immunoblotting analysis of PDGFR-β in +/+ and UBR4-/- MEFs at passages 10, 11, 13, and 15. β-Actin was probed for loading control. (F) Immunoblotting analysis of EGFR, PDGFR-β, and UBR4 in siControl (siCtrl) and siUBR4-treated SH-SY5Y cells. (G) Immunostaining of EGFR and UBR4 in siControl (siCtrl) and siUBR4-treated differentiated SH-SY5Y. Retinoic acid-derived differentiated SH-SY5Y cells were transiently transfected with siRNA for 48 h. Total 12 images were collected using the confocal microscope at 0.36 μm intervals to create a stack in the Z axis. The fluorescence intensity of EGFR was significantly down-regulated in UBR4 knockdown SH-SY5Y cells. White dotted lines delineate the cell boundary (scale bar = 20 μm). Enlarged views show the areas indicated by yellow rectangles and numbers. (H) Immunostaining analysis of EGFR on the cross sections of +/+ and UBR4-/- YSs at E9.5. EGFR was significantly down-regulated in the YS of UBR4-/- embryos at E9.5 (scale bar = 10 μm). Ed, endodermal layer; Md, mesodermal layer. (I) Immunostaining analysis of EGFR on the cross-sections of +/+ and UBR4-/- embryos at E9.5. EGFR was markedly down-regulated in various embryonic tissues, such as the neural tube, and paraximal mesoderm (scale bar = 5 μm).</p

The null phenotypes of <i>UBR4</i>^<i>-/-</i> embryos are not due to cell death or misregulation in cell cycle or proliferation.

(A) H&E staining of the midbrain of +/+ and UBR4-/- mouse embryos at E9.5. Insets: magnified image of area enclosed by lined boxes. cm, cephalic mesenchyme tissue; mv, mesencephalic vesicle; ne, neuroepithelium of midbrain region (scale bar = 200 μm). (B) TUNEL assay of +/+ and UBR4-/- embryos at E9.5 (scale bar = 150 μm). (C) Cytochemical staining of senescence-associated β-galactosidase (SA-βgal) activity in +/+ and UBR4-/- MEFs. For a positive control, UBR4-/- MEF were treated with 50 nM doxorubicin for 7 days. White dotted line delineates the cell boundary (scale bar = 30 and 15 μm, respectively). N, nucleus. (D) BrdU incorporation assay of +/+ and UBR4-/- embryos at E9.5. S-phase indices were determined to be 39.3% and 33.4% for +/+ and UBR4-/- embryos, respectively. nt, neural tube; ht, heart (scale bar = 150 μm). (E) Immunostaining of F-actin (filamentous actin) and Ki67 in +/+ and UBR4-/- MEFs. For a negative control (G0 phage), UBR4-/- MEF were treated with 50 nM doxorubicin for 7 days. (scale bar = 15 μm). (F) Quantitative of Ki67-positive signals in +/+ and UBR4-/- MEFs. The percentage of Ki67-positive cells among DAPI-positive cells was calculated in 5 randomly selected fields at a magnification of 200x. Statistically no significant difference was noted between +/+ and UBR4-/- MEFs (n.s., not significant; p = 0.8591). Data are presented as mean percentage ± SEM. Statistical significance was determined using one-way analysis of variance (ANOVA) and Tukey test as a post hoc comparison.</p

UBR4 is associated with MVBs and required for the formation of MVBs.

(A) Immunostaining of UBR4 and the MVB marker CD63 on siControl- and siUBR4-treated +/+ and UBR4−/− MEFs (scale bar = 15 μm and 300 nm, respectively). (B) Transmission electron microscopy (TEM) of HEK293 cells stably expressing UBR4-V5. Fixed cells were incubated with primary antibody against V5 followed by secondary IgG antibody labelled with 12 nm gold beads. Arrows indicate UBR4-V5 molecules located at an MVB (scale bar = 100 nm for left panel and 20 nm for right panel).</p

Supplementary Figure legend from Secretory TRAIL-Armed Natural Killer Cell–Based Therapy: <i>In Vitro</i> and <i>In Vivo</i> Colorectal Peritoneal Carcinomatosis Xenograft

Supplementary Figure legend</p

UBR4 depletion causes neurite shortening in differentiated SH-SY5Y cells.

(A) Immunostaining analyses of UBR4 in differentiated SH-SY5Y cells treated with siCtrl (siControl)- and siUBR4. SH-SY5Y cells were differentiated by retinoic acid treatment for 5 days, followed by siRNA treatment for 48 h. White dotted lines delineate the cell boundary. Enlarged views show the areas indicated by yellow rectangles (scale bar = 30 μm). (B) Quantification of A. Total 25 control and 18 UBR4 knockdown SH-SY5Y cells were observed. ***p .0001. Data are presented as mean ± SEM. Statistical significance was determined using unpaired t-test. (C) Average neurite diameters (in μm) of control and UBR4 knockdown SH-SY5Y cells. Linear regression (solid line) was applied to this data. The slope indicates that the immunofluorescence intensity of UBR has no relationship with the neurite diameter. n.s., not significant. (D) Average neurite length (in μm) of control and UBR4 knockdown SH-SY5Y cells. Linear regression (solid line) was applied to this data. The slope indicates that the immunofluorescence intensity of UBR4 had a correlation with neurite length.</p

Proteomic analysis of cell surface proteins.

(A) Coomassie brilliant blue (CBB) staining of purified surface proteins separated using SDS-PAGE. (B) Molecular functions of identified cell surface proteins by the Ingenuity Pathway Analysis (IPA) tool. (C) Schematic illustration of mass spectrometric data of 93 membrane proteins whose abundance significantly altered in UBR4-/- MEFs as compared with +/+ MEFs. (D) Immunostaining analyses of VTI1A, SLC7A1 and VAT1 (scale bar = 10 μm). (E) Quantification of D, representing the relative fluorescence intensities for VTI1A, SLC7A1, and VAT1. *p = 0.0325 vs VTI1A of +/+ (n = 13–14 in each group), ***p .0001 vs SLC7A1 of +/+ (n = 13–23 in each group), and **p = 0.0070 vs VAT1 of +/+ (n = 16–22 in each group). Data are presented as mean percentage ± SEM of +/+. Statistical significance was determined using unpaired t-test.</p

Mouse embryos lacking UBR4 die at midgestation associated with multiple developmental abnormalities.

(A) Gross morphology of +/+ and UBR4-/- mouse littermates at E8.5-E9.5. UBR4 mutants exhibit embryonic growth retardation and malformed pharyngeal arches. The red and blue lines represent the first PA and second PA, respectively (scale bar = 1 mm). (B) Anatomical illustration of the pharyngeal arch (PA) and heart of the mouse embryo at E9.0. First PA mesodermal cells (red) move to the developing heart tube for the right ventricular myocardium at E7.5–8.0, while second PA mesodermal cells (blue) migrate to the outflow tract (oft) of the myocardium at around E9.5-E10. (C) Hematoxylin and eosin (H&E) staining on cross sections of +/+ and UBR4-/- embryos at E9.5. nt, neural tube; at, atrium; vt, ventricles; 4th, fourth ventricle (scale bar = 100 μm). (D) H&E staining of the placenta of +/+ and UBR4-/- mouse littermates at E9.5 (scale bar = 500 μm).</p