Stimuli-responsive membrane activity of cyclic-peptide–polymer conjugates

Cyclic peptide nanotubes were coupled to poly(oxazoline)s using a cleavable connection. Upon stimuli responsive detachment of the polymer an on-demand membrane activity could be achieved.

1 H-NMR spectra were measured using a Bruker DPX-300 or DPX-400 NMR spectrometer which operated at 300.13 and 400.05 MHz, respectively. The residual solvent peaks were used as internal references.
For SEC measurements of polymers in chloroform, an Agilent 390-LC MDS instrument with differential refractive index (DRI), viscometry (VS), dual angle light scatter (LS) and two wavelength UV detectors was used. The system was equipped with 2 x PLgel Mixed D columns (300 x 7.5 mm) and a PLgel 5 µm guard column. The eluent was CHCl 3 with 2 % TEA (triethylamine) additive. Samples were run at 1 mL min -1 at 30 °C. Poly(methyl meth-acrylate), and polystyrene standards (Agilent Easy Vials) were used for calibration.
Cyclic peptide conjugates were analyzed on a Polymer Laboratories PL-GPC 50 Plus system using a PolarGel-M guard column (7.5 × 50 mm) followed by two PolarGel-M columns (7.5 × 300 mm). DMF (0.1% LiBr) was used as eluent at 1.0 mL min −1 at 50 °C. Commercial narrow linear poly(methyl methacrylate) standards in range of 2.0 × 10 2 −1.0 × 10 6 g mol −1 were used to calibrate the DMF SEC system. Analyte samples were filtered through a nylon membrane with 0.22 μm pore size before injection. Respectively, experimental molar mass (M n , SEC) and dispersity (Đ) values of synthesized polymers were determined by conventional calibration using Agilent GPC/SEC software.
Electrospray Ionisation (ESI) spectra were obtained using a Bruker MicroToF and the results analysed using Bruker Data Analysis. Samples were dissolved in methanol at a concentration of 1 µg mL -1 .
The fluorescent intensity was monitored using Agilent Technologies Cary Eclipse Fluorescence Spectrophotometer. The solutions of vesicles were introduced in polystyrene cuvettes for the measurements.

Static light scattering
Light scattering measurements were obtained using an ALV-CGS3 system operating with a vertically polarized laser with wavelength λ = 632 nm. The measurements were taken at 20 °C, over a range of scattering wave vectors (q = 4πn sin(θ/2)/λ, with θ the angle of observation and n the refractive index of the solvent). The measurements determined the relative excess scattering (I) which is defined as, where I solution , I solvent and I toluene are the scattering intensities of the solution, solvent and reference (toluene) respectively, and the Rayleigh ratio of toluene ( = 1.35 x 10 -5 cm -1 for λ = 632.8 nm). I is then expressed in 1/Å and is related to the apparent weight-average The optical constant, K, is defined for light scattering by eq. 2, where N a is Avogadro number and dn/dC is the incremental refractive index, n s is the refractive index of toluene and n of the solvent. (n water = 1.333, n toluene = 1.496).
At a given concentration the Rayleigh ratio, R θ , is related to the apparent molecular weight of the sample, given by eq. 3. It is only at infinite dilutions, where the interactions between scattering particles are negligible, that the apparent molecular weight is equal to the true molecular weight. 1 Multiple concentrations were measured and a plot of linear regression used to determine the apparent molecular weight at a concentration of 0 mg mL -1 .
where d is the density of the solution. ρ solute is the scattering length density for the polymer and has been computed according to its chemical structure.
The obtained reduced data was analyzed with the open access software SASfit. 3 For the conjugates 25, 26 and 27 a form factor of a diblock copolymer micelle with rod-like core was applied. 4 The best fit for the conjugate 24 was obtained using a form factor of a micelle with a flexible cylindrical core. 4 Form factor for a micelle with a rod-like core The best model used to fit the SANS data for the conjugates 25, 26 and 27 in D 2 O was that of a hairy rod-like micelle.  Table S1 in the following. 9.45 × 10 -7 1.10 × 10 -7 1.10 × 10 -7 ρ corona d (cm -1 ) 9.45 × 10 -7 1.10 × 10 -7 1.10 × 10 -7 ρ solvent d (cm -1 ) 6.38 × 10 -6 6.38 × 10 -6 6.38 × 10 - Form factor for a micelle with a flexible cylindrical core From equation 5, a flexible cylindrical core was described using Kholodenkos approach to reproduce the rigid rod limit and the random-coil limit. Defining (L: contour length, l: and and The following values were estimated from the scattering length densities:  solv = 6.38 10 -6 Å -2 ,  s = 9.45 10 -7 Å -2 ,  c = 9.45 10 -7 Å -2 The fit ( Figure S25) was performed with R, Rg, N agg , l and N as adjustable parameter. The values afforded by the fit are gathered in Table S2 in the following.

Hemolysis assay
Defibrinated donor sheep blood was purchased from Thermo Fisher and red blood cells (RBC) were purified as follows. 2 mL of blood was distributed over Eppendorf tubes and centrifuged at 4500 rpm for 1 min. The supernatant was removed and the remaining RBCs were diluted with 1 mL of sterile PBS (pH 7.4). After mixing the mixture was centrifuged again and the supernatant was removed. The process was repeated until the supernatant remained colorless.
RBC were diluted 1:150 in PBS. In Eppendorf tubes, 20 µL of compound solution (at a 20-fold of the final concentration) was mixed with 380 µL of purified RBC solution. The mixture was incubated for 1 h at 37°C and the RBC were subsequently removed by centrifugation as described above. In the case of DTT addition, a final concentration of 30 mM was added. The absorbance of the remaining solution was measured at 414 nm to determine hemolysis levels.
PBS was used as negative control and a 1% solution of triton X-100 was used to lyse all RBCs.

Cell Culture
CaCo2 human colorectal adenocarcinoma cells were grown in a 50:50 mixture of Ham's F12 and DMEM medium supplemented with 10% of fetal calf serum, 1% of 2 mM glutamine and 1% penicillin/streptomycin. They were grown as adherent monolayers at 310 K in a 5% CO2 humidified atmosphere and passaged at approximately at 70-80% confluence.

In vitro growth inhibition assays
The antiproliferative activity of the cyclic peptides carrying or not different polymeric arms was determined in CaCo2 colorectal cancer cells. Briefly, 96-well plates were used to seed 10000 cells per well. The plates were left to pre-incubate with drug-free medium at 310 K for 24 h before adding different concentrations of the compounds to be tested (1 mg ml -1 -10 ng ml -1 ). A drug exposure period of 72 h was allowed. The SRB assay was used to determine cell viability. 6 The experiment was performed as duplicates of triplicates in two independent sets of experiments and their standard deviations were calculated.

Conjugation of cyclic peptide and NHS activated polymers
For polymer conjugation, cyclic peptide (21, 52 mg, 0.039 mmol) was dissolved in DMF (1 mL) and NMM (11 mg, 0.11 mmol, 2.5 eq.) was added. The solution was stirred for 30 min at room temperature and NHS functionalized polymer (0.099 mmol. 2.5 eq.) was added to the mixture. After 3 d the reaction mixture was diluted to 25 mL using water and the conjugate was isolated using centrifuge filter tubes (Amicon, Ultracel -10K). After freeze drying the * DP could not be obtained from 1 H-NMR due to overlap of end group signals. Values were adopted from precursor polymers.