‘Multicopy Multivalent’ Glycopolymer-Stabilized Gold Nanoparticles as Potential Synthetic Cancer Vaccines

Mucin-related carbohydrates are overexpressed on the surface of cancer cells, providing a disease-specific target for cancer immunotherapy. Here, we describe the design and construction of peptide-free multivalent glycosylated nanoscale constructs as potential synthetic cancer vaccines that generate significant titers of antibodies selective for aberrant mucin glycans. A polymerizable version of the Tn-antigen glycan was prepared and converted into well-defined glycopolymers by Reversible Addition–Fragmentation chain Transfer (RAFT) polymerization. The polymers were then conjugated to gold nanoparticles, yielding ‘multicopy-multivalent’ nanoscale glycoconjugates. Immunological studies indicated that these nanomaterials generated strong and long-lasting production of antibodies that are selective to the Tn-antigen glycan and cross-reactive toward mucin proteins displaying Tn. The results demonstrate proof-of-concept of a simple and modular approach toward synthetic anticancer vaccines based on multivalent glycosylated nanomaterials without the need for a typical vaccine protein component.

Solvents were purchased from Fischer Scientific and dried by passage through two alumina columns using an Innovative Technology Inc. solvent purification system and stored under N 2 . TLC was performed on aluminium-backed silica plates (Merck), with visualisation using H 2 SO 4 (5 %) in ethanol.
2-Hydroxyethyl methacrylate was passed through a short column of basic alumina in order to remove MEHQ inhibitor prior to polymerisation. Flash column chromatography was performed using a Biotage SP1 automated purification system using pre-packed silica columns. (4-cyanopentanoic acid)-4dithiobenzoate (CPADB) was synthesised according to a previously described procedure 1 ; analytical data were in agreement with literature values.

Instrumentation
NMR spectra were recorded on a Varian Inova-700 spectrometer at 700MHz ( 1 H) and 176MHz ( 13 C), a Varian Inova 500 spectrometer at 499.87 ( 1 H) and 125.67 MHz ( 13 C, 1 H decoupled at 500 MHz) or a Bruker Avance 400 spectrometer at 400.13 MHz ( 1 H) or 100.26 MHz ( 13 C, 1 H decoupled at 400 MHz), at ambient temperature in CDCl 3 , DMSO, D 2 O or MeOD. NMR spectra were analysed using MestReNova v6.04 software and referenced internally to the protons of the residual solvent. IR spectra were recorded on a Nicolet Nexus FT-IR spectrometer as thin films on KBr discs cast from a suitable solvent. Mass spectral analyses were performed on a Thermal-Finnigan LTQ using a positive or negative ionisation electrospray mode. Elemental analysis was conducted on an Exeter Analytical E-440 elemental analyser.
SEC was performed using a triple detection method (with angular correction) and measurements were performed on a Viscotek TDA 301 triple detection SEC fitted with two (300 x 7.5 mm) GMPWxl methacrylate-based mixed bed columns with an exclusion limit of 5,107 g mol -1 , having refractive index, viscometer and RALLS detectors. The eluent was DMF with added LiBr salt at a flow rate of 1.0 ml min -1 . Thermogravimetric analyses were performed on a Perkin Elmer Pyris 1 TGA under argon gas; heating from 20 °C to 800 °C at 10 °C min -1 . Dynamic light scattering measurements were acquired using a Brookhaven Instruments 90 Zeta-Plus particle size analyser; samples were passed through a 0.22 µm S3 syringe filter prior to analysis. Samples for TEM analysis were prepared by deposition of a drop of the particle solution on to a carbon-coated copper grid and the excess solution removed using filter paper, leaving a thin film of the particles. The samples were imaged using a Hitachi H7600 microscope.

N-(2-Hydroxyethyl)-2-methacrylamide (HEMAm)
N-(2-hydroxyethyl)-2-methacrylamide (HEMAm) was prepared according to the method described by Chan et al. 2 Freshly distilled methacryloyl chloride (1.30 cm 3 , 13.4 mmol) was dissolved in anhydrous dry chloroform (15 cm 3 ) and added slowly to a solution of ethanolamine (1.62 cm 3 , 26.8 mmol) at 0 °C in anhydrous chloroform (20 cm 3 ). The reaction was stirred for a further 2 h at 0 °C, after which the precipitated salt was removed by filtration, and the solvent removed in vacuo to yield crude HEMAm as a colourless oil. This was dissolved in chloroform and stirred overnight with basic alumina. After filtration, the solvent was removed in vacuo to give the final product as a pale yellow oil, which was stored with BHT at ~4 °C in order to prevent unwanted polymerisation (1.50 g, 11.4 mmol, 90 %). 1

Glyconanoparticle Synthesis
Glyconanoparticles were synthesized using quantities of reagents detailed in Table S3. Separate solutions of HAuCl 4 (0.5 mM), glycopolymer (5.0 mM) and sodium borohydride (50 mM) were prepared in UHQ water (resistivity < 18.0 M). The HAuCl 4 and glycopolymer solutions were combined and then treated with NaBH 4 solution. An immediate solution colour change from yellow to pale brown was observed in S9 all cases. Stirring was continued for 2.5 h after which the nanoparticle solutions were purified by centrifugal filtration (Sartorious Vivaspin 15R, MWCO 30 kDa) and washing with UHQ water.

Preparation of Asialo-Bovine Submaxillary Mucin
Bovine submaxillary mucin (BSM) was desialylated following the procedure described by O'Boyle et al. 3 Briefly, BSM was heated in 0.1N sulfuric acid solution for 1 h, dialysed against ultrapure water (3 x 2L) then freeze-dried and stored at -18 o C.

Nanoparticle Characterisation
TEM images of glyconanoparticle samples are shown in Figure S1.