Polyvalent Display of Biomolecules on Live Cells

Abstract

Surface display of biomolecules on live cells offers new opportunities to treat human diseases and perform basic studies. Existing methods are primarily focused on monovalent functionalization, that is, the display of single biomolecules across the cell surface. Here we show that the surface of live cells can be functionalized to display polyvalent biomolecular structures through two-step reactions under physiological conditions. This polyvalent functionalization enables the cell surface to recognize the microenvironment one order of magnitude more effectively than with monovalent functionalization. Thus, polyvalent display of biomolecules on live cells holds great potential for various biological and biomedical applications.

Keywords: DNA self-assembly; cell surface engineering; molecular recognition; polyvalent display.

Figure 1

Polyvalent engineering for surface display of branched DNA polymers. a) Schematic illustration of in situ formation of polyvalent DNA polymers on the cell surface. b) Characterization of DI-functionalized nanoparticles (NP) with UV-vis absorption spectra (left). Analysis of DNA polymerization on the particle surface using dynamic light scattering (right). c) Fluorescence images of the cells after unimolecular (left) or polyvalent (right) functionalization. Scale bars, 50 μm. d) Flow cytometry analysis of the engineered cells. Data were presented as mean±s.d.

Figure 2

Comparison between unimolecular and polyvalent display in promotion of cell-microenvironment interactions. a) Examination of the cell surface in nanomaterials recognition with STEM imaging. Bright spots: ~10 nm of quantum dots. b) Recognition of cell. Representative images of the constructs formed with the engineered CCRF-CEM cells. The engineered cells were separated into two groups and labeled with either DiO (green) or DiD (red), respectively. Scale bars, 100 μm. Schematic illustration was used for clear legibility. c) The difference between polyvalent and unimolecular functionalization in promoting cell-cell recognition. For measurement and calculation, the cell constructs were assumed to be spheres. Data were presented as mean±s.d.

Figure 3

Kinetic analysis of cell assembly. a) Flow cytometry analysis of the flower-shaped constructs. DiO and DiD stained cells were mixed at a ratio of 1:25 and analyzed by flow cytometry. The gated blue population represented the flower-shaped constructs. b) Kinetic profiles of cell assembly for the formation of the constructs. c) Representative flow cytometry histograms showing the conversion of DiO-labeled cells into the constructs. The time point for analysis was set at 180 s. d) Comparison of the initial conversion rates (within the first 10 sec) after the mixing of the two cell populations. e) High-speed microscopic images of cell constructs during flow cytometry analysis (left). Representative images of different constructs were given (right). f) Analysis of the percentages of the constructs. Data were presented as mean±s.d.

Figure 4

Generation and growth of hMSC-NHAC constructs. a) Fluorescence images of the cells engineered with the unimolecular or polyvalent functionalization methods. Blue: DAPI. Green: Calcein-AM. Red: DNA or polyvalent DNA polymer. Scale bars, 100 μm. b) Flow cytometry analyses of the engineered cells. c) Size analysis of the hMSC-NHAC constructs during the culture. hMSC expressed RFP for clear observation. Representative images acquired at different time points are shown to demonstrate the growth of the constructs. Scale bars, 100 μm. d) Immunofluorescence staining of the constructs for analyzing the expression of type II collagen and aggrecan. Scale bars, 50 μm.