Article pubs.acs.org/Macromolecules
One-Pot Synthesis for Biocompatible Amphiphilic Hyperbranched Polyurea Micelles Fei Xiang,† Marc Stuart,§ Joop Vorenkamp,† Steven Roest,† Hetty Timmer-Bosscha,‡ Martien Cohen Stuart,∥ Remco Fokkink,∥ and Ton Loontjens*,† †
Department of Polymer Chemistry, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands ‡ Department of Medical Oncology, University Medical Center Groningen, Hanzeplein 1, 9713 GZ, Groningen, The Netherlands § Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands ∥ Laboratory of Physical Chemistry and Colloid Science, University of Wageningen, Droevendaalsesteeg 4, 6708PB, Wageningen, The Netherlands ABSTRACT: Here we report, for the first time to our knowledge, a method to synthesize AB2 monomers, the corresponding hyperbranched and the corresponding amphiphilic hyperbranched polymers in a one-pot procedure, starting from two commercial available compounds. Since the B groups were blocked isocyanates (BIs), the end groups of the hyperbranched polyurea were BIs as well. Coupling of a range of monomethoxy-poly(ethylene glycol)s onto the BIs yielded a platform of amphiphilic hyperbranched polymers, with controllable hydrophobic cores and hydrophilic shells. After the three consecutive reaction steps, without intermediate purification, the final polymers were purified by precipitation in a nonsolvent, in which the polymer precipitated and the excess PEG remained dissolved. Pyrene inclusion experiments showed the formation of micelles above a critical concentration. Both cryo-EM and DLS revealed the presence of two distinct particle populations, being the primary micelles and aggregates thereof. All micelles showed a LCST behavior, with transitions close to body temperature. The low cytotoxicity of the micelles make them promising for drug delivery.
1. INTRODUCTION The effectiveness of hydrophobic drugs is generally low due to the very fast clearance by the kidney.1,2 The concentration of drugs drops rapidly below the efficacy level. Administration of a higher concentration of drugs is not acceptable due to toxicity. Hence, there is only a narrow application window in which drugs are effective. Encapsulation of drugs has therefore attracted a growing interest in pharmaceutical community to prolong the drug circulation times and to reduce toxicity.3−6 Micelles of linear amphiphilic block copolymers were regarded as promising candidates to encapsulate hydrophobic drugs for use in aqueous environment (the body).7 Although, polymeric micelles are much more stable than low molecular surfactants (soaps), they still can dissociate too early into free polymer chains on dilution, when administered into body fluids. Because of dilution, the concentration will drop below the critical micelle concentration (cmc). Moreover, the limited stability is further put to the test by various in vivo parameters, like temperature and pH.8 Various strategies have been adopted to improve the stability of micellar structures. In one route the polymeric micelles were stabilized by intramicellar cross-linking.9,10 However, crosslinking reduces the biodegradability, which will hamper the © XXXX American Chemical Society
necessary excretion. Amphiphilic dendrimers are elegant alternatives, as these perfect monodispersed polymers have micelle-like properties by their own, i.e., without self-assembling steps.11−14 Unfortunately, syntheses of dendrimers are laborious, multistep processes. Moreover, most of the dendrimers are not biodegradable. Restani et al., however, reported recently that polyurea dendrimers are biodegradable.15 Micelles from hyperbranched polymers (HBPs) could be very attractive drug-encapsulating carriers because of their convenient synthetic routes. Wang and co-workers16 reported spherical micelles obtained by self-assembly of toluene-4sulfonyl or benzoyl-terminated hyperbranched polyesters. Frey et al. described the synthesis and encapsulation properties of amphiphilic molecular nanocapsules comprising a hydrophilic hyperbranched polyglycerol core.17,18 Haag et al. studied the self-assembly mechanism of micelles from hyperbranched star copolymers with a hydrophilic poly(ethylenimine) core, an inner shell of long aliphatic chains and a poly(ethylene glycol) corona.19 Most of the reported hyperbranched polymers have a Received: March 14, 2013 Revised: May 17, 2013
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dx.doi.org/10.1021/ma400552x | Macromolecules XXXX, XXX, XXX−XXX
Macromolecules
Article
2.6. Cryogen Transmission Electron Microscope (Cryo-TEM). The morphologies of OAHBP micelles were visualized by cryo-TEM. The micelle solutions were prepared by dissolving 2 mg of OAHBPxxx sample into 1 mL of double distilled water through ultrasonication. Micelle solution was applied to a glow-discharged holey carbon support film (Quantifoil 3.5/1) with a pipet. The specimen grid was blotted with filter paper and vitrified in ethane using the Vitrobot (FEI, Eindhoven The Netherlands). After that, the samples were transferred into a Gatan type 626 cryo specimen holder. Cryo-TEM recordings of the samples were carried out at an operating voltage of 120 kV with a Philips CM120 TEM. The imaging was performed on a Gatan type ultrascan 4000SP CCD camera under low-dose conditions. 2.7. Dynamic Light Scattering (DLS). Size and size distribution of the micelles in water were measured by DLS. The micelle solution was prepared by solving 1 mg OAHBPxxx sample into 1 mL of double distilled water through ultrasonication (Bransonic, from VWR) for 0.5 h and diluted to obtain a concentration range from 10−5 to 1.0 mg/ mL. DLS was carried out on an ALV-125 goniometer light scattering setup with an ALV5000/60X0 external correlator and equipped with an ALV/SO SIPD single photon detector with ALV static and dynamic enhancer fiber optics (ALV, Langen, Germany). The light source was a 300 mW Cobolt Samba-300 DPSS laser, operating at a wavelength of 532 nm. All experiments were performed at a scattering angle of 90°. Temperature was controlled by using a Haake C35 thermostat, providing an accuracy of ±0.1 °C. The hydrodynamic radius was calculated from cumulant38 fits or a CONTIN multiexponential fit.39,40 All OAHBP micelles samples were measured without filtration treatment. 2.8. Lower Critical Solution Temperature (LCST). The LCST study was carried out by placing a cuvette with a polymer solution in a RC6 CP LAUDA water bath, furnished with a thermostat to control the temperature. IR measurements, with light of a wavelength of 950 nm, were carried out in a 10 mm quartz cuvette. A Turbidity U047 apparatus measured the 0° emission and the 90° scattering of the incident light beam. The turbidity was calculated by homemade software. 2.9. MTT Assay. The MTT assay measures the activity of enzymes that reduce MTT to formazan dyes, giving a purple color. It allows to assess the viability and proliferation of cells. MTT (3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole) is reduced to purple formazan in living cells.41 A solvent (either dimethyl sulfoxide, an acidified ethanol solution, or a solution of the detergent sodium dodecyl sulfate in diluted hydrochloric acid) is added to dissolve the insoluble purple formazan product. The absorbance of this colored solution is quantified by measuring the absorption at a wavelengths between 500 and 600 nm by a UV−vis spectrophotometer. SKOV-3 cells (American Type Culture Collection HTB-77) were plated in 100 μL culture medium (Dulbecco’s modification of eagles medium, DMEM, with 10% fetal bovine serum) in a 96-well plate at a cell density of 30 000 cells per well and allowed to attach for 24 h in an incubator in a humidified atmosphere at 37 °C and 5% CO2. After 24 h, 100 μL of culture medium with the desired amount of micelles was added, and cells were cultured for another 24 h, after which the metabolic activity of cells was measured. For that, 20 μL of MTT solution (2 mg/mL in phosphate buffered salt) was added to each well. After 4 h plates were centrifuged (15 min at 180g, room temperature), supernatant was aspirated, and formazan crystals were dissolved in dimethyl sulfoximine (100%). Absorbance was read at a microplate reader (Biorad) at 520 nm. Survival percentage was calculated relative to cells cultured in culture medium without micelles. 2.10. Synthesis of Amphiphilic Hyperbranched Polymers. To a solution of bis(hexamethylene)triamine (1.079 g, 5.01 mmol) in dry DMF (50 mL) carbonyl biscaprolactam (CBC) (2.528 g, 10.02 mmol) was added, and the resulting mixture was stirred at 80 °C for 8 h under a N2 atmosphere. Then the reaction temperature was increased to 145 °C, and the solution of mixture was stirred under a N2 atmosphere for 6 h. After a polymerization, a solution of MPEG (∼12 mmol) in dry DMF in the presence of 0.5−1 wt % dibutyltin
hydrophilic core, although an inverse topology is more preferred, as most of the drugs are hydrophobic. HBPs from A2 and B3 monomers are easily accessible due to the abundant availability of these kinds of monomers. However, stoichiometric mixtures form during the polymerization at high conversions insoluble gels. In contrast, AB2 monomers cannot form gels but are generally more difficult to prepare. Here, we report a convenient synthetic route to prepare novel AB2 monomers, the corresponding hyperbranched polyurea and the corresponding amphiphilic hyperbranched polyurea in a one-pot procedure, in high yields and without intermediate purification steps. These polymers comprised a hydrophobic polyurea core and a hydrophilic poly(ethylene oxide) shell, potentially suitable to encapsulate hydrophobic drugs in aqueous environments. Micelle formation of these biocompatible polymers is demonstrated with various techniques, such as cryo-electron microscopy (cryo-TEM), AFM, DLS, and pyrene inclusion experiments. The biocompatibility is demonstrated with SKOV-3 cells.
2. EXPERIMENTAL SECTION 2.1. Materials. Carbonyl biscaprolactam (CBC, ALLINCO) was kindly obtained from DSM Innovation Center and used without purification (>99% pure according to HPLC). Monomethyl-poly(ethylene glycol)s (MPEG350, 550, 750, and 2000) were obtained from Sigma-Aldrich and used without purification. Magnesium sulfate (MgSO4, 97%) was obtained from Acros Organics and used without purification. Bis(hexamethylene)triamine (BHTA, 99+%), chloroformd (CDCl3-D, 99.8 atom % D), dimethylformamide (DMF), and dimethyl-d6 sulfoxide (DMSO-d6, 99.5 atom % D) were obtained from Aldrich Chemical Co. and used without purification. In all cases, the highest available purity of the solvents was used. HPLC grade toluene (
