Obtaining control of cell surface functionalizations via Pre-targeting and Supramolecular host guest interactions

Abstract

The use of mammalian cells for therapeutic applications is finding its way into modern medicine. However, modification or "training" of cells to make them suitable for a specific application remains complex. By envisioning a chemical toolbox that enables specific, but straight-forward and generic cellular functionalization, we investigated how membrane-receptor (pre)targeting could be combined with supramolecular host-guest interactions based on β-cyclodextrin (CD) and adamantane (Ad). The feasibility of this approach was studied in cells with membranous overexpression of the chemokine receptor 4 (CXCR4). By combining specific targeting of CXCR4, using an adamantane (Ad)-functionalized Ac-TZ14011 peptide (guest; K_D = 56 nM), with multivalent host molecules that entailed fluorescent β-CD-Poly(isobutylene-alt-maleic-anhydride)-polymers with different fluorescent colors and number of functionalities, host-guest cell-surface modifications could be studied in detail. A second set of Ad-functionalized entities enabled introduction of additional surface functionalities. In addition, the attraction between CD and Ad could be used to drive cell-cell interactions. Combined we have shown that supramolecular interactions, that are based on specific targeting of an overexpressed membrane-receptor, allow specific and stable, yet reversible, surface functionalization of viable cells and how this approach can be used to influence the interaction between cells and their surroundings.

Figure 1

(a) Schematic representation of the supramolecular functionalization of cell surfaces via targeting of the membrane-receptor CXCR4 (green). As first step, cellular specificity is introduced by using Ac-TZ14011-Ad (1) peptide to target CXCR4 (step 1). This provides an Ad-functionality on the surface that can be used as basis for more generic functionalization with β-CD polymers containing variable fluorescent labels and β-CD; Cy5_0.5CD₁₀PIBMA₃₉(3). Cy3_1.5CD₇₂PIBMA₃₈₉ (4) (step 2; x = 10 or 72). The then artificially generated CD-surfaces can be used to drive cellular interactions with entities containing matching guest functionalities. Hereby a third generation of functionalization can be introduced such as Ad-functionalized fluorescent dye (step 3) or cell-cell interactions can be induced with Ad-functionalized cells (step 4). (b) Chemical structures of the key compounds; Ac-TZ14011-Ad (1), Cy5_0.5CD₁₀PIBMA₃₉(3), Cy3_1.5CD₇₂PIBMA₃₈₉ (4) and Cy5_0.4PIBMA₃₉(2), the polymer-units containing different functionalities are randomly distributed within the polymer. In Ac-TZ14011-Ad the pharmacophore of Ac-TZ14011 is indicated in purple. The main guest for β-CD; Ad is indicated by a pink solid line, together with a possible second guest: Tyr10 (pink dotted line).

Figure 2

Supramolecular surface modification of viable MDAMB231 × 4 (with CXCR4-linked GFP-Tag) and MDAMB231 cells (without GFP-Tag) in mixed cell culture. Modification was accomplished via specific functionalization of the CXCR4 receptor with Ac-TZ14011-Ad, followed by host-guest interaction between β-CD molecules on fluorescent Cy5_0.5CD₁₀PIBMA₃₉ or Cy3_1.5CD₇₂PIBMA₃₈₉ polymers and the Ad functionality. Functionalization mainly occurs on the CXCR4 overexpressing MDAMB231 × 4 cells. For clarity, both the (overlay) image and the same image at the individual channels are displayed, with GFP in green, Cy5 (Cy5_0.5CD₁₀PIBMA₃₉) in red, and Cy3 (Cy3_1.5CD₇₂PIBMA₃₈₉) in blue.

Figure 3

Host-guest interaction dependent cellular functionalization: (a) Binding of Cy5_0.5CD₁₀PIBMA₃₉increases in a statistically significant manner when the guest moieties Ac-TZ14011 and Ac-TZ14011-Ad become available at the cell surface. (b) Cy5_0.4PIBMA₃₉ functionalization is not influenced by the availability of guest moieties. These values remain at baseline. The degree of functionalization was quantified by Flow cytometry (blue) or Confocal microscopy (red). Graphs show the normalized data with the error bars indicating the standard deviations (n = 6) and the significance of differences marked with *(p < 0.05) or **(p < 0.01).

Figure 4

(a) Schematic illustration of introducing a third-generation of surface modification, e.g. Cy5-Ad₂. The host-guest interaction of CD-Ad is dynamic and after functionalizing the cell surface with CD_nPIBMA_m polymers, e.g. Cy3_1.5CD₇₂PIBMA₃₈₉ (step 1,2), non-bound β-CD groups should be available to host the second fluorescent label (step 3). (b) Confocal images visualizing the introduction of Cy5-Ad₂ on Cy3_1.5CD₇₂PIBMA₃₈₉ functionalized MDAMB231 × 4 cells. For clarity, both the (overlay) image and the same image at the individual channels are displayed, with GFP in green, Cy3 (Cy3_1.5CD₇₂PIBMA₃₈₉) in blue and Cy5 (Cy5-Ad₂) in red.

Figure 5

(a) Schematic overview of inducing cell-cell interactions (3) between β-CD polymer (Cy3_1.5CD₁₀PIBMA₃₈₉) functionalized cells (1) and Ad (Ac-TZ14011-Ad) functionalized cells (2) with Hoechst staining (white) (b) Representative confocal images of inducing supramolecular cell-cell interactions between variable functionalized MDAMB231 × 4 cells. With GFP in green, Cy3 in blue and Hoechst in white. (c) Average values of the fraction of cell-cell interactions in each test condition. Significance of differences is marked with *(p < 0.05) or **(p < 0.01).