Hyperbranched polydendrons: a new nanomaterials platform with tuneable permeation through model gut epithelium† †Electronic supplementary information (ESI) available: Materials, full experimental details and characterisation. See DOI: 10.1039/c4sc02889a Click here for additional data file.

Highly branched vinyl polymers (hyperbranched polydendrons), displaying combinations of dendritic and PEG end groups, have been synthesised using a mixed initiator approach. Nanoprecipitated polydendron particles have exhibited controlled permeation through a gut epithelium model.

-Triple detection Gel Permeation Chromatography (GPC) overlays of A) refractive index detector and B) right angle light scattering detector for G 2 Dendron initiated ATRP of HPMA -target DP n = 50 monomer units. The figures show analysis of both linear dendritic polymer hybrids (dotted lines) and branched polymerisations (solid line). A small shoulder is seen in the RALS signal for the linear-dendritic polymer analogue, possibly due to a small amount of bifunctional monomer within the commercial sample as purchased.

Characterisation
Molecular weights and molecular weight distributions (i.e. dispersity, Ð) were measured using a Malvern Viscotek instrument equipped with a GPCmax VE2001 auto-sampler, two Viscotek T6000 columns (and a guard column), a refractive index (RI) detector VE3580 and a 270 Dual Detector (light scattering and viscometer) with a mobile phase of THF at 35ºC and a flow rate of 1.0 mLmin -1 .
NMR spectra were recorded using a Bruker DPX-400 spectrometer operating at 400 MHz for 1 H NMR and 100 MHz for 13 C, in CDCl 3 , D 2 O or DMSO.
Dynamic light scattering (DLS) measurements were performed using a Malvern Zetasizer Nano ZS instrument (laser wavelength; 630 nm), ran at 25 °C.
Scanning electron microscopy (SEM) images of nanoparticles were obtained using a Hitachi S-4800 FE-SEM. The aqueous nanoparticle samples were dropped on a glass cover slide mounted on an aluminium stub with a carbon tab and left to dry over several hours or overnight. They were Au sputter coated at 20 mA for 2 min prior to imaging.
Fluorescence spectra were obtained on a PerkinElmer Luminescence spectrofluorophotometer LS55. Emission spectra for Nile red containing samples were recorded between 550 nm and 700 nm, exciting at 552 nm. The slit widths for emission and excitation were 5 nm and 10 nm, respectively, with a scan rate of 100 nm/min. Emission spectra for pyrene labelled samples were recorded between 330 nm and 500 nm, exciting at 335 nm. The slit widths for emission and excitation were 5 nm and 5 nm, respectively, with a scan rate of 100 nm/min.

Linear dendritic polymer synthesis with G 2 dendron initiator
In a typical experiment, G 2 dendron initiator,1,(0.648 g, 0.69 mmol)and HPMA, 2, (targeted DP n = 50) (5.0 g, 34.7 mmol) were weighed into a round bottom flask. The flask was equipped with magnetic stirrer bar, sealed and degassed by bubbling with N 2 for 20 minutes and maintained under N 2 at30 °C. Anhydrous methanol was degassed separately and subsequently added to the monomer/initiator mixture via syringe to give a 50 wt% mixture with respect to the monomer. The catalytic system; Cu(I)Cl (0.069 g, 0.69 mmol) and 2,2'bipyridyl (bpy) (0.217 g, 1.39 mmol), were added under a positive nitrogen flow in order to initiate the reaction. The polymerisations were stopped when conversions had reached over 98 % determined by 1 H NMR using the vinyl CH 2 peaks and protons of the polymer backbone. The polymerisation was stopped by diluting with a large excess of tetrahydrofuran (THF), which caused a colour change from dark brown to a bright green colour.

G 2 polydendron synthesis
In a typical experiment, G 2 dendron initiator, 1,(0.648 g, 0.69 mmol)and HPMA, 2, (targeted DP = 50) (5.0 g, 34.7 mmol) were weighed into a round bottom flask. EGDMA, 3, (105 μl, 0.55 mmol) was added and the flask was equipped with magnetic stirrer bar, sealed and degassed by bubbling with N 2 for 20 minutes and maintained under N 2 at30 °C. Anhydrous methanol was degassed separately and subsequently added to the monomer/initiator/brancher mixture via syringe to give a 50 % v/v mixture with respect to the monomer. The catalytic system; Cu(I)Cl (0.069 g, 0.69 mmol) and 2,2'-bipyridyl (bpy) (0.217 g, 1.39 mmol), were added under a positive nitrogen flow in order to initiate the reaction. The polymerisations were stopped when conversions had reached over 98 % determined by 1 H NMR using the vinyl CH 2 peaks and protons of the polymer backbone. The polymerisation was stopped by diluting with a large excess of tetrahydrofuran (THF), which caused a colour change from dark brown to a bright green colour.

Mixed initiator branched polymerisations via ATRP
In a typical reaction, G 2 dendron initiator (0.324 g, 0.35 mmol) and 2K PEG initiator (0.745 g, 0.35 mmol) (for a targeted ratio of G 2 dendron:2000PEG of 50:50 mol%) were weighed into a round bottom flask, followed by HPMA (5.0 g, 34.7 mmol, targeted DP=50). EGDMA (112 μL, 0.59 mmol, 0.85 mol%) was added and the flask was equipped with magnetic stirrer bar, sealed and degassed by bubbling with N 2 for 20 minutes and maintained under N 2 at30 °C. Anhydrous methanol was degassed separately and subsequently added to the monomer/initiator/brancher mixture via syringe to give a 50 % v/v mixture with respect to the monomer. The catalytic system; Cu(I)Cl (0.069 g, 0.69 mmol) and 2,2'-bipyridyl (bpy) (0.217 g, 1.39 mmol), were added under a positive nitrogen flow in order to initiate the reaction. The polymerisations were stopped when conversions had reached over 98 %. The polymerisations were stopped by diluting with a large excess of tetrahydrofuran (THF), which caused a colour change from dark brown to a bright green colour. The catalytic system was removed using Dowex ® Marathon TM MSC (hydrogen form) ion exchange resin beads and basic alumina. The resulting polymer was isolated by precipitation from the minimum amount of THF into cold hexane.

Kinetics experiments
Kinetics experiments were conducted at 30 °C. To determine the kinetic parameters of polymerisations samples (~0.1 mL) were taken at regular intervals and analysed by 1 H NMR and gel permeation chromatography (GPC). Approximately one third of each sample taken was diluted into 0.8 mL of deuterated DMSO (for 1 H NMR analysis) and the other two thirds was diluted into 4mL THF (for GPC analysis). Oxidation of Cu(I) to Cu(II) was observed by a colour change from brown to turquoise in DMSO and from brown to green in THF indicating termination of the reaction. Conversion of monomer to polymer was determined by 1 H NMR as previously discussed. The samples diluted in THF were prepared for GPC analysis by removal of the catalytic system with Dowex ® Marathon TM MSC (hydrogen form) ion exchange resin beads, transferred into a pre-weighed vial, removal of THF from the polymer solution by evaporation to give dry polymer residue of a known weight. The dry polymer residues of known weights were then diluted with HPLC grade THF to give polymer solutions with concentrations around 5.0 mg/mL. These were analysed by triple detection GPC consisting of refractive index (RI), light scattering (LS) and viscometer detectors.

Aqueous nanoparticle formation
Polymers were dissolved in THF at various concentrations. Once fully dissolved, polymer in THF (0.1 or 1 mL at 5 or 10 mg/mL) was added quickly to a vial of water (5 or 10 mL) stirring at ambient temperature. The solvent was allowed to evaporate overnight in a fume cupboard to give a final concentration between 0.01 -2 mg/mL polymer in water. By adjusting the starting concentration and the volume of water used, the size of the corresponding nanoparticles were controlled.

Encapsulation of guest molecules in nanoparticles
Polymers were dissolved in THF at various concentrations. The fluorescent dyes, Nile red and pyrene, were dissolved in THF to give a stock solution (0.1 mg/mL). The stock solution (50 µL, 0.1 mg/mL) was added to an empty vial and the THF was allowed to evaporate to leave 5 µg of dye. The polymer dissolved in THF (1 mL, 5 mg/mL) was added to the dry Nile red or pyrene to give a mixture containing 5 mg polymer, 5 µg dye dissolved in 1 mL of THF. This was then added quickly to a vial of water (5 mL) stirring at ambient temperature.
The solvent was allowed to evaporate overnight in a fume cupboard to give a final concentration of 1 mg/mL polymer and 1 µg/mL Nile red or pyrene (0.1 w/w%) in water.

Characterisation
Cell count and viability was determined using a Countess automated cell counter (Invitrogen).
Absorbance was read using a Tecan Genosis plate reader at 560 nm (Tecan Magellan, Austria).
Luminescence was then measured using a Tecan Genios plate reader (Tecan Magellan, Austria).

Cytotoxicity studies (Nile red loaded hyp-polydendrons)
Caco-2 cells were seeded at a density of 1.0 x 10 4 cells / 100 µL in DMEM supplemented with 15 % FBS into each well of a 96 well plate (Nunclon, Denmark) and incubated at 37 °C and 5 % CO 2 . Cells from 4 separate flasks of biological replicates of each cell type were used (N1-4) to improve statistical power. Media was then aspirated from column 1 and replaced with media containing each hyp-polydendron or aqueous Nile red solution at an equivalent 1 µM Nile red concentration then diluted 1:1 in media across the plate up to column 11. Column 12 served as a negative control and consisted of media and untreated cells.
Following hyp-polydendron addition, the plates were incubated for 24 hours or 120 hours at 37 °C, 5 % CO 2 prior to assessment of cytotoxicity.
MTT assay -Following incubation of treated plates for 24 hours or 120 h, 20 µL of 5 mg/mL MTT reagent was added to each well and incubated for 2 hours. Subsequently, 100 µL MTT lysis buffer (50% N-N-Dimethylformamide in water containing 20% SDS, 2.5% glacial acetic acid and 2.5% hydrochloric acid, pH 4.7) was added to each well to lyse overnight at 37 °C, 5% CO 2 . Following incubation the absorbance of each well was read using a Tecan Genosis plate reader at 560 nm (Tecan Magellan, Austria).
ATP assay -Following incubation of treated plates for 24 hours or 120 hours, cells were equilibrated to room temperature for approximately 30 minutes. All but 20 µL of media was removed from each well and 20 µL CellTiter-Glo® (Promega, UK) reagent was added. All reagents were made fresh and in accordance with the manufacturer's instructions. Plates were put on an orbital shaker for 10 minutes to mix contents and allow for stabilisation of luminescence signal. Luminescence was then measured using a Tecan Genios plate reader (Tecan Magellan, Austria).

Caco-2 permeation studies (Nile red loaded hyp-polydendrons)
Transwells were seeded with 3.5 x 10 4 cells per well and propagated to a monolayer over a 21 day period, during which media in the apical and basolateral wells was changed every other day. Trans-epithelial electrical resistance (TEER) values were monitored until they were >800 Ω. 1 µM of Nile red hyp-polydendron or 1 µM aqueous Nile red was added to the apical chamber of 4 wells and the basolateral chamber of 4 wells to quantify transport in both Apical to Basolateral (A>B) and Basolateral to Apical (B>A) direction and sampled on an hourly basis over a 4 h time period. Apparent permeability coefficient (P app ) was then determined by the amount of compound transported over time using equation (1) below.
Where (dQ/dt) is the amount per time (nmol/sec), A is the surface area of the filter and C 0 is the starting concentration of the donor chamber (1 µM).

Aqueous Nile Red solution for cellular studies
An aqueous Nile Red solution was prepared in dimethyl sulfoxide (DMSO) at 1 mg/mL final concentration and used to spike either complete growth media or transport buffer. The resulting 1 µM final concentration Nile Red solution was subsequently used in cytotoxicity assays or for transcellular permeability assessment respectively. Transport buffer consisted of; Hanks buffered Saline Solution (HBSS), 25 mM4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), and 1 mg/mL Bovine Serum Albumin (BSA), adjusted to pH 7.4.

Routine cell culture/cell maintenance
Caco-2 cells were purchased from American Type Culture Collection (ATCC, USA) and maintained in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 15% filtered sterile foetal bovine serum. Cells were incubated at 37°C and 5% CO 2 and were routinely sub-cultured every 4 days when 90% confluent. Cell count and viability was determined using a Countess automated cell counter (Invitrogen).

Extraction and quantification of Nile Red
100 µl of each collected sample was mixed with 900 µl acetone, vortexed, sonicated for 6 minutes and centrifuged at 13300 rpm for 3 minutes. The supernatant was completely dried in a vacuum centrifuge at 30°C until the dry solid sample was left. This was reconstituted in 150 µl acetonitrile, transferred to a 96-well black walled, flat bottomed plate and measured for fluorescence intensity excitation wavelength 480 nm, emission wavelength 560 nm using a Tecan Genios plate reader (Tecan Magellan, Austria).