Tuneable peptide cross-linked nanogels for enzyme-triggered protein delivery

of the scattering object obtained for data fitted with the Unified power Rg model; R a , R b , R c are the minor equatorial radius, major equatorial radius and polar radius respectively obtained from the triaxial ellipsoid fitting model; R the sphere radius determined by fitting the data with the sphere model

The macro-RAFT agent 2 (2 g, 0.38 mmol) was added in a 15 mL Schlenk tube equipped with a magnetic stirrer and a septum. MAA (0.327 g, 3.8 mmol), TMSPMA (1.88 g, 9.6 mmol) and 11.4 mL of 1,4 dioxane were charged to the Schlenk tube to a final monomer concentration of 1M. AIBN (6.24 mg, 0.038 mmol) and mesitylene (100 μL) were added to the reaction mixture as initiator and reference for calculating monomer conversion respectively. After 3 freeze-pump-thawing cycles, the reaction mixture was transferred to a pre-heated oil bath at 65 °C and stirred overnight. After 16 h, the reaction was quenched by introducing oxygen.
MAA monomer conversion was calculated by 1    Fmoc-L-Lys-OH (3.69 g, 10 mmol), Na2CO3 (3.18 g, 30 mmol), imidazole-1-sulfonyl azide hydrochloride (2.51 g, 12 mmol) and CuSO4·5H2O (25 mg, 0.1 mmol) were dissolved in methanol (125 mL). A few drops of water were added to aid dissolution. The resulting reaction S10 mixture was stirred overnight at r.t. and then the solvent removed in vacuo. The crude material was partitioned between water (50 mL) and EtOAc (50 mL) and acidified to pH 4 by dropwise addition of 37 % HCl. The organic phase was washed with water (50 mL) and brine (50 mL) and dried over MgSO4. The solvent was removed in vacuo to obtain a pale-yellow oil with a yield of 75 %. Spectroscopic data matched those previously reported in the literature. The peptide was purified using reverse phase HPLC under acidic conditions using a acetonitrile/water gradient at a flow rate of 15 mL/min, using a Shimadzu prominence system equipped with UV detector at 220 and 280 nm and a Phenomenex C18 Gemini NX column (5 μm pore size, 110 Å particle size, 150 x 21.2 mm). Table S5 reports details of the HPLC method S11 adopted for the purification of the peptide. The retention time of the MMP-7-cleavable peptide was 9.5 min.

LC-MS analysis
Following degradation, the solutions containing the peptide were diluted to 250 μL with methanol and analysed by liquid chromatography-mass spectrometry (LC-MS) on an Agilent S12 Phenomenex C18 Gemini NX column (5 μm pore size and 100 Å particle size). Mass-to-charge ratios (m/z) of treated peptides were compared with expected fragments and those of initial peptides.

Optimised protocol for peptide cross-linked nanogel preparation
The desired block copolymer (1.76 · 10 -2 μmol) was dissolved at r.t. in 125 μL of PBS in a 1.5 mL Eppendorf tube and the resulting solution was heated up to a temperature T ≥ LCST(Polymer) + 10°C, and mixed at 350 rpm for 10 minutes by using an AccuTherm TM microtube shaking incubator with 40 x 1.5 mL block to induce nanogel self-assembly. Following self-assembly, a 15 mg/mL solution of the peptide cross-linker in PBS (3.52 · 10 -1 μmol) was added to the pre-

Characterisation of nanogels by DLS
A Zetasizer Nano ZS (Malvern) was used for DLS measurements. For all the DLS measurements, the scattering angle was fixed at 173° and disposable micro cuvettes were used.

Study of temperature-triggered self-assembly behaviour
A 1 mg/mL solution of each block copolymer was prepared in PBS prior to analysis. Each measurement, consisting of 15 runs, was repeated three times at the desired temperature.
Averaging the results from the three repeated measurements, an average size distribution and an average size value (number mean) was obtained. The temperature-responsive behaviour of the triblock copolymers and their temperature of cloud point (Tcp) was determined by DLS. Solutions of the final triblock copolymers (1mg/mL) in PBS were subjected to a 1 °C temperature ramp (from 25 to 60 °C and from 60 to 25 °C) with 5 min equilibration time between each temperature-controlled measurement. The reported derived count-rate (kcps) is proportional to the total intensity of the scattered light and therefore to the selfassembly of the copolymers. The inflection points of each curve represent the Tcp of the corresponding copolymer.

Cargo labelling
The protein (BSA) was dissolved in 50 mM HEPES buffer, pH 8.5. The NHS-functionalized dye of (NHS-Oregon Green, 1.5 μmol) was dissolved in DMSO and added to the protein solution prior to stirring at room temperature overnight. The labelled-protein was then purified and buffer exchanged by using PD-10 minitrap desalting columns containing Sephadex G-25 resin and PBS as running buffeer. The labelled protein was subsequently concentrated to 10 mg/mL with Amicon Ultra 0.5 mL centrifugal filter units (MWCO: 10kDa).

Cryo-TEM sample preparation and imaging
Holey Carbon on Cu-200 mesh EM Grids were used for sample deposition and were glowdischarged on a Gatan SOLARIS plasma cleaner for 15 seconds with O2/H2 1:1. Block copolymer nanogel solutions with a concentration of 3-6 mg/mL in PBS were incubated in a block heater at 50 °C for 15 minutes. 4 μL of the pre-heated cryo-specimen solution was loaded onto the carbon side of the plasma-treated grid, which was incubated for 30 sec at 60 % humidity and 50 °C temperature using a Leica EM GP plunge-freezer. Following sample deposition, the grid was blotted twice for 1 sec using filter paper and immediately vitrified.
The grids were then stored under liquid nitrogen until imaging. For imaging, grids were inserted in a Gatan 914 cryo-holder and images were collected using a JEOL 2100 Plus electron microscope under low dose conditions using Minimum Dose System software settings.
Images were acquired using a Gatan Orius SC 1000 camera over 5 sec of exposure time.
Magnification of 15, 25 and 30 k and a defocus of -5 and -10 μm were used. From the cryo-TEM pictures of each block copolymer, analysis of the size distribution was performed by manually measuring the size of about 100 nanogels using the line drawing tool in Fiji-Image J to obtain the diameters.

SANS sample preparation and measurements
Deuterated PBS was prepared by dissolving the appropriate amount of Gibco PBS tablets (manufacturer's composition: 10 mM sodium phosphates, 2.68 mM potassium chloride, 140 mM sodium chloride) (ThermoFisher Scientific) in D2O. Solutions of copolymers were S16 prepared in deuterated PBS at a concentration of 6 mg/mL immediately prior to measurement to avoid any labile hydrogen/deuterium exchange. 5c1-based cross-linked nanogels were formed by cross-linking the corresponding self-assembled nanostructure with the MMP-7-specific peptide using the methodology reported above. It must be noted that in this case, both polymer and reagent solutions for the preparation of cross-linked nanogels were prepared in deuterated PBS to avoid incoherent scattering from H2O and increase scattering contrast. Following cross-linking, the nanogels were purified through 100 kDa

SANS data fitting
SANS data reduction was performed with MantidPlot and SasView v4.1. was used to fit the experimental data. The analysed block copolymers/nanogels were shown to have temperature-and cross-linking-dependent structural transition. Therefore, the analysis of their scattering behaviour required the use of several models of fitting to best represent the different temperature and solvent conditions. The fitting models were selected on the basis of the system information previously collected by DLS and cryo-TEM. All the fitted data were plotted using Prism 6. For all the graphs, since axes are logarithmic, only values greater than S17 zero can be plotted using Prism 6. For this reason, the down error bar that would go to a negative Y value are missing from some data points.
Some of the SANS data was fitted using a power law model. This is a shape-independent function where the scattering intensity I(q) is calculated as simple power law with a flat background using the following: Other SANS data was fitted using a Guinier-Porod model. 8,9 This is an empirical model that can be used to determine the size and dimensionality of a generalised Guinier/power law object including both spherical and non-spherical objects such as rods, platelets and intermediate shapes as long as the q-range collected is sufficient to cover the Guinier (overall shape of the scatterer) and Porod (internal/surface configuration of the scatterer) regimes. 8 The scattering intensity I(q) results from the contributions of the two regimes as shown here: q is the scattering vector, is the radius of gyration, s is the dimension variable, G and D are the Guinier and Porod scale factors respectively, and m is the Porod exponent. The Guinier form is used for ≤ 1 and the Porod form is used for ≥ 1 where 1 is the value at which S18 the slopes of the Guinier and Porod terms are connected. 1 is calculated using the following equation: For 3D globular objects such as spheres s = 0. For a 2D symmetry such as rods s = 1 and for 1D symmetries such as lamellae and platelets s = 2. Therefore, a dimensionality parameter (3 -s) is defined and is 3 for spheres, 2 for rods and 1 for lamellae and platelets. Regarding the Porod exponent, 1 ≤ m ≤ 3 indicates mass fractal (q -5/3 for swollen chains, q -2 for Gaussian chains and q -3 for clustered networks) while 3 ≤ m ≤ 4 characterises surface fractals such as spheres and cylinders (q -3 for rough surfaces and q -4 for smooth surfaces). The Guinier-Porod model is completely empirical and provides a shortcut for collecting useful information from the scattering of non-spherical objects and systems with complex phase behaviour rather than having to use multiple models for each phase structures. 8 Some SANS data was fitted using a sphere model. 10 The 1D scattering intensity I(q) is calculated from the following equation: were scale is a volume fraction, V is the volume of the scattering object, r is the sphere radius and background is the background level. Δ is the difference between scattering length density of the scattering object (sld) and the scattering length density of the solvent (sld_solvent).
Another model that was used for SANS data fitting was a Unified Power Rg model. 9,11 This is a shape-independent model where an empirical multiple level unified Exponential/Power law fit method is employed. Similar to the Guinier-Porod model, this method, also known as The radius of gyration for this system is calculated as 2 = ( ) 2 /5. The contrast is defined as SLD(ellipsoid) -SLD(solvent).

Enzyme-triggered nanogel degradation for FCS
For the analysis of nanogel degradation by FCS, nanogels were first cross-linked and then labelled with Cy5-azide.

MMP-7-triggered cargo release for FCS study
The release of OG-BSA from the cross-linked nanogels was tested by FCS upon addition of MMP-7 and compared to diffusion-based release. Prior to enzyme degradation, the solution containing the loaded nanogels was buffer exchanged to enzyme buffer using Amicon Ultra-0.5 mL centrifugal filter units with a MWCO of 100 kDa (5 times x 5 min, 5500 g This experiment was performed with three independent particle samples (N = 3). and is the structural parameter defined as the ratio of height to width of the confocal volume (fixed to 5). CPP is counts per particle in kHz. NR(0) is the number of cargo per particle averaged for each independent particle sample across all time points that show no release.

Fluorescence correlation spectroscopy (FCS)
NR(t) is the number of cargo per particle at timepoint t. In the few cases when NR(t) was higher than NR(0) due to a large variability in measurements, NR(t)/NR(0) was set to 1.