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Polydepsipeptide Block Stabilized Polyplexes For Efficient Transfection of Primary Human Cells Weiwei Wang, Toufik Naolou, Nan Ma, Zijun Deng, Xun Xu, Ulrich Mansfeld, Christian Wischke, Manfred Gossen, Axel Thomas Neffe, and Andreas Lendlein Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01034 • Publication Date (Web): 27 Sep 2017 Downloaded from http://pubs.acs.org on September 28, 2017
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Polydepsipeptide Block Stabilized Polyplexes For Efficient Transfection of Primary Human Cells
Weiwei Wang a, 1, Toufik Naolou a, 1, Nan Ma a, b, Zijun Deng a, b, Xun Xu a, b, Ulrich Mansfeld a, Christian Wischke a, Manfred Gossen a, Axel T. Neffe a,c and Andreas Lendlein a, b, c,*
a
Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies,
Helmholtz-Zentrum Geesthacht, Teltow, Germany b
Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
c
Institute of Chemistry, University of Potsdam, Potsdam, Germany
*Correspondence to: Andreas Lendlein Institute of Biomaterial Science, Helmholtz-Zentrum Geesthacht, Kantstr. 55, 14513, Teltow, Germany Email:
[email protected] Phone: +49 (0)3328 352-450 Fax: +49 (0)3328 352-452 1
These authors contributed equally to this work.
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1
Abstract
Rational design of polyplex gene carrier aims to balance maximal effectiveness of nucleic acids transfection into cells with minimal adverse effects. Depsipeptide blocks with an Mn ~ 5 kDa exhibiting strong physical interactions were conjugated with PEI moieties (2.5 or 10 kDa) to diand triblock copolymers. Upon nanoparticle formation and complexation with DNA, the resulting polyplexes (sizes typically 60-150 nm) showed remarkable stability compared to PEIonly or lipoplex, and facilitated efficient gene delivery. The intracellular trafficking was visualized by observing fluorescence-labelled pDNA and highlighted the effective cytoplasmic uptake of polyplexes and release of DNA to the perinuclear space. Specifically, a triblock copolymer with a middle depsipeptide block and two terminal swallowtail structure of PEIs (10 kDa) mediated the highest levels of transgenic VEGF secretion in mesenchymal stem cells with low cytotoxicity. These nanocarriers form the basis for a delivery platform-technology especially for gene transfer to primary human cells. Keywords: polyethylenimine, polydepsipeptide, gene delivery, mesenchymal stem cells, molecular interactions
2
Introduction
Gene transfer technology is one of the principle tools to predictively modify cellular gene expression for basic research applications, in biotechnology, as well as for gene therapy. A principal distinction can be made between viral and non-viral gene delivery 1. Despite its efficacy in delivering genetic payloads especially into various primary cells, the use of recombinant viral vectors faces challenges with respect to immunogenicity, mutagenicity, cytotoxicity, limited DNA packaging capacity, and difficulties of vector production
2-4
. Thus,
non-viral approaches to mediate the uptake of nucleic acids like plasmid DNA (pDNA), siRNA, miRNA, and mRNA are preferred for many, especially biomedical applications. Polymer particles are, besides other approaches 5, of extensive interest for non-viral nucleic acid delivery as they may interact with nucleic acids forming carriers systems, protect them from extra- and intracellular degradation, and mediate their cellular entry. For this task, a proper chemical design of the carrier is essential to balance desired features like charge compensation,
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condensation and colloidal properties vs. e.g. toxicity, and to eventually enable transfection. Polycationic polyethylenimine (PEI) has been extensively studied for this purpose
6
due to the
high ability for DNA/RNA complexation, the slightly positive zeta potential of the polyplexes to induce uptake by endo-, pino-, or phagocytosis, the cargo protection and promoted endosomal escape
7, 8
. However, toxicity associated with disruption of cell membranes by electrostatic
interactions remains a relevant issue particularly for branched polyethylenimine 7, 9-12. Since low molecular weight PEI is less efficient as a gene transfection vector
13
, chemical and physical
association of polycations by crosslinking or self-assembly employing linear PEI, low molecular weight variants, or alternative degradable polycations are strategies to increase the transfection efficiency of PEI while decreasing or eliminating its cytotoxicity. Examples include grafting hydrophilic chains, e.g. poly(ethylene glycol) (PEG) or gelatin onto PEI chains 14, 15, crosslinking low molecular weight PEI using acid-labile linkages to form degradable high molecular weight polycations
16
, attaching targeting peptides and hydrophobic groups
17, 18
, or the formation of
amphiphilic block copolymers comprising degradable hydrophobic polyester chains 19-24. The degradable and hydrophobic segments in the core of nanoparticles in this approach have to fulfill two tasks. On the one hand, strong interactions between these hydrophobic polymer chains have to support efficient self-assembly and ensure the stability of the carrier in the extracellular milieu. On the other hand, upon cellular uptake, they should facilitate the intracellular dissociation of polyplexes required for functional nucleic acid delivery. Thus, the nature of the core plays a critical role in the design of potent transfection systems. Important structural elements that could support both tasks include hydrogen bonds and stable secondary structures, which often goes hand-in-hand with specific incorporation of water molecules 25. The sequences adopting secondary structures may furthermore engage in hydrophobic interactions, which are enhanced through a repetitive pattern. Both these interaction-determining features should be susceptible to conditions encountered inside the cell, such as lower pH, triggering core destabilisation and release of the polyplex cargo. A core-forming polymer class that fulfills such requirements for structural elements are homodepsipeptides, as these contain amide bonds that may participate in hydrogen bonds, and they have a clearly defined, alternating sequence structure of α-amino acid and α-hydroxy acid subunits. Polydepsipeptides (PDP) have recently been shown to display strong chain interactions, phase separation phenomena, adoption of highly regular secondary structures, and high shape fixity, the latter being unusual in thermoplastic 3 ACS Paragon Plus Environment
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shape-memory polymers and correspond to strong physical netpoints
26, 27
. When used as
transfection agents, copolymers based on lactide and 3(S)-methyl-2,5-morpholinedione (MMD) 22
or on glycolide, PEG, and MMD
28
, in both cases containing less than 30 mol% MMD,
showed increased micellar stability contributing to the observed transfection efficacy. However, these copolymers with irregular sequence structure and low ability to form hydrogen bonds cannot exhibit the required strong physical interactions. Here, it was hypothesized that the choice of a pure depsipeptide block in a block copolymer having a tailored architecture with swallowtail PEI segments would support the formation of polyplexes through van-der-Waals and hydrogen bonding. Taking advantage of the strong physical interaction of depsipeptide segments, a highly stabilized polyplex would be formed with a delayed collapse of the core, even upon ester hydrolysis. Further, after the internalization of polyplex, the endosomal escape would be facilitated through the decrease in hydrogen bond strength and reduced secondary structural stability upon decrease of pH while moving along the endocytotic route. In particular, the penetration of water molecules into the hydrophobic core would result in depsipeptide core destabilization due to the weakened hydrogen bonding. In this way, the particles may become easier to degrade or sensitive to enzymes 29. The sustained gene delivery could be achieved with the gradually decreasing interaction of depsipeptide segments, potentially through a mechanism different to other environment-sensitive gene carriers, such as the temperature- and reduction-sensitive polymers
30, 31
. The PEI segments in the block
copolymers with a wide buffering range is expected to induce the “proton sponge” effect, which would disrupt the endosome for polyplex release 32. The attached linear PEI after self-assembly of the carriers is considered to resemble a branched structure. After eventual hydrolytic and potentially also enzymatic degradation of the core, linear PEI with its lower toxicity than higher molecular weight branched PEI remains. Here, the depsipeptide based copolymer vectors were explored as an efficient tool for in vitro transfection. The use of cell-based assays provides a possibility to evaluate physicochemical parameters gene carrier designs systematically with a dynamic and quantitative readout, while allowing to follow polyplex uptake with subcellular resolution. This is of particular relevance when aiming at the genetic modification of human primary stem cells, such as mesenchymal stem cells (MSCs). MSCs are a promising cell source for regenerative therapies, but are relatively difficult to transfect and sensitive to various transfection stress. Therefore, a non-viral gene delivery vector with low or negligible toxicity 4 ACS Paragon Plus Environment
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and highly efficient activity for in vitro or ex vivo transfection of human MSCs would be of great utility to improve the therapeutic efficacy of MSCs in regenerative medicine 33, 34. In this study, the preparation and characterization of amphiphilic, cationic di- and triblock copolymers from hydroxy mono- and difunctional oligo(3-(S)-sec-butylmorpholine-2,5-dione) (OBMD) with a number average molar mass Mn = 4500-4900 g·mol-1 and linear PEI of molar masses of 2500 and 10000 g·mol-1 (PEI2.5 and PEI10, respectively) and their self-assembling to nanoparticles is described. OBMD with large hydrophobic side chains was chosen as the stability of secondary structures adopted by depsipeptides increases with the size of the hydrophobic substituent, and such secondary structures were hypothesized to contribute to particle stability. The molecular weight of the OBMD block is suitable to allow effective interchain interactions supporting the self-assembly 35. Subsequently, polyplexes with plasmid DNA coding for reporter genes (GFP, luciferase) as well as the cellular growth factor VEGF were formed and their stability was evaluated by gel electrophoresis and in a turbidity assay. Polyplexes were used for transfection of established as well as primary human cells, and the transfection efficacy, toxicity, and overall transgene expression levels were quantified.
3
Materials and Methods
3.1 3.1.1
Synthesis and characterization of block copolymers Materials
1,8-Octanediol 98%, dimethylformamide (anhydrous) 99.8%, hexane (anhydrous) ≥99%, butyl glycolate ≥90%, dichloromethane (anhydrous) ≥99.8%, linear polyethylenimine Mn = 10000 g·mol-1 (PEI10; actual Mn determined by 1H NMR: 8800 g·mol-1), and toluene (anhydrous) 99.8% were purchased from Sigma-Aldrich (Steinheim, Germany) and used as received. L-isoleucine, chloroform 99%, and diethyl ether ≥99.5% were purchased from Roth (Karlsruhe, Germany). Dibutyltin dilaurate (DBTDL) >95.0% and isophorondiisocyanat (IPDI) >95.0% were obtained from TCI (Eschborn, Germany). Tin(II) 2-ethylhexanoate (Sn(Oct)2) 92.5-100.0% was bought from Sigma-Aldrich (Steinheim, Germany) and was dried by azeotropic distillation from toluene. Linear polyethylenimine PEI Mw= 2500 g·mol-1 (PEI2.5; actual Mn determined by 1H NMR: 2450 g·mol-1), PDI = 1.56, was purchased from Polysciences, Inc. (Hirschberg an der 5 ACS Paragon Plus Environment
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Bergstrasse, Germany). Branched PEI Mw= 25000 g·mol-1 (PEI25) was purchased from SigmaAldrich (St. Louis, MO, USA).
3.1.2 1
General Instrumentation and Methods Preparations
H-NMR spectra were recorded at room temperature on a DRX 500 Avance spectrometer (500
MHz, Bruker, Rheinstetten, Germany; software Topspin version 1.3) using deuterated dimethylsulfoxid (DMSO-d6), deuterated chloroform (CDCl3), or deuterated methanol (CD3OD) as solvents. Mass spectra were measured on an ultrafleXtreme MALDI-ToF spectrometer (Bruker, Bremen, Germany).
Trans-2-[3-(4-tert-Butylphenyl)-2-methyl-2-propenylidene]malononitrile
(DCTB)
was used as matrix. The polydispersity of OBMD was determined on a multidetector GPC, which consisted of a GRAM VS1 precolumn (40 mm x 4.6 mm), a GRAM 30Å 5091312 and a GRAM 1000Å 71111 column (both 250 mm x 4.6 mm) (all PSS, Mainz, Germany), a CO-200 column oven (W.O. electronics, Langenzersdorf, Austria), an isocratic pump 980, an automatic injector 851-AS, a LG 980-02 ternary gradient unit, a multiwave length detector MD-910, a RI detector RI-930 (all Jasco, Gross-Umstadt, Germany), a differential viscometer (WGE Dr. Bures, Dallgow-Doeberitz, Germany), a Wyatt miniDawn Tristar light scattering detector (Wyatt Technology Corporation, Santa
Barbara,
USA),
a
degasser
ERC-3315
(Ercatech,
Berne,
Switzerland),
and
dimethylformamide (0.4 wt% toluene as internal standard, 35 °C, 1.0 mL·min-1) as eluent by universal calibration with polystyrene standards using WINGPC 6.2 (PSS) software. Fourier transform infrared spectroscopy (FT-IR) was performed on a Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific, Dreieich, Germany). Measurements performed ranged from 600 cm-1 to 4000 cm-1. The thermal transitions of the utilized oligomers were investigated by differential scanning calorimetry (DSC) using Netzsch DSC 204 (Selb, Germany). About 5 mg of the sample were analyzed in an aluminum pan. The experiments were conducted by heating the sample from 0 °C to 200 °C, then cooling down to -50 °C, and again warming up to 200 °C with a constant heating
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and cooling rate of 10 K·min-1. The glass transition temperature (Tg) was determined from the second heating run.
3.1.3
Synthesis of block copolymers
Synthesis of oligo((S)-3-((S)-sec-butyl)morpholine-2,5-dione) (OBMD-ol): 0.56 mL (0.17 mmol) of freshly prepared 0.3 M solution of Sn(Oct)2 in anhydrous THF was transferred to a 20 mL oven dried Schlenk tube. The tube was sealed with a rubber septum and the solvent was removed by applying vacuum for 20 min. This was followed by addition of 90 µL (0.86 mmol) of benzyl alcohol and 4.5 g (25 mmol) of BMD. The reaction tube was then evacuated and refilled with nitrogen several times then placed in a preheated oil bath at 135 °C. The polymerization process was ceased by addition of chloroform and the resulting product was then purified by three times precipitation from chloroform in diethyl ether. The final product was dried under vacuum to yield a colorless powder (2.85 g, 63%) Mn, NMR: 4900 g·mol-1, Mn, GPC: 8300 g·mol-1, PDI: 1.3, Mn, ;MALDI: 2040 g·mol-1 . OBMD-diol was synthesized in a similar procedure, except that 1,8 octanediol was used as initiator instead of benzyl alcohol. The resulting powder was colorless, Yield: 74%, Mn, NMR: 4500 g·mol-1, Mn, GPC: 7900 g·mol-1, PDI: 1.2, Mn, MALDI: 2200 g·mol-1.
Synthesis of oligo((S)-3-((S)-sec-butyl)morpholine-2,5-dione)-diisocyanate: 1.86 mL (8.8 mmol) of IPDI and 20 µL of DBTDL were dissolved in 10 mL of anhydrous toluene. The resulting solution was degassed by purging nitrogen for 15 min. 1 g (0.44 mmol) of OBMD was dissolved in 10 mL of anhydrous chloroform and dropwise added to the previous solution under nitrogen. The resulting solution was stirred overnight at ambient temperature. The solvent was partially evaporated at 40 °C using a rotary evaporator. The resulting polymer was then purified by precipitation in anhydrous hexane three times, collected and dried under vacuum for two days to yield a colorless powder (0.8 g, 80%), Mn, GPC: 9700 g·mol-1, PDI: 1.3, Mn, MALDI: 2300 g·mol-1. The polymer was stored at 4 °C under argon.
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Oligo((S)-3-((S)-sec-butyl)morpholine-2,5-dione)-isocyanate was prepared from OBMD-ol using a similar synthetic route to yield a colorless powder. Yield: 65%, Mn, MALDI: 2370 g·mol-1. The polymer was kept in the fridge under argon
Synthesis
of
oligo((S)-3-((S)-sec-butyl)morpholine-2,5-dione)-block-polyethlyleneimine
(t-
OBMD-PEI2.5) (Scheme S1, II) : (306 mg, 122 µmol) of PEI2.5 was added to a 25 mL oven dried Schlenk tube. After several evacuation-refilling cycles, 10 mL of anhydrous DMF were added to the tube via a degassed syringe. The mixture was then heated up to 70 °C to dissolve the PEI; 20 µL of DBTDL were then added dropwise to the solution over a time period of 1 h. 0.3 g (66 µmol) of OBMD was dissolved in 5 mL of anhydrous DMF and added to the solution in the tube. The solution was stirred for 24 h at 70 °C. The final solution was concentrated under vacuum at 65 °C using a rotary evaporator and then dialyzed against chloroform using a Spectra/Por® regenerated cellulose dialysis membrane with a MWCO of 3500 Da for three days, then precipitated in diethyl ether and dried at 65 °C for one day to yield a yellowish polymer of (d-OBMD- PEI2.5 208 mg, 70 %). The other block copolymers were synthesized using a similar protocol except that a regenerated cellulose dialysis membrane with a MWCO of 14000 Da was used in the case of d-OBMDPEI10 and t-OBMD-PEI10.
3.2
Preparation and characterization of nanocarriers and polyplexes from di- and triblock copolymers
3.2.1
Nanoparticle preparation
Typically, 35 mg of copolymer were dissolved in 7 mL of DMSO. The solution was stirred for 1 h and was then transferred into a regenerated cellulose dialysis bag (MCWO: 3500 Da). The polymer phase was dialyzed against Milli-Q water for 2 days. The obtained suspension was adjusted to a volume of 35 mL and filtered through 0.22 µm filter. The final concentration was determined by lyophyllization of 1.5 mL aliquots of the suspension in pre-weighed Eppendorf tubes. 8 ACS Paragon Plus Environment
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Polyplex preparation
The polymer/DNA ratio (N/P ratio), where ‘N’ is the molar amount of nitrogen in PEI and ‘P’ is the molar amount of phosphate in plasmid DNA, was calculated by taking into account that 1 µg DNA contains 3 nmol of phosphate and that 43 ng PEI (1 nmol of C2H5N repeat units) holds 1 nmol of nitrogen. To prepare polyplexes with defined N/P ratios, plasmid DNA was diluted with 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer to a final concentration of 20 µg·mL-1, and the polymer solution (varing concentration in 20 mM HEPES) with the double volume to DNA solution was added dropwise. After a vortex for 30 seconds, the mixture was incubated at room temperature for 30 minutes. The commercial product Lipofectamine® 2000 (Lipo2000, Thermo Fisher Scientific, Schwerte, Germany) was used as a reference transfection reagent for comparison. The lipoplex with a fixed Lipo2000/DNA ratio was prepared according to the given protocol. In brief, the DNA was first diluted with serum free Dulbecco's minimum essential medium (DMEM) to a final concentration of 20 µg·mL-1, followed by mixing with the equal volume of diluted Lipo2000 (6 µL diluted with serum free DMEM to a final volume of 100 µL). Then, the mixture was incubated at room temperature for 5 minutes. For hADSCs, following this protocol resulted in the highest transfection rates among the different conditions tested.
3.2.3
Polyplex characterization
The particle size and the polydispersity index (PDI) as well as the zeta potential were determined using ZetasizerNano SZ instruments (Malvern, Worcestershire, UK). The measurements were performed at 25 °C. Each sample was measured in disposable cuvettes at least three times with several sub-runs each time. For analysis of the polyplexes (polymer/pcDNA3.1-Luc), the samples were standardized by first dilution to the final DNA concentration of 1.5 µg·mL-1 using 20 mM HEPES buffer. For the gel electrophoresis assay, the prepared polyplex (polymer/pcDNA3.1-Luc) with various N/P ratios were mixed with glycerol (20% in PBS) followed by loading onto the agarose gel (1.5%). DNA retardation capacity was analysed by electrophoresis at room temperature in Tris-
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acetic acid-EDTA (TAE) buffer at a voltage of 100 Volt. After staining with ethidium bromide solution, the gel was put into an UV illuminator (ChemiDoc™ XRS+, Bio-Rad, Hercules, CA, USA) to visualize the DNA bands. The polyplex morphology was investigated with cryo-transmission electron microscopy (TEM). The polyplexes (polymer/pCEP4-VEGF165), prepared at N/P ratio 16 with the final DNA concentration of 6.7 µg·mL-1, were kept for 120 min at room temperature prior to cryo-fixation. A Cu grid with a lacey carbon film (400 mesh, Plano GmbH, Wetzlar, Germany) was ionized by glow discharge and 3 µL of the polyplex solution was deposited on it within 20 min. The grid was blotted at 5 °C and plunge-freezed in liquid ethane using a Vitrobot Mark IV (FEI, Eindhoven, The Netherlands). After preparation, the samples were stored in liquid nitrogen and measured at a temperature below -176 °C to avoid devitrification of the sample. Measurements were performed at 200 kV on a Talos F200X (FEI, Eindhoven, The Netherlands) equipped with a high-brightness electron source (X-FEG). Bright-field images were recorded with a bottommounted Ceta 16M pixel CMOS-based camera with 4096×4096 pixels using low-dose imaging with electron doses < 20 e/Ų to minimize beam damage. Images were post-processed by ImageJ 1.46r using the implemented FFT bandpass filter plugin. The polyplex stability was evaluated with a polyanion competition assay. In brief, 10 µL of heparin solution prepared in 20 mM HEPES buffer was added into 30 µL of polyplex (polymer/pcDNA3.1-Luc, N/P ratio 16). The final heparin/DNA weight ratio (w/w) was 5:1. After 2 hours of incubation at room temperature, the released DNA from the polyplex was detected with gel electrophoresis as described above. The pure DNA solution served as control, and the samples incubated with 10 µL of 20 mM HEPES without heparin were include for comparison.
3.3 Serum stability assay The serum stability of pcDNA3.1-Luc complexes was determined via a turbidity assay
36
. In
brief, fetal bovine serum (FBS) was added to the prepared complexes with a final DNA (pcDNA3.1-Luc) concentration of 6.7 µg·mL-1 to reach a FBS concentration of 10%, followed by shaking on a plate shaker at 400 rpm for 5 minutes. Then, 100 µL of the mixture was added into each well of a 96-well tissue culture plate (TCP) and incubated at 37 °C. The turbidity of the 10 ACS Paragon Plus Environment
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mixture was recorded at different time points by measuring the absorbance at 595 nm using a microplate reader (Infinite 200 PRO®, Tecan Group Ltd., Mannedorf, Switzerland). Notably, HEPES buffer (20 mM) was used as diluent instead of serum free DMEM to prepare the Lipo2000/DNA lipoplex in this test, in order to eliminate the influence of DMEM on absorbance at 595 nm.
3.4
DNA cellular uptake and intracellular trafficking
The pDNA pCEP4-VEGF165 was first labelled using a nucleic acid labeling kit (Ulysis™ Alexa Fluor® 647, Thermo Fisher Scientific, Schwerte, Germany) according to the given protocol. Then, the DNA polyplexes (N/P ratio 16) and lipoplex were prepared and added into hADSC cultures as described above. After 8 hours of incubation, the medium was carefully aspirated and the cells were washed 3 times using PBS, followed by fixation with 4% paraformaldehyde. Then, the cells were stained with Alexa Fluor® 555 Phalloidin (Thermo Fisher Scientific, Schwerte, Germany) and Hoechst 33342 (NucBlue® Live Reagent, Thermo Fisher Scientific, Schwerte, Germany) to detect the F-actin and nuclei, respectively. To investigate the endosomal routing of the DNA, after 8 hours of transfection, the cells were stained with carboxyfluorescein succinimidyl ester (CFSE), Hoechst 33342 and LysoTracker® (Thermo Fisher Scientific, Schwerte, Germany) to visualize the cell outline, nuclei and lysosomes, respectively. The stained cells were then imaged with a confocal laser scanning microscope (LSM 780, Carl Zeiss, Jena, Germany). Further biological methods (Plasmid DNA amplification and purification, cell culture, cytotoxicity assay, and DNA transfection) are reported in the supporting information.
3.5
Statistics
The number of replicates for experiments was ≥3 as indicated in the figure legends of the respective assays, and data were expressed as mean ± standard deviation. The two-tailed independent-samples t test was used for statistical analysis and a significance level (Sig.) lower than 0.05 was considered to be statistically significant.
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4 4.1
Results and Discussion Synthesis of OBMD-PEI di and triblock copolymers
The hydroxyl-terminated oligodepsipeptide precursors (OBMD-ol and OBMD-diol) were synthesized via ring-opening polymerization (ROP) of BMD in presence of Sn(Oct)2 as a catalyst and either benzyl alcohol or 1,8-octanediol as initiator. The molecular weights determined by NMR, GPC, and MALDI differ somewhat from each other. While MALDI tends to give too low Mn, GPC may overestimate molar mass. Therefore, Mn determined by NMR (error of integration: ~10%) was used for further calculations (e.g. required amount of reagents), Linear PEI2.5 and PEI10 were attached to OBMD via an IPDI linker using a two-step synthetic route as shown in Scheme S1. The functionalization of OBMD-ol and OBMD-diol at the end group(s) with IPDI was quantitative, i.e. no species were found by MALDI corresponding to non-functionalized OBMD, and NMR integration was ≥100%. MALDI analysis was helpful to identify side products of this reaction (see Figure S1). Partial decarboxylation occurred at the end groups to give amines that are unreactive in the further coupling step (
