Journal: Nano letters

Article Title: Bifunctional Janus Particles as Multivalent Synthetic Nanoparticle-Antibodies (SNAbs) for Selective Depletion of Target Cells

doi: 10.1021/acs.nanolett.0c04833

Figure Lengend Snippet: Schematic illustration of SNAbs and their hypothetical mechanism of action. (a) SNAbs are Janus nanoparticles bearing two distinct chemically modified faces. One of the two faces presents targeting ligands to perform the function of Fab domains in mAbs, and the other displays Fc-mimicking ligands to crosslink Fc receptors on the effector cells as Fc fragments in mAbs. (b) Once administered into patients or animals with diseases, the SNAbs circulate and recognize target cells in blood or organs of interest by binding onto their surface proteins and engaging with effector cells (e.g. macrophages, NK cells) to induce antibody-like cellular cytotoxicity or phagocytosis.

Article Snippet: 19 Briefly, streptavidin (SA)-coated Au nanoparticles (SA-AuNP-SAs, 30 nm spheres, with an average hydrodynamic diameter of 70 nm) were bound onto aminomethyl resin (200–300 μm) via a reducible crosslinker, sulfo-NHS-S-S-Biotin ( Supplementary Fig. 1a ).

Techniques: Modification, Binding Assay

Journal: Nano letters

Article Title: Bifunctional Janus Particles as Multivalent Synthetic Nanoparticle-Antibodies (SNAbs) for Selective Depletion of Target Cells

doi: 10.1021/acs.nanolett.0c04833

Figure Lengend Snippet: Fabrication and characterization of the Janus Au nanoparticles and molecular dynamic modeling of the interaction between G3/G3* and S100A8/A9. (a) Aminomethyl resins were functionalized with sulfo-NHS-S-S-biotin crosslinker (step 1) and then bound with streptavidin-coated, 30nm Au nanoparticles (step 2). The cleavage of the disulfide bonds in the crosslinker by TCEP releases the Janus Au nanoparticles (step 3), which has a streptavidin face with open biotin-binding pockets and a thiol face with available free thiols. (b) STEM-BF, STEM-HAADF, and EDS mapping images of 10–12 nm biotinylated quantum dot (QD)-conjugated nonJanus (SA-AuNP-SA, left column) and Janus (SA-AuNP-SH, right column) gold nanoparticles demonstrates the asymmetric distribution of open biotin-binding sites on the Janus nanoparticles fabricated using the method described in a. Colors: red-gold (nanoparticles), blue-oxygen (streptavidin), cyan blue- Cd (biotinylated QDs). (c) Available thiol groups on either Janus nanoparticles or unmodified nanoparticles (to compare here, labeled as SA-AuNP-SA in the graph, named as AuNP in other parts of the paper) shown by conjugation of Alexa Fluor 647-maleimide dye, where signal is normalized to the concentration of nanoparticles. Data are presented as mean ± s.d. N=2 independent samples with n=3 technical replicates. (d-g), Molecular dynamics simulation of G3 or G3* binding onto human S100A8/A9 heterotetramer. Ten short (100 ns each) molecular dynamics simulations with human S100A8/A9 heterotetramer demonstrate the capability of both G3-biotin and G3*-biotin to interact with the S100A8/A9 proteins. (d-e), The superposition of the final frames of the five 100-ns G3 simulations (d) and G3* simulations (e). f-g, The contact probability (defined as coming within 3.5 Å) determined from all five 100 ns G3 simulations (f) and G3* simulations (g). The sequence along the x axis is that of each peptide.

Article Snippet: 19 Briefly, streptavidin (SA)-coated Au nanoparticles (SA-AuNP-SAs, 30 nm spheres, with an average hydrodynamic diameter of 70 nm) were bound onto aminomethyl resin (200–300 μm) via a reducible crosslinker, sulfo-NHS-S-S-Biotin ( Supplementary Fig. 1a ).

Techniques: Binding Assay, Labeling, Conjugation Assay, Concentration Assay, Sequencing

Journal: Nano letters

Article Title: Bifunctional Janus Particles as Multivalent Synthetic Nanoparticle-Antibodies (SNAbs) for Selective Depletion of Target Cells

doi: 10.1021/acs.nanolett.0c04833

Figure Lengend Snippet: Ligand modification on Janus Au nanoparticles and evaluation of the binding of SNAbs onto mouse MDSCs and macrophages by photoacoustic (PA) imaging. (a) Following fabrication of Janus nanoparticles, the Fc-mimicking ligands, cp33, was conjugated onto the thiol face of the Janus nanoparticles via thiol-maleimide reaction, and the targeting ligands, G3, was modified onto the streptavidin face via biotin-streptavidin interaction. (b,c) Alexa Fluor 680-NHS-estertagged MDSC-targeting peptide (G3) and Alexa Fluor 680-NHS-ester-tagged Fc-mimicking peptide (cp33) were reacted with SA-AuNP-SH or SA-AuNP-SA. The conjugation level of each peptide was assessed by measuring fluorescence of the samples and normalizing against nanoparticle concentrations. Background fluorescence of the untagged-peptide-modified nanoparticles was subtracted from the measured values of the samples. Data are presented as mean ± s.d. N=2 (left) or 3 (right) independent samples with n=3 or n=6 technical replicates. (d) Hydrodynamic sizes of nonJanus SA-AuNP-SA, Janus nanoparticles SA-AuNP-SH and SNAbs after modification with ligands were measured by dynamic light scattering on a Malvern Zetasizer. (e) The photoacoustic (PA) and ultrasound (US) images of nanoparticle-treated samples of mouse MDSCs. Top: PA images of the cell inclusions of SNAb, NonJanus AuNP-cp33, NonJanus AuNP-cp33 and AuNP at a wavelength of 532 nm. Bottom: US images of the cell inclusions of SNAb and AuNP. (f,g) The relative amount of nanoparticles bound to mouse MDSCs (f) or RAW 264.7 macrophages (g) based on the average PA signals of each cell inclusion (0.5 million cells/40𝜇L). PA signals shown in the graphs were normalized against the laser energy and backscattered ultrasound signals. Data are presented as mean ± s.d. of at least six cross-section images of two or more technical replicates of corresponding independent samples.

Article Snippet: 19 Briefly, streptavidin (SA)-coated Au nanoparticles (SA-AuNP-SAs, 30 nm spheres, with an average hydrodynamic diameter of 70 nm) were bound onto aminomethyl resin (200–300 μm) via a reducible crosslinker, sulfo-NHS-S-S-Biotin ( Supplementary Fig. 1a ).

Techniques: Modification, Binding Assay, Imaging, Conjugation Assay, Fluorescence

Journal: Nano letters

Article Title: Bifunctional Janus Particles as Multivalent Synthetic Nanoparticle-Antibodies (SNAbs) for Selective Depletion of Target Cells

doi: 10.1021/acs.nanolett.0c04833

Figure Lengend Snippet: MDSC-SNAbs induce antibody-like killing of mouse and rat MDSCs in the presence of effector cells, such as macrophages. (a-h) In a mouse splenocyte-suspension assay, the single cell suspension from the spleens of 4T1-tumor-bearing mice were treated with equal concentration of various nanoparticle formulations for 24 hours. The cells were then stained with fluorescent antibodies and fixable viability dye and analyzed by flow cytometry for the total percentage (a) of live and dead MDSC (PMN-MDSC and monocytic MDSCs), the percentage of dead MDSCs (b), dead PMN-MDSCs (CD11b+Ly6G+Ly6Clow) (c) or dead M-MDSCs (CD11b+Ly6G-Ly6Chigh) (d) out of corresponding cell populations, and the percentages of CD11b+F4/80+macrophages (e), CD3-B220+B cells (f), CD11b+CD11c+DCs (g), and CD3+T cells (h) out of total cells. (i) Rat MDSC and CD11b+ monocytes sorted from peripheral blood mononuclear cell (PBMC) of rats with femoral segmental defects were cultured ex vivo at 1:10 ratio and treated with MDSC-SNAbs or control treatments for 24 hours. The percentages of MDSCs (CD11b+His48+) in the co-culture after treatment were measured by flow cytometry. Data are presented in box plots (n=6) or presented by individual values with mean and standard deviation (n=6). Significance was determined using one-way ANOVA with Tukey post-hoc test (**** p<0.0001, *** p<0.0002, ** p<0.0021, * p<0.0332). AuNPs are nonJanus streptavidin coated Au nanoparticles. NonJanus AuNP-G3/cp33 nanoparticles are AuNPs coated with randomly distributed G3 and cp33. G3-SNAbs and G3*-SNAbs are Janus G3-AuNP-cp33 or G3*-AuNP-cp33 nanoparticles targeting MDSCs.

Article Snippet: 19 Briefly, streptavidin (SA)-coated Au nanoparticles (SA-AuNP-SAs, 30 nm spheres, with an average hydrodynamic diameter of 70 nm) were bound onto aminomethyl resin (200–300 μm) via a reducible crosslinker, sulfo-NHS-S-S-Biotin ( Supplementary Fig. 1a ).

Techniques: Suspension, Concentration Assay, Staining, Flow Cytometry, Cell Culture, Ex Vivo, Control, Co-Culture Assay, Standard Deviation

Journal: Nano letters

Article Title: Bifunctional Janus Particles as Multivalent Synthetic Nanoparticle-Antibodies (SNAbs) for Selective Depletion of Target Cells

doi: 10.1021/acs.nanolett.0c04833

Figure Lengend Snippet: Biodistribution and therapeutic effects of MDSC-SNAbs in a 4T1 mouse breast cancer model. (a,b) Biodistribution of SNAbs after intravenous injection in tumor-bearing mice. G3-SNAbs were administered through tail vein injection on day 9 post tumor inoculation. Lung, liver, spleen, kidney, tumor and blood were harvested after 6, 24, or 48 hours for ICP-MS analysis. Changes in the biodistribution over time, presented as concentration of Au per ug of tissue (a) and calculated percentage of SNAbs in each organ out of total injected amount of nanoparticles (b), were plotted with mean ± s.d. of 3 biological samples (n = 3). (c-g) Therapeutic effects of MDSCs in the 4T1 murine model. (c) The schedule of tumor inoculation and systemic injection (i.v. via the tail vein) of G3-SNAbs (Janus G3-AuNP-cp33), IrrelPep-SNAb (Janus scAHNP-AuNP-cp33), Janus control NP (Janus biotin-AuNP-NEM), AuNP and PBS (n=7, 7.5×1010/20𝜇L per injection). The percentage of total MDSCs (PMN-MDSCs and M-MDSCs) out of total cells in the blood (d) and spleens (e), the percentage of CD3+CD8+ cytotoxic T cells (f) and CD3-CD49b+NK cells (g) infiltrated in the tumors after treatment on day 11 are presented in box plots with box showing median, 25 and 75 percentile and whiskers showing min and max. After removing outliers, n=6 for untreated groups, n=6 in (d) and n=7 in (e) and (f-g) for AuNP group, N=7 for Janus control NP, n=4 in (e) and (g), n=5 in (d) and n=6 in (f) for IrrelPep-SNAb group, and n=7 for G3-SNAb single injection group, n=4 in (d),(e),(g) and n=5 in (f) and for G3-SNAb 3-injection groups. Significance was determined using one-way ANOVA with Tukey post-hoc test (**** p<0.0001, *** p<0.0002, ** p<0.0021, * p<0.0332).

Article Snippet: 19 Briefly, streptavidin (SA)-coated Au nanoparticles (SA-AuNP-SAs, 30 nm spheres, with an average hydrodynamic diameter of 70 nm) were bound onto aminomethyl resin (200–300 μm) via a reducible crosslinker, sulfo-NHS-S-S-Biotin ( Supplementary Fig. 1a ).

Techniques: Injection, Concentration Assay, Control

Journal: Nano letters

Article Title: Bifunctional Janus Particles as Multivalent Synthetic Nanoparticle-Antibodies (SNAbs) for Selective Depletion of Target Cells

doi: 10.1021/acs.nanolett.0c04833

Figure Lengend Snippet: Summary of the nanoparticles used in this paper.

Article Snippet: 19 Briefly, streptavidin (SA)-coated Au nanoparticles (SA-AuNP-SAs, 30 nm spheres, with an average hydrodynamic diameter of 70 nm) were bound onto aminomethyl resin (200–300 μm) via a reducible crosslinker, sulfo-NHS-S-S-Biotin ( Supplementary Fig. 1a ).

Techniques: Control