*2.1. Synthesis and Characterisation*

Charged CyD polymers were synthesised in water, starting from the polypeptide N-butyl-polyglutamate (PGA), ArgOCH<sup>3</sup> and CyD 3-amino derivatives, using 4-(4,6- Dimethoxy-1,3,5-triazin 2-yl)-4-methylmorpholinium chloride (DMTMM) as the condensing agent, as reported elsewhere [26]. We found that this method gave high conjugation yield using a green synthetic route. This procedure is appropriate for modulating the number of CyD cavities and charged groups of Arg in the PGA backbond [27].

We synthesised various polymers with different amounts of Arg and CyD units to explore the effect on polymer properties.

All the new polymers were characterised by NMR (Figures S1–S11). In Figure 2 the NMR spectra of PGAβCyDArg1 and PGAβCyDArg4 are reported. <sup>1</sup>H NMR spectra of all the derivatives show common patterns; the protons of CyD resonate at 5 ppm (H-1), and 4.0-3.4 ppm (H-3,-6,-5,-4,-2). Protons of arginine and the glutamic acid side chain of PGA resonate at 3.3 ppm and 2.5-1.8 ppm region. Butyl protons of PGA are also evident

between 1.5 ppm and 1.0 ppm. We determined the number of CyD units linked to the PGA backbone for each polymer derivative by calculating the integral ratios of the signal of Hs-1 of CyD, the signal of the ethylene chain protons of PGA or the N-buthyl chain protons. Moreover, the integral ratio of signal due to the γ-CH<sup>2</sup> of Arg moieties at 3.3 ppm and the signals of PGA ethylenic protons or the N-buthyl protons was used to value the number of Arg moieties grafted to the polymer. The results obtained from NMR for each bioconjugate are reported in Table 1.

**Figure 2.** <sup>1</sup>H NMR spectra of PGAβ-CyDArg 1 (top), PGAβCyDArg4 (bottom) (D2O, 500 MHz).


**Table 1.** Features of PGA-CyD-Arg derivatives.

The <sup>13</sup>C NMR spectra of the derivatives show signals due to guanidium carbons at about 160 ppm and signals around 174 ppm due to the carboxyl group of PGA and arginine methyl ester, in addition to the signals of CyD units in the aliphatic region.

CyD polymers were also characterised by dynamic light scattering (DLS) and Zeta potential values were also measured (Table 1). The hydrodynamic diameters increase with the number of cavities linked to the PGA backbone. The Z potential values increased when the number of Arg units increased from negative values (−58 mV for PGA alone) to positive values, in keeping with the progressive increase in positive charges due to the guanidinium groups.

Spectrometric measurements were also carried out further to characterise the structural features of the new polymers. The MALDI spectra recorded in linear mode (Figure S12) mainly contain a wideband; the *m*/*z* values of the highest peaks match to those obtained by the NMR studies (Table 1), within the experimental errors, thus confirming the calculated molecular weight (Mw) of the new CyD polymers.

As for PGAβCyDArg1, the MALDI spectrum is resolved into several components (Figure 3). The average difference between two successive relative peaks is 1280 ± 20. This value suggests that the repeat unit contains both the CyD (MW 1135) and glutamic acid (MW 147) moieties, as expected.

**Figure 3.** MALDI-TOF MS spectrum of PGAβCyDArg1. The raw spectrum (gray line) was properly smoothed (black line) in order to obtain the *m*/*z* values of all the relative peaks.
