Dendrimer end-terminal motif-dependent evasion of human complement and complement activation through IgM hitchhiking

Complement is an enzymatic humoral pattern-recognition defence system of the body. Non-specific deposition of blood biomolecules on nanomedicines triggers complement activation through the alternative pathway, but complement-triggering mechanisms of nanomaterials with dimensions comparable to or smaller than many globular blood proteins are unknown. Here we study this using a library of <6 nm poly(amido amine) dendrimers bearing different end-terminal functional groups. Dendrimers are not sensed by C1q and mannan-binding lectin, and hence do not trigger complement activation through these pattern-recognition molecules. While, pyrrolidone- and carboxylic acid-terminated dendrimers fully evade complement response, and independent of factor H modulation, binding of amine-terminated dendrimers to a subset of natural IgM glycoforms triggers complement activation through lectin pathway-IgM axis. These findings contribute to mechanistic understanding of complement surveillance of dendrimeric materials, and provide opportunities for dendrimer-driven engineering of complement-safe nanomedicines and medical devices.


Supplementary methods
General dendrimer synthesis and characterisation procedures. Unless otherwise stated, all starting materials were obtained from commercial suppliers and used as received.
Solvents were HPLC grade and used as received. Methanol was dried over molecular sieves. Thin-layer chromatography was carried out using silica plates on aluminum (Merck, Silica 60-F254, 0.2 mm layer thickness) with detection under UV light and, if required treatment with 1 % solution of ninhydrin in ethanol. UV/Vis spectra were recorded on a Perkin Elmer apparatus using 1 cm quartz cuvettes. 1 H-NMR and 13 C-NMR spectra were performed on a 300 MHz NMR (Bruker) apparatus (300 MHz 1 H-NMR, 75 MHz 13 C-NMR) or on a 500 MHz NMR (Bruker) apparatus . Chemical shifts are reported in parts per million (ppm) downfield of TMS (tetramethylsilane) using the resonance of the deuterated solvent as internal standard. Proton couplings are described as s (singlet), d (doublet), t (triplet), q (quartet), br (broad) and m (multiplet), coupling constants are reported in Hertz. NMR data was analysed using Mnova 10.0 software (Mestrelab).
A Jasco V-650 spectrophotometer (Jasco, Japan) and a Jasco Model FP-6200 spectrofluorometer (Jasco, Japan) were used to collect absorption and emission spectra, respectively. Spectra were measured using 1 cm path quartz cuvettes and Milli-Q water as solvent. The final concentration of all PAMAM dendrimers was 0.4 mM.
A size-exclusion chromatography system (HPLC: Dionex Ultimate ® 3000, Column: TSKgel® GMPWXL HPLC Column) coupled with a multiangle light scattering (miniDAWN TREOS-AQUEOUS) and refractive index (RI Detector Refractomax 521) detectors were used to acquire the RI and LS signals. The number and weight average molecular mass values were obtained using ASTRA software. The SEC-MALS-RI system and ASTRA software were purchased from Wyatt technology Europe (Dernbach, Germany), and the column was from Sigma Aldrich (Denmark). Citrate buffer (pH 2.9) was used as solvent to dissolve amine-terminated dendrimers, while pyrrolidone-and carboxy-Tris-terminated dendrimers were dissolved in phosphate buffer (pH 7.4). A 50 μL volume of sample solution prefiltered with 0.2 μm Acrodisc ® Syringe Filter (Supor ® Membrane) was injected into the system with a flow rate of 0.5 mLmin -1 .
MALDI was measured on a Bruker SolariX XR instrument in positive mode with a SA matrix. For HPLC-MS analysis a Dionex Ultimate 300 PLC connected to an ESI-MS (MSQ Plus Mass Spectrometer, Dionex) was used.
IR spectra were recorded on an FT-IR instrument using the attenuated total reflectance (ATR) sampling technique, and the measurements were carried out on a thin film of each sample obtained by evaporation from a solution of deuterated chloroform or methanol.
TLC was used to monitor full conversion. The excess methyl acrylate and methanol were removed using a rotary evaporator. The final product was gained as colourless oil with a yield of 97% (71.55 g, 0.167 mol).
G0: DAB-PAMAM-(NH2)4 [2]. DAB-PAMAM-Core (71.55 g, 0.167 mol) was dissolved in methanol (850 mL). Ethylene diamine (EDA) (524 g, 8.7 mol, 13 eq. per surface group) was dissolved in methanol (110 mL) and cooled to 0 °C, using an ice bath. The dendrimer solution was added to this solution dropwise over 1 h under nitrogen atmosphere. The reaction was stirred for 4 days at room temperature and kept under vacuum. After that, azeotropic distillation with a mixture of methanol/toluene 1/9 was performed until all EDA was removed in vacuum. The excess toluene was removed by azeotropic distillation with methanol. The final compound was a colourless oil (91.5 g, 0.167 mol).
General Synthesis of DAB-PAMAM dendrimer of generation (G) 0.5 to G5. The PAMAM dendrimer synthesis consists of a repeating series of two reactions, first a branching of the amine unit to two branches using a Michael addition of two methyl acrylate molecules per amine, this leads to the half-generation dendrimers G0.5; G1.5; G2.5; G3.5 and G4.5. This step is always followed by an "activation" step where EDA forms an amide, substituting the outer methyl esters, which yields to new primary amines as the outer dendrimer layer (full generation dendrimers G1; G2; G3; G4 and G5).
General synthesis of half generation dendrimers. Methyl acrylate (3 eq. per dendrimer amine surface group) was dissolved in methanol (typical same volume as the methyl acrylate) and cooled with an ice bath to 0 °C. Under nitrogen atmosphere, DAB-PAMAM-Gn (1 eq.) dissolved in methanol (10 w/w %) was added dropwise over 1 h. The reaction was stirred for two days at room temperature. Full conversion was checked by KAISER test (1 % ninhydrin in ethanol) for remaining amines. The solvent and excess methyl acrylate was removed on a rotary evaporator, followed by high vacuum. This gave the halfgeneration dendrimer in form of a slight yellowish oil.
General synthesis of full generation dendrimers. DAB-PAMAM-half-generation (1 eq.) was dissolved in methanol (10 w/w %). EDA (25 eq. per ester surface group) was dissolved in methanol (typical 25% of the EDA volume) and cooled to 0 °C, using an ice bath. The dendrimer solution was added to this solution dropwise over 1 h under nitrogen atmosphere. The reaction was stirred for 4 days at room temperature. Methanol and EDA was removed under vacuum. After that, azeotropic distillation with a mixture of methanol/toluene 1/9 was performed until all EDA was removed under vacuum. The excess toluene was removed by azeotropic distillation with methanol. Dendrimers of generations G2-5 were further purified by dialysis against first water and then methanol.
The full generation dendrimer was typically gained in form of white foam.

Surface functionalization of PAMAM dendrimers
General procedure for 1-(4-carbomethoxypyrrolidone) terminated PAMAM dendrimers. A 10 w/w% solution of dendrimer was added slowly to a 70 w/w% solution of dimethyl itaconate in methanol (1.05 equivalents per amine surface group), while cooling to 0 °C. Upon completion of the addition, the reaction mixture was left in the cooling bath to slowly heat to ambient temperature. The reaction was stirred until the KAISER-test (1% ninhydrin in ethanol) was negative, usually 2-4 days depending on the generation. The functionalized dendrimer was isolated by removing methanol, dissolving the dendrimer in a minimum amount of water, and multiple extraction times with diethyl ether until any remaining dimethyl itaconate had been removed. Higher dendrimer generations (G2-G5) were dialysed against first methanol then water. The aqueous phase was lyophilized to give the pure and quantitative covered 1-(4-carbomethoxypyrrolidone) terminated PAMAM dendrimers as white solids.
Dimethyl itaconate [13]. Itaconic acid (30 g, 0.23 mol) was dissolved in methanol (400 mL) and acetyl chloride (89 mL, 1.2 mol, 5 eq.) was added carefully, while cooling the reaction mixture with a water/ice bath. After complete addition, the reaction was heated to reflux for 1 h and then stirred for 72 h at room temperature. Volatiles were removed in vacuum and the solid material was dissolved in diethyl ether (300 mL) and extracted three times with slightly basic water (300 mL, pH approximately 10). The ether phase was dried over MgSO4 and evaporated. After 24 h vacuum the compound was gained in form of colorless crystals (18.1 g, 11.4 mmol, 50 %).
General procedure for carboxy-Tris terminated PAMAM dendrimers. The PAMAM dendrimer was dissolved in methanol (10 wt%) and reacted with 2,5-dioxopyrrolidin-1-yl methyl succinate ("NHS activated methylsuccinic acid", 1.05 eq. per dendrimer-NH2 surface group) which was directly added to the solution. The reaction mixture was stirred for two days. To this solution (10 wt%) of the methyl succinate functionalized dendrimer in dry methanol was added 2-amino-2-hydroxymethyl-propane-1,3-diol (1.20 eq. per dendrimer methyl ester surface group). Anhydrous potassium carbonate was added in catalytic amounts (10 wt%) to promote the reaction. After two days water was added to the reaction mixture to hydrolyze the remaining succinic methyl esters. The potassium carbonate, NHS and the excess 2-amino-2-hydroxymethyl-propane-1,3-diol were removed by dialysis. After removal of the solvent the dendrimer was gained as a white solid. It needs to be noted that the introduction of TRIS groups is purely statistical.
Distribution of Tris groups on the dendrimer surface is statistical. The number of attached Tris groups was calculated by comparing the integral of the Tris signals with the integral of the PAMAM dendrimers. It was calculated to be 6 Tris groups for the G4-Dendrimer and 12 Tris groups for the G5-Dendrimer.
2,5-Dioxopyrrolidin-1-yl methyl succinate [20]. Freshly distilled methyl 4-chloro-4oxobutanoate (5g, 33.2 mmol) was dissolved in THF (50 mL) and cooled to 0 °C with a water/ice bath. Triethyl amine (4.85 mL, 1.08 eq) was slowly added, followed by the addition of N-hydroxysuccinimide (3.82 g, 33.5 mmol, 1.01 eq.). The reaction mixture was slowly allowed to warm to ambient temperature and stirred overnight. The triethyl ammonia salt was removed by filtration followed by removal of volatiles in vacuum. The final product was gained after recrystallization from 2-propanol in form of a white crystalline solid (4.8 g, 21 mmol, 63 %).
Covalent attachment of phthalocyanine (Pc) to G4 pyrrolidonated dendrimers. Pc (173 mg, 0.225 mmol) was dissolved in dichloromethane (15 mL). A solution of Nhydroxysuccinimide (52 mg, 0.45 mmol, 2 eq.) in anhydrous DMSO (30 mL) was added to the Pc solution followed by the addition of N,N'-dicyclohexylcarbodiimide (65.8 mg, 0.315 mmol) to the reaction mixture. The reaction was stirred overnight at room temperature and under nitrogen and the formed 1,3-dicyclohexylurea side product was filtered of the reaction mixture followed by a removal of the solvent under reduced pressure. The product was then dissolved in DMSO. This solution was added slowly to an ice bath cooled solution of G4-PAMAM dendrimer (1.60 g, 0.1125 µmol à 2Pc per dendrimer) in dry methanol (20 mL). The reaction was stirred 2 days, followed by a removal of insoluble side products by filtration . The dendrimer-Pc solution was then directly used for dendrimer surface functionalization without further purification.
The PAMAM dendrimer solution from the previous reaction (Pc coupling) was directly taken and added drop wise to an ice bath cooled solution of dimethyl itaconate (1.68 g, 10.6 mmol, 1.47 eq. per dendrimer surface group) dissolved in methanol (4 mL).
The solution was cooled with an ice bath during the addition. The reaction was stirred for four days. The excess of dimethyl itaconate was removed by dialysis: 1. DMSO overnight (solution gets very blue à removed Pc), 2. Methanol for 2 days changing to fresh methanol every 24 hrs, 3. Water for 2 days changing to fresh water every 24 hrs, 4. The content of the dialysis bag was filtered through a 200 nm Millipore filter to remove any remaining particles and freeze-dried.
G4 Carboxy58-Tris6 dendrimers with trapped phthalocyanine (Pc). The SuccTRIS G4-PAMAM dendrimer (259 mg, 9.5 µmol) was dissolved in methanol (25 mL) and the Pc (39 mg, 47 µmol, 5 eq. per dendrimer) dissolved in dichloromethane (7 mL) was added. After 20 min incubation time, a small amount of water (5 mL) was added and the mixture was stirred for another 15 minutes. The solvent was removed using a rotary evaporator. The dark blue compound was then taken up in water, filtered and freeze-dried.