Fluorescent Magnetopolymersomes: A Theranostic Platform to Track Intracellular Delivery

We present a potential theranostic delivery platform based on the amphiphilic diblock copolymer polybutadiene-block-poly (ethylene oxide) combining covalent fluorescent labeling and membrane incorporation of superparamagnetic iron oxide nanoparticles for multimodal imaging. A simple self-assembly and labeling approach to create the fluorescent and magnetic vesicles is described. Cell uptake of the densely PEGylated polymer vesicles could be altered by surface modifications that vary surface charge and accessibility of the membrane active species. Cell uptake and cytotoxicity were evaluated by confocal microscopy, transmission electron microscopy, iron content and metabolic assays, utilizing multimodal tracking of membrane fluorophores and nanoparticles. Cationic functionalization of vesicles promoted endocytotic uptake. In particular, incorporation of cationic lipids in the polymersome membrane yielded tremendously increased uptake of polymersomes and magnetopolymersomes without increase in cytotoxicity. Ultrastructure investigations showed that cationic magnetopolymersomes disintegrated upon hydrolysis, including the dissolution of incorporated iron oxide nanoparticles. The presented platform could find future use in theranostic multimodal imaging in vivo and magnetically triggered delivery by incorporation of thermorepsonsive amphiphiles that can break the membrane integrity upon magnetic heating via the embedded superparamagnetic nanoparticles.

Methods 1 H-NMR measurements: 1 H -solution spectra were collected on a Bruker DPX spectrometer operating at 300MHz. Chemical shifts were recorded in ppm and referenced to residual protonated solvent (CDCl3: 7.26 ppm ( 1 H).
ESI-MS measurements: Mass spectra were collected using a Q-Tof Ultima ESI (Waters, USA) mass spectrometer in positive ion mode (range 100-1500 Da). Samples were dissolved in MeOH and diluted to 100µg/ml. ATR-FTIR measurements: Mid-IR powder spectra of the lyophilized samples were collected using a Bruker Tensor 37 FTIR spectrometer with a Bruker Platinum Diamond single reflection ATR equipment at a resolution of 4cm -1 by averaging 32 scans.
UV-Vis measurements: UV-Vis absorption spectra were collected at a scan speed of 400nm/min on a Hitachi UV-2900 spectrophotometer.
Fluorescence measurements: Fluorescence spectra were collected with a PerkinElmer LS 55 luminescence spectrometer with a scan speed of 400nm/min and a slit width of 2.5 nm.
TGA/DSC measurements: Thermograms were recorded on a Mettler-Toledo TGA/DSC 1 STAR System in the temperature range 25-650°C with a ramp of 10K/min under 80mL/min synthetic air gas flow. The mass loss was evaluated by horizontal step setting.

Synthesis & Characterization
N-palmityl-6-nitrodopamide capped superparamagnetic iron oxide nanoparticles (P-NDA SPIONs) Oleic acid coated magnetite nanoparticles were prepared according to Hyeon et al via thermal decomposition of iron pentacarbonyl in hot surfactant solution. 1 The as-synthesized particles were purified by repeated precipitation from minimal toluene into excess ethanol.
N-palmityl-6-nitrodopamide was synthesized via COMU mediated peptide coupling according to Bixner et al. 2 Ligand exchange and purification of the particles was conducted as in Bixner et al. 2 The coating exchange was performed in CHCl3:DMF:MeOH=6/3/1 by sonicating equal weight amounts of ligand and as-synthesized SPION under inert atmosphere for 3h. The crude mixture was evaporated to the DMF fraction and precipitated in MeOH. The sample was washed thrice with hot MeOH and collected via magnetic precipitation. Post-coating was conducted in minimal 2,6-lutidine in an excess of ligand at 50°C under nitrogen gas for 48h. P-NDA coated SPIONs were purified by repeated precipitation from hot MeOH. DEAC-CA was prepared by Knoevenagel condensation of para-substituted ortho-hydroxybenzaldehyde with alpha-C-H acidic Meldrum's acid according to Song et al. 3 In brief, a mixture of 4-(diethylamino)salicylaldehyde (20 mmol), Meldrum's acid (2,2-dimethyl-1,3-dioxane-4,6-dione; 2.89 g, 20 mmol), piperidinium acetate (58 mg, 0.4 mmol) and ethanol (10 mL) was stirred at room temperature for 30 min and refluxed for 3h. The reaction mixture was allowed to cool down to room temperature, followed by chilling in an ice bath for 1h. The product was filtered, washed three times with and recrystallized from EtOH. DEAC-CA was obtained as bright orange crystals in ~ 85% yield. Poly(butadiene(1200)-block-ethyleneoxide(600))-O-(7-(diethylamino)-coumarin-3-carboxylic ester) (PBDb-PEO-DEAC) 100mg PBD-b-PEO were dissolved in 10ml N2-saturated, anhydrous CH2Cl2 (DCM) under sonication. and subsequently activated for 15 min with 1eq. of 1,4-Diazabicyclo[2.2.2]octane (DABCO). Next, 1.5eq. of 7-(Diethylamino)-coumarin-3-carboxylic acid (DEAC-CA) and 0.2eq. 4-Dimethylaminopyridine (DMAP) were added and the 10% polymer solution was purged with N2 gas for 15min before cooling to 0°C in an ice-bath. N,N-Dicylcohexylcarbodiimide (DCC, 1.7eq) in 5ml DCM was dropwise added to the magnetically stirred polymer solution at 0°C. The reaction mixture was allowed to slowly warm to room-temperature and reacted in the dark for 3 days under inert atmosphere. The crude reaction mixture was reduced in volume to approx. 5ml, cooled to -20°C and precipitated DCU was filtered off. The filtrate was diluted with DCM, extracted thrice with 1M HCl, 5% NaHCO3 and washed with Milli-Q water and brine. The combined organic phases were dried over Na2SO4, and the cooling-filtration procedure was repeated from minimal acetonitrile (MeCN). The organic phase was loaded onto a SiO2-column (Silica 60) and washed with several volumes of MeCN to remove excess dye and by-products. The fluorescently labeled target compound was finally eluted in THF:MeOH=10:1. Lyophilization from THF:Milli-Q (1:10) yielded PBD-b-PEO-DEAC as a yellow viscous residue (functionalization approx. 10%). The crude product was diluted with DCM, extracted thrice with 1M HCl, 5% NaHCO3, washed with Milli-Q and brine. The organic phases were dried over Na2SO4, evaporated and dried in high vacuum overnight to yield ~ 95 % of a transparent viscous residue (functionalization approx. 75%). Cytotoxicity chart for different preparation methods