Covalent cell surface recruitment of chemotherapeutic polymers enhances selectivity and activity

Synthetic macromolecular chemotherapeutics inspired by host defence peptides can disrupt cell membranes and are emerging as agents for the treatment of cancer and infections. However, their off-target effects remain a major unmet challenge. Here we introduce a covalent recruitment strategy, whereby metabolic oligosaccharide engineering is used to label targeted cells with azido glycans, to subsequently capture chemotherapeutic polymers by a bio-orthogonal click reaction. This results in up to 10-fold reduction in EC50 and widening of the therapeutic window. Cell death is induced by not only membrane leakage, but also by apoptosis due to the conjugated chemotherapeutic being internalised by glycan recycling. Covalent recruitment also lead to increased penetration and significant cell death in a 3-D tumour model in just 3 hours, whereas doxorubicin required 24 hours. This conceptual approach of ‘engineering cells to capture polymers’ rather than ‘engineering polymers to target cells’ will bring new opportunities in non-traditional macromolecular therapeutics.

(Gibsco) and reseeded in 175/75 cm 2 cell culture flasks at appropriate seeding densities. All azido sugar and polymer solutions were prepared in the respective cell media.  were dissolved in dioxane (10 mL) at ratios presented in Table S1 to obtain 3 degrees of polymerisation (DP). Mesitylene (150 µL) was used as an internal reference and an aliquot was S6 taken in CDCl3 for NMR analysis. The reaction mixture was stirred under N2 for 30 min at RT and a further 16 h at 70 o C. An aliquot of the post-reaction mixture was taken for NMR analysis in CDCl3, allowing percentage conversion calculations. The polymer was reprecipitated into hexane from THF three times, yielding a yellow polymer product. The resulting product was dried under vacuum and an aliquot was taken for NMR analysis in CDCl3. NMR percentage conversion and SEC results are presented in Table S1.  IR v / cm -1 : 3025 -2689 cm -1 (w, C-H stretch); 2820 cm -1 (w, asym. C-H stretch (N-CH3));

Functionalisation of PFP-pDMAEMA n with DBCO-NH 2 and Thiocarbonate Reduction
An aliquot was also taken for NMR analysis in CDCl3.    DBCO-pDMAEMAn (0.10 g, 1 Eq), and N-(5-fluoresceinyl)maleimide (1.5 Eq) were dissolved in DMF (2 mL), degassed and left to stir for 24 h. The yellow mixture was reprecipitated into cold hexane from THF three times, yielding a yellow fluorescent polymer product. DMF SEC analysis was completed with the UV-Vis detector set at 494 nm to demonstrate size separation and absorbance overlap, Table S3 and Fig. S12. Polymer: dye ratios were calculated using UV-Vis.  nm using a BioTek Synergy HT microplate reader to monitor the reduction of resazurin to S12 resorufin by viable cells. Each sample was incubated for 4 h (or until resazurin reduction reached max 70%) with alamarBlue® solution at 37 ºC and 5% CO2 with readings obtained every 30 min / 1 h. Negative control cells untreated and treated with Ac4ManNAz (40 µM)

General Protocol for Metabolic Labelling of Cell Lines
were treated with alamarBlue® solution to provide a maximum resazurin reduction value of viable cells. A solution containing media alone and alamarBlue® solution was also measured for subtraction of percentage resazurin reduction values contributed from phenol red in media.
Percentage cell viability was reported relative to either cells grown solely in cell culture media alone or Ac4ManNAz. Five biological repeats were completed.
Calculating Percentage Resazurin Reduction: Calculating Percentage Viability Relative to Control Cells: Values were reported as %haemolysis (four biological repeats were completed).

General Protocol for Metabolic Glycan Labelling of Spheroids
A549 spheroids were metabolically labelled using two approaches: (1) spheroid formation in Ac4ManNAz (4 days) and (2) pre-formed spheroids (for 3 days) were subsequently treated with Ac4ManNAz (4 days). For method (1), A549 cells were seeded in an ultralow attachment Ubottom plate at a seeding density of (2k cells per well) in the presence and absence of Ac4ManNAz (40 µM). The plate was centrifuged at 2k RPM for 10 min and spheroids were allowed to grow for 96 h in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. For method (2), A549 cells were seeded in an ultralow attachment U-bottom plate at a seeding density of (1k cells per well) centrifuged at 2k RPM for 10 min and allowed to form for 72 h.
Following removal of media, the preformed spheroids were treated with or without Ac4ManNAz (40 µM) supplemented media for 96 h. Spheroids made using both approaches were subsequently treated with polymer solutions, or doxorubicin, and subjected to cell viability and cytotoxicity assays (see section 1.9).
To confirm Ac4ManNAz recruitment with both methods, spheroids were stained with DBCO-

Doxorubicin Spheroid Viability Assay
A549 spheroids produced using both protocols from section 1.8 were incubated with doxorubicin (31.3 -500 μg.mL -1 , 100 μL) for 24 h. The solutions were removed and spheroids were washed with DPBS (x3). Following replacement of media with fresh media (100 μL), an equal volume of CellTiter-Glo® was subsequently added and the protocol was completed as described above (section 1.9). S20 2 Additional Results

DBCO-pDMAEMAn-Fl
Previously, SEC results of PFP-pDMAEMAn and DBCO-pDMAEMAn demonstrated no absorbance at 494 nm (Fig. S14). Following fluorescein conjugation to the thiol terminated region of DBCO-pDMAEMAn, SEC results demonstrated a dramatic increase in absorbance readings at 494 nm which also overlapped the size distribution profile (Fig. S12), providing confirmation of successful fluorescein attachment. Polymer:dye ratios (Table S3)

Statistical analysis was performed comparing all polymer treatments against blood incubated
in DPBS alone (3 h Fig. 2A)