Characterization of 1,2-Distearoyl-<i>sn</i>-glycero-3-phosphoethanolamine–<i>N</i>‑[Methoxy(polyethylene glycerol)-2000] and Its Complex with Doxorubicin Using Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics

Polyethylene glycol (PEG) lipid nanoparticles (LNPs) spontaneously assemble in water, forming uniformly sized nanoparticles incorporating drugs with prolonged blood clearance compared to drugs alone. Previously, 1,2-distearoyl-<i>sn</i>-glycero-3-phosphoethanolamine–<i>N</i>-[methoxy­(polyethylene glycerol)-2000] (DSPE–PEG₂₀₀₀) and several drug adducts, including doxorubicin, were analyzed by a combination of physical and molecular dynamic (MD) studies. In this study, a complete chemical shift assignment of DSPE–PEG₂₀₀₀ plus or minus doxorubicin was achieved using nuclear magnetic resonance (NMR), one-dimensional selective nuclear Overhauser spectroscopy (1D-selNOESY), NOESY, correlation spectroscopy (COSY), total correlated spectroscopy (TOCSY), heteronuclear single quantum coherence (HSQC), and HSQC–TOCSY. Chemical shift perturbation, titration, relaxation enhancement, and NOESY analysis combined with MD reveal detailed structural information at the atomic level, including the location of doxorubicin in the micelle, its binding constant, the hydrophilic shell organization, and the mobility of the PEG₂₀₀₀ tail, demonstrating that NMR spectroscopy can characterize drug–DSPE–PEG₂₀₀₀ micelles with molecular weights above 180 kDa. The MD study revealed that an initial spherical organization led to a more-disorganized oblate structure in an aqueous environment and agreed with the NMR study in the details of the fine structure, in which methyl group(s) of the stearic acid in the hydrophobic core of the micelle are in contact with the phosphate headgroup of the lipid. Although the molecular size of the LNP drug complex is about 180 kDa, atomic resolution can be achieved by NMR-based methods that reveal distinct features of the drug–lipid interactions. Because many drugs have unfavorable blood clearance that may benefit from incorporation into LNPs, a thorough knowledge of their physical and chemical properties is essential to moving them into a clinical setting. This study provides an advanced basic approach that can be used to study a wide range of drug–LNP interactions

NSCs target breast carcinoma and can deliver anti-HER2 antibody <i>in vivo</i>.

<p>Confocal fluorescence micrographs of tumor sections from MCF7/HER2 xenografts. First three panels in the upper row show the presence of CM-DiI-labeled red NSCs (HB1.F3, HB1.F3.Ad-H2IgG, HB1.F3.Lenti-H2IgG, respectively) in tumors 4 days after intravenous injection. The fourth panel of the upper row shows tumor with no red NSCs in mice treated with trastuzumab alone (sporadic small red dots not associated with cells are visible as autofluorescence background). Middle row shows tumor sections stained with FITC-conjugated anti-human IgG (green). Bar, 50 µm. Insets are 2× magnification (Bar, 20 µm). Bottom row shows confirmation of the presence or absence of HB1.F3 NSCs within tumors by PCR detection of a DNA amplicon (293 bp) of the v<i>-myc</i> transgene, a unique identifier of the HB1.F3 cell line.</p

NSC-secreted human IgG specifically binds HER2.

<p>Transfected NSCs were co-cultured with CM-DiI-labeled (red) MCF7 (<b>A</b>) or MCF7/HER2 cells (<b>B</b>) and stained with FITC-conjugated anti-human IgG (green) and DAPI (blue). Arrows indicate NSCs expressing human IgG that does not bind to adjacent MCF7 control cells (<b>A</b>) or areas of NSC-secreted human IgG bound to MCF7/HER2 target cells (<b>B</b>). Bar, 50 µm. Flow cytometric analysis of BT474, MCF7/HER2, or MCF7 target cells after incubation with supernatant from HB1.F3.H2IgG, HB1.F3.Adeno-H2IgG, or HB1.F3.Lenti-H2IgG (<b>C</b>), followed by incubation with FITC-conjugated anti-human IgG (blue histograms). Red histograms on each graph show target cells incubated with supernatant from unmodified HB1.F3 NSCs as a negative control.</p

NSC-secreted anti-HER2 antibody is functionally equivalent to trastuzumab.

<p>Flow cytometric analysis (<b>A</b>) of MCF7, MCF7/HER2, and BT474 cells labeled with F3-IgG, trastuzumab, or a human IgG isotype control antibody. Graphs show mean fluorescence intensity (MFI) of labeled cells. Inhibition of cell proliferation (<b>B</b>) of MCF7, MCF7/HER2, or BT474 cells treated for 6 days with F3-IgG, trastuzumab, or isotype control antibody. Graphs show proliferation as a percentage of untreated cells.</p

<i>In vitro</i> migration of NSCs to breast carcinoma conditioned media.

<p>Migration of parental NSCs and anti-HER2-transfected HB1.F3 NSCs to breast tumor-conditioned media in an <i>in vitro</i> chemotaxis assay. In this assay, bovine serum albumin (BSA) was used as a negative control for chemotaxis. Both parental and transfected NSC lines preferentially migrated to MCF7/HER2 compared to negative control (2% BSA) (<i>p</i><0.01).</p

Expression of human IgG in NSCs.

<p>Fluorescence micrograph of parental HB1.F3 NSCs (<b>A</b>), HB1.F3.H2IgG (<b>B</b>), HB1.F3.Adeno-H2IgG NSCs (<b>C</b>), and HB1.F3.Lenti-H2IgG (<b>D</b>). Cells were stained with anti-human IgG (green) and DAPI nuclear stain (blue). Expression was confirmed by intracellular flow cytometry of fixed and permeabilized NSCs (<b>E</b>). Fluorescence of fixed and permeabilized parental NSCs (indicated by gray histogram) was used to set the marker for each graph.</p