Surface Distribution and Biophysicochemical Properties of Polymeric

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Surface Distribution and Biophysicochemical Properties of Polymeric Micelles Bearing Gemini Cationic and Hydrophilic Groups Zhicheng Pan, Danxuan Fang, Nijia Song, Yuanqing Song, Mingming Ding, Jiehua Li, Feng Luo, Hong Tan, and Qiang Fu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14339 • Publication Date (Web): 28 Dec 2016 Downloaded from http://pubs.acs.org on December 29, 2016

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ACS Applied Materials & Interfaces

Surface Distribution and Biophysicochemical Properties of Polymeric Micelles Bearing Gemini Cationic and Hydrophilic Groups Zhicheng Pan, Danxuan Fang, Nijia Song, Yuanqing, Song, Mingming Ding*, Jiehua Li, Feng Luo, Hong Tan* and Qiang Fu

College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, China

*

Corresponding author. Fax: +86-28-85405402; Tel: +86-28-85460961; E-mail: [email protected], [email protected]

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ACS Paragon Plus Environment


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Abstract Polymeric micelles containing cationic gemini quaternary ammonium (GQA) groups have shown enhanced cellular uptake and efficient drug delivery, while the incorporation of polyethylene glycol (PEG) corona can potentially reduce the absorption of cationic carriers by opsonic proteins and subsequent uptake by mononuclear phagocytic system (MPS). To understand the interactions of GQA and PEG groups and their effects on the biophysicochemical characters of nanocarriers, a series of polyurethane micelles containing GQA and different molecular weight of PEG were prepared and carefully characterized. It was found that the GQA and PEG groups are unevenly distributed on the micellar surface to form two kinds of hydrophilic domains. As a result, the particle surface with some defects cannot be completely shielded by the PEG corona. Despite this, the longer PEG chains with a brush conformation provide superior stabilization and steric repulsion against the absorption of proteins, and thus can reduce the cytotoxicity, protein absorption and MPS uptake of micelles to some extent. This study provides a new understanding on the interactions between PEG chains and cationic groups and a guideline for the design and fabrication of safe and effective drug delivery systems.

Keywords: surface distribution, Gemini cation, PEG, polyurethane micelles, cellular uptake, phagocytosis

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1. Introduction Polymeric micelles with physically entrapped or chemically bonded drugs play an important role in medical and biotechnology applications1-4. The shared task of carriers is the targeted delivery and controlled release of therapeutics and imaging agents to specific sites in the body5-7. However, when the polymeric micelles contact biological systems, their surface could be rapidly adsorbed by proteins to form the so-called “protein corona”, which immediately gives the micelles a “biological identity” distinct from their original surface chemistry, size, and shape after synthesis8-11. These proteins, especially the opsonins such as fibrinogen, immunoglobulins and complement proteins, are rapidly recognized and sequestered by the mononuclear phagocyte system (MPS), leading to a rapid blood clearance and accumulation of nanoformulations in the liver and spleen12-14. To suppress the nonspecific interactions of nanocarriers with physiological environment and reduce the blood clearance, polymeric micelles should be “stealthy” and not be recognized by the environment15-16. One strategy is to passivate the surface of nanomaterials by modification with charge-neutral and highly hydrophilic polymers17. Such anti-fouling polymers render protein adsorption thermodynamically unfavorable. For example, grafting poly(ethylene glycol) (PEG) onto micellar surface (PEGylating) can suppress the protein adsorption by blocking protein-binding sites and creating a thermodynamic shield to reduce nonspecific protein adsorption10, 18. The PEGylated nanocarriers are hardly recognized by macrophages, therefore, they have the ability to

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evade the interception from the immune system, and to prolong the circulation time and improve the pharmacokinetics in vivo19. The surface charge is very important for determining the fate of nanocarries in vivo, such as the efficiency and mechanism of cellular uptake, circulation time and biodistribution20-22. Cationic gemini quaternary ammonium (GQA) has been proposed as a promoter to increase the cell internalization of polymer micelles for efficient drug delivery23-24. The cationic head groups of GQA can interact with negatively charged cell surface, which may facilitate the penetration and diffusion of polymeric nanocarriers through the cell membrane25. However, nanocarries with positive surfaces can be rapidly absorbed by opsonins when contacting the biological environment, leading to the removal of nanoparticles by the reticuloendothelial system (RES)26. Furthermore, a high density of positive surface charge could also break the integrity of cell membrane and result in cytotoxicity27. It is known that PEGylation can shield the positive charge of polymeric micelles, thus reduce the nonspecific interaction of micelles with opsonins and improve their biocompatibility in vivo28. Nevertheless, PEG corona has also been reported to hamper the interaction of cationic nanocarriers with the targeted cell membranes, resulting in less efficient cellular uptake and undesirable therapy effect29. Interestingly, according to our previously report, PEGylated polyurethane micelles could enter cancer cells efficiently, even though the incorporation of PEG outer corona to the cationic micelles do screen the positive charge and reduce the cytotoxicity of nanocarriers to some extent23. As revealed by recent studies, the 4


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protein adsorption, blood circulation time and interaction with the cellular membrane of nanocerries strongly depend on the density and the molecular weight of the PEG chains employed15, 30-31. Therefore, it is noteworthy to systematical investigate the role of PEG chain length and their spatial configuration on the architecture of micelles and their interaction with proteins and cells. On the other hand, PEG and GQA are different hydrophilic moieties which are both located on the surfaces of micelles with a competitive distribution. The former makes polyurethane micelles more inert while the latter makes them more active. However, it remains unclear that how PEG chains interact with GQA cation groups and what the distributions of the two hydrophilic groups are on the surface of polyurethane micelles. Moreover, the impacts of the interactions and distributions of these moieties on the biophysicochemical characters of nanocarriers, including protein adsorption, macrophage removal and tumor cell uptake, have yet to be understood. To better understand the correlation between the molecular weight of the PEG chains and GQA groups, as well as their effects on the physicochemical and biological properties of polyurethane micelles, a series biodegradable multiblock polyurethanes containing varied amounts of GQA groups and PEG with different molecular weights were

synthesized

from

methoxy

poly(ethylene

glycol)

(mPEG),

poly(ε-caprolactone)-diol (PCL), L-Lysine ethyl ester diisocyanate (LDI) , 1,3-Propanediol (PDO) and GQA. The self-assembly, stability, drug loading and release properties of the micelles were fully characterized. Moreover, the distribution of GQA and PEG and the surface structure of the micelles were investigated by proton 5



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nuclear magnetic resonance spectra (1H NMR) and dissipative particle dynamics (DPD) simulation. In addition, methyl tetrazolium (MTT) test and confocal laser scanning microscopy (CLSM) were performed to evaluate the cytotoxicity, cell internalization and phagocytosis of these polyurethane micelles.

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2. Materials and methods 2.1 Materials Paclitaxel (PTX, 99.5%) was purchased from Shanghai Jinhe Bio-Technology Limited Company, China. The commercially available Taxol was obtained from West China Hospital, Sichuan University, China. A 3.5 kDa MWCO dialysis bag was purchased from Solarbio. Fluorescein isothiocyanate isomer I (FITC, 90%) was obtained from Acros Organics, USA. 4', 6-diamidino-2-phenylindole (DAPI, 98%) was purchased from Roche Diagnostics, Germany. 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide (MTT, 98%) was obtained from Sigma-Aldrich, USA. Gemini quaternary ammonium salt (GQA), an L-lysine-derivatized diamine, was synthesized according to our previous work32. In this paper the GQA has long alkyl chain with eight alkyl groups. 1, 3-Propanediol (PDO) and LDI (L-Lysine ethyl ester diisocyanate) were purified by vacuum distillation. N, N-dimethylacetamide (DMAc) were dried with CaO for 2 d, and distilled under reduced pressure and stored in the presence of 4Å molecular sieves. mPEG (Mn = 500, 1900, 5000) and PCL (Mn = 2000, Dow Chemical Company) were used after dehydration at 90 ºC for 2 h under high vacuum.

2.2 Synthesis of polyurethanes with different PEG chain lengths Polyurethanes were synthesized from LDI, PCL, mPEG, PDO and GQA, the feed ratios of the monomers were listed in Table 1. Firstly, PCL was copolymerized with 7



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LDI at 60 °C for 1 h in the presence of a dry nitrogen atmosphere and 0.1% stannous octoate catalyst. Secondly, chain extender PDO was added at 80 °C for 2 h to form high molecular weight copolymers. Then GQA was added and the reaction was kept for 1 h at room temperature, then the temperature was raised to 60 °C for another 2 h reaction. Finally, mPEG with different molecular weights (Mn 500, 1900 and 5000) was used to terminate the polymers at 90 °C for about 6 h. The resultant polyurethanes were precipitated from the mixture of diethyl ether and methanol to remove remaining stannous octoate for three times.

2.3 Bulk properties 400 MHz 1H NMR spectrum was obtained on a Varian UNITY INOVA400 spectrometer using tetramethylsilane (TMS) as an internal standard and perdeuterated dimethyl sulfoxide (DMSO-d6) as the solvent. Fourier transform infrared (FTIR) spectroscopy was analyzed using the KBr tablet method at room temperature. All the spectra were recorded on a Nicolet 6700 Fourier-transform infrared spectrometer (Thermo Electron Corporation). The molecular weights and molecular weight distributions of polymers were estimated by Gel Permeation Chromatography (GPC) on a Waters-1515. N, N-dimethylformamide (DMF)–0.05% LiBr (40 °C) was used as a continuous phase at a flow rate of 1 mL/min. The columns were calibrated by the polymethyl methacrylate (PMMA) standards. The sample concentration was 3 mg/mL.

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2.4 Preparation of nanomicelles Polyurethane micelles were prepared by a dialysis method. Briefly, polyurethane (25 mg) was dissolved in 5 ml DMAc. Then the solution was added dropwise into 15 mL distilled water. The organic solvent was removed by dialysis against distilled water for 72 h. The resulting solution was centrifugalized for 20 min at 3000 r/min, then the supernatant was taken out and filtered through a 0.45 µm pore-sized syringe filter (Milipore, Carrigtwohill, Ireland).

2.5 Characterization of polyurethane micelles The size and zeta potential of polyurethane micelles was determined by the dynamic light scattering (DLS) measurement at 25 °C at an angle of 90° using a Malvern zetasizer Nano ZS ( (Zen 3690, Malvern, U.K.). The physical stability of the nanocarriers was assessed by monitoring the change of micellar size under simulative physiological conditions. In brief, the micellar solution was mixed with an equal volume of phosphate buffer solution (PBS, 0.2 M), medium containing 10% fetal bovine serum (FBS), and distilled water as a control. The mixtures were incubated at 37 °C for different times and determined by DLS. The critical micelle concentration (CMC) of polyurethane micelles was determined from the measurements of fluorescence of pyrene as a probe33. A known weight of pyrene was dissolved in acetone and added into the vials. The acetone was evaporated and the vials were added with a series of micellar solution with different concentrations (the concentration of pyrene was 5 × 10−7 mol/L). The mixture was 9



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sonicated at room temperature for 4 h. The excitation spectra was recorded from 206 to 406 nm with the emission wavelength at 372 nm (emission slit = 2 nm) using 970CRT fluorescence spectrophotometer (Shanghai Precision & Scientific Instrument, China). The ratios of the fluorescence intensity at 337 nm to that at 334 nm in the excitation spectra were plotted versus the logarithmic concentrations of polyurethane micelles. Then the diagram was properly fitted by a sigmoid function of the Boltzmann type and the CMC values were derived from the cross-point when extrapolating the intensity ratio I337/I334 at low and high concentration regions. The morphology of polyurethane micelles was examined by transmission electron microscopy (TEM). The sample solution was negative stained by phosphotungstic acid (1%) and dropped onto the copper grid. The solution was then blotted off and air dried before observation on a Hitachi model H-600-4 transmission electron microscope operated at 75 kV. The lyophilized micelle solution was dispersed in D2O (TMS as reference) and analyzed on a Bruker AV II-400 MHz spectrometer at 25 °C.

2.6 PTX loading and controlled release PTX-loaded polyurethane micelles were prepared as follows: PTX was first dissolved in acetone and transferred into a glass vial. After the acetone was evaporated completely, the micelles were added and ultrasonated at room temperature for 2 h. Then the resulting solution was centrifugalized for 10 min at 3000 r/min. The supernatant was taken out and filtered through a 0.45 µm pore-sized syringe filter. 10


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The release profiles of PTX from polyurethane micelles were studied with a dialysis method. The dialysis bag containing drug-loaded micelles was immersed in PBS (10 mM, pH 7.4) in the presence of 1 M sodium salicylate at 37 °C in an incubator shaker.34. 1 mL of release media was taken out and an equal amount of fresh buffer solution was added at selected time intervals. The PTX concentration in the sampled release media was determined using a High Performance Liquid Chromatography (HPLC) system (Agilent 1260 series, USA) with a reverse-phase C18 column (4.6 × 100 mm) at 227 nm by UV detection. The eluent was composed of acetonitrile and water (60/40 v/v), and the flow rate was 1 mL/min. All the measurements were conducted in triplicate.

2.7 Cell culture Human cervical carcinoma cell line (HeLa), murine fibrosarcoma cell line (L929) and murine macrophage cell line (Raw264.7) were cultivated in RPMI 1640 containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. All the cells were cultivated at 37 °C in a humidified atmosphere of 5% CO2.

2.8 Cytotoxicity assay To evaluate the cytotoxicity of drug-free micelles and the therapeutic efficacy of drug-loaded micelles against normal cells and cancer cells, L929 and Hela cells were seeded into 96-well plates with a density of 5 × 103 cells/well and incubated for 12 h to allow for cell attachment. The L929 and Hela cells were then incubated for 1 d and

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3 d with various concentrations of drug-free and drug-loaded micelle solutions, respectively. All the samples were sterilized by filtration with a 0.22 µm filter before measurements. At determined time, the formulations were replaced with RPMI 1640 containing MTT (5 mg/ml) and cells were then incubated at 37 °C for additional 4 h. The MTT solution was aspirated off and the formed formazan crystals were dissolved in 150 µL of DMSO. The samples were determined using a spectrophotometer at 492 nm. Untreated cells in culture media were set as negative control with 100% viability. All the measurements were performed in triplicate.

2.9 In vitro cellular uptake of micelles Hela and Raw264.7 cell lines were seeded at 1×105 cells/well in 6-well chamber slides for 24 h. The culture media were replaced by 1 mL of fresh RPMI 1640, and FITC-encapsulated micellar solutions were added and incubated with the cells for 30 min and 60 min. Afterward, the cells were washed with cold PBS for three times, fixed with 4% paraformaldehyde at 4 ºC for 30 min, and stained with DAPI for 10 min. The cells were observed with a confocal laser scanning microscopy (Zeiss 510 LSM microscope).

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3. Results and Discussion 3.1 Synthesis of polyurethanes with different PEG chain lengths The polyurethanes containing GQA and mPEG with different molecular weights (500, 1900, and 5000) were successfully synthesized as illustrated in Figure 1. The resultant multiblock polymers are named as G8mEX, where X denotes the molecular weight of mPEG segment. Polyurethane without PEG was also prepared as a control (G8mE0). As shown in Table 1, all the polyurethanes show moderate molecular weights (Mw 16000-33000) and narrow molecular weight distributions (PDI 1.20-1.64). The 1H NMR spectra of polyurethane are shown in Figure 2. The peak near 3.1 ppm is assigned to the methyl and methylene protons of the GQA units (-N(CH3)2 and -N(CH2)2). That signal appears very weak because the molar content of GQA is only about 7.5%. The peaks at 1.31 ppm (-CH2-CH2CH2-), 1.55 ppm (-CH2CH2CH2-), 2.27 ppm (-CH2COO-) and 3.98 ppm (-CH2O-) are assigned to the methylene groups of the PCL segment. The signals at 4.46 ppm (-CH2-CH (-N)-CO-), 4.07 ppm (-CH2OCO-) and 1.17 ppm (-CH3) are attributed to the chemical shifts of ethoxyl group in LDI. The peaks at 4.33 ppm (-O-CH2-) and 3.51 ppm (-CH2CH2O-) are ascribed to the methylene protons of PDO units and PEG chain, respectively. The FTIR spectra of polyurethanes indicate the appearance of a strong peak around 3410 cm-1 arising from N–H vibration and the disappearance of the peak at 2200 cm-1 corresponding to N=C=O stretching vibration (Figure S1), suggesting that the isocyanate groups of LDI have been completely reacted. The peaks at 2890 cm-1 13



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and 1110 cm-1 are assigned to the methylene CH2 and ether C–O–C stretching vibration of mPEG, the two peaks gradually increase with the PEG molecular weight. A strong band at approximately1723 cm-1, corresponding to the ester carbonyl groups, is overlapped by the LDI and PCL soft segments, as well as the hydrogen-bonded and free carbonyl of urethane. The peak around 1650 cm-1 attributed to the urea carbonyl derived from GQA, which further demonstrates the successful synthesis of the polyurethanes.

3.2 Properties of polyurethane micelles Polyurethanes containing GQA groups have shown extraordinary self-assembly property in aqueous solution20. To investigate the effect of the varying molecular weights of mPEG segments on the self-assembly behavior, the particle size, zeta potential and morphology of polyurethane micelles were measured using DLS and TEM. All the micelles show positively charged surfaces and favorable hydrodynamic sizes ranging from 57 to 90 nm with narrow size distributions (PDI 0.2-0.3) and (Table 2). The average size decreases significantly and the particle morphology appears more regular with the incorporation of mPEG segments with higher molecular weights (Figure 3), owning to the fact that the improvement of the hydrophilicity of polyurethanes can enhance the steric stabilization and decrease the size of micelles35. Moreover, the zeta potentials of polyurethane micelles exhibit varying degree of decrease due to the screening of GQA groups by long PEG chains (Table 2). In particular, it was found that longer PEG chains (Mn≥1900) shield the 14


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cationic surface more effectively than shorter ones. Nevertheless, G8mE5000 with the longest PEG chain (Mn 5000) still display a positively charged surface, with a zeta potential of 24.5 mV, implying that the outer corona formed by high molecular weight of PEG cannot fully screen the cationic surface of micelles. A possible explanation is that both the hydrophilic GQA8 and PEG chain are distributed on the micellar surfaces and they possess significant polarity difference and poor compatibility36, which may result in some defects on the micellar surface and partial exposure of cationic shell25,

37-38

. In addition, the low molar content of mPEG (10%) is also

responsible for the positive surface charge of micelles, since a low density of PEG chains on the particle surface can hardly cover the cationic shell completely18. To better understand the distribution and interaction of PEG and GQA on the micellar surface, the lyophilized polyurethane micelles were re-dispersed in heavy water (D2O) and determined with 1H NMR. As shown in Figure S2, the peak near 3.1 ppm (peak A) is assigned to the methyl protons of GQA, and the peak at 3.51 ppm (peak B) is ascribed to PEG. The integrated area ratios of peak B to peak A were calculated to be 9.64, 52.80 and 113.64, respectively, for G8mE500, G8mE1900 and G8mE5000, suggesting that the surfaces of micelles are protected by the PEG corona to different extent. Interestingly, the signal of GQA groups is still observed in the 1H NMR spectrum of G8mE5000 micelles with the longest PEG segments (Figure S2). The result further demonstrates that PEG corona does not fully shield the GQA groups on the surface of micelles, which is in agreement with zeta potential analysis (Table 2). It is worth noting that there is no evident signal in the spectrum of G8mE0, because 15



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the micelles without PEG corona cannot be well resuspended in water after freeze-drying process. The morphology of micelles and the distribution of GQA groups and PEG chains were further investigated by a DPD simulation. As shown in Figure 4, all the polyurethane micelles appear as dispersed individual particles, and the sizes of micelles become smaller as the molecular weight of PEG increases, which is consistent with TEM and DLS results. Of interest, GQA groups and PEG chains were found nonuniformly distributed on the surface of the micelles, forming two kinds of hydrophilic domains and thus resulting in some defects on the micellar surface (Figure 4). As a result, the entire surface of micelles could not be covered by PEG chains due to these defects. In addition, PEG segments with lower molecular weight (500) exhibit a mushroom conformation on the micellar surface, while those with higher molecular weights (Mn>1900) form a brush conformation. This phenomenon is potentially helpful for the high stability and long circulation time of these nanocarriers in the body, since it is known that PEG chains with a brush conformation can provide a superior stabilization and steric repulsion against the absorption of proteins39. To verify this potential, the stability of polyurethane micelles under different conditions was assessed. Firstly, CMC was determined to evaluate the thermodynamic stability of micellar formations using pyrene as a fluorescent probe. As shown in Table 2, the CMC values of G8mE0, G8mE500, G8mE1900 and G8mE5000 micelles were determined to be 1.07, 3.35, 0.93 and 3.31 µg/mL, respectively, which are much lower than those reported for many other block copolymers40-42, owning to the 16


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enhanced surface activity with the incorporation of GQA groups28. Further, the stability of polyurethane micelles under a simulative physiological condition was investigated by monitoring the change of particle size in PBS. It was found that the micelles containing only GQA groups (G8mE0) exhibit a dramatic change of size (over 50 times), with visible precipitates formed in micelle solution instantaneous after addition of PBS (movie S1). This phenomenon may be due to the disturbance of the stabilizing effect of GQA surfactant by buffer solutions43. On the other hand, as PEG groups are introduced to polyurethane structure, the micelles are more stable in PBS solution. The size of G8mE500 increases by 10 times, and those of G8mE1900 and G8mE5000 remain almost unchanged (Figure 5). These results indicated that PEG corona can provide good steric stabilization for cationic polyurethane micelles, and the protection effects are further improved with the increased molecular weight of PEG segments. To evaluate the effect of PEG chain length and micellar architecture on the stability of polyurethane micelles against proteins, the changes of particle size and zeta potential were monitored in the medium containing 10% FBS at 37 °C for 48 h. As shown in Figure 6, all the micelles show an increase of hydrodynamic diameter and decrease of zeta potential over time, suggesting the absorption of negatively charged bovine serum onto the micellar surfaces. In particular, the sizes of G8mE0 and G8mE500 increase by 2-3 fold, while those of G8mE1900 and G8mE5000 grow slightly. Moreover, after 1 h of incubation in protein-containing medium, the size and zeta potential of G8mE5000 micelles are almost unchanged (Figure 6). These results 17



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demonstrate that long PEG chains could provide better shielding effect for cationic polyurethane micelles and steric repulsion against the absorption of blood proteins, which is beneficial to achieving high stability and prolonged circulation times in vivo44. However, considering the negative charged surface and initial size increase of G8mE5000 micelles after incubation with FBS, the shielding effect of long chain PEG is still limited due to the defects on the shell of micelles. Hence, the further optimization of macromolecular structure and the distribution of functional groups on the particle surfaces are clearly warranted.

3.3 Drug loading and release studies The drug loading ability of PU micelles was evaluated using chemotherapeutic drug PTX as a model. The drug loading content (LC) and the drug encapsulation efficiency (EE) of PTX-loaded PU micelles are shown in Figure 7. The maximum LC and EE are 25% and 99%, respectively, indicating that all the polyurethane micelles can encapsulate PTX efficiently. Interestingly, as the molecular weight of PEG increases, the maximum drug loading content increases significantly from 6.6% to 25.0%. This may be due to that once PTX is encapsulated into the micelles by hydrophobic interaction, the hydrophilic shell formed by PEG with long chains could hinder drug desorption from the micellar core. The release of PTX from drug loaded micelles was studied in PBS solution containing 1 M sodium salicylate. As depicted Figure 8, a strong correlation between the molecular weights of PEG and the release rates of PTX is observed. In particular, 18


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the accumulative release rate of PTX in 120 h decreases gradually from 74% to 54% as the repeating unit number of PEG increases from 0 to 113. There are probably two reasons to account for the change of release rate. One is that longer hydrophilic PEG on the micelles surface would hamper the PTX release. The other reason is that the micelles with short PEG chain or without PEG corona are unstable in PBS solution (Figure 5), resulting in rapidly release of PTX.

3.4 In vitro cytotoxicity The cytotoxicity of drug-free and PTX-loaded micelles toward L929 cells and HeLa cells was evaluated by MTT assay. The cell viability of all the PU micelles is above 90% after 24 h of incubation, demonstrating that the drug-free PU micelles show good cytocompatibility (Figure 9). However, after 72 h of incubation, the cell viability of G8mE0 micelles in a high concentration (0.1 mg/ml) is even lower than 80% while that of micelles with PEG corona is still higher than 90%. The cytotoxicity of G8mE0 micelles is attributed to the strengthened surface positive charges of GQA groups that disturb the integrity of cell membrane45-47, and the improved cytocompatibility of PEGylated polyurethane micelles is resulted from the effective shielding of GQA groups by the PEG outer corona23. The antitumor efficacy of PTX-loaded PU micelles was studied using clinical antitumor agent Taxol as a positive control. As shown in Figure 10, the cytotoxicity of drug-encapsulated polyurethane micelles is dose-dependent. The formulations with higher molecular weight of PEG exhibit relative higher cell viability compared to 19



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those with shorter PEG chains or without PEGylation. The half maximal inhibitory concentrations (IC50) of G8mE0 and G8mE500 are 9.40 and 8.94 µg/ml, respectively, which are lower than those of G8mE1900 and G8mE5000 (28.66 and 33.35 µg/ml, respectively). The lowered cytotoxicity of G8mE1900 and G8mE5000 can be associated with the screened surface charge as well as hindered release of therapeutics as demonstrated above.

3.5 In vitro cellular uptake To further investigate the effect of PEG molecular weight and the surface character of polyurethane micelles on the cellular uptake of nanovehicles, HeLa cells were cultured with fluorescently labeled micelles and observed by CLSM. Evidently, G8mE0 micelles without PEG corona exhibits the fastest and highest uptake in HeLa cells, with maximal fluorescence intensity observed in tumor cell after 0.5 h of incubation (Figure S4). With the incorporation of PEG segments into polyurethanes, the intracellular signal of micelles increases with time. There is no much different between micellar formulations with shorter PEG chains, which is in good agreement with our previous work23. However, as the molecular weight of PEG increase to 5000, the fluorescence intensity of micelles in Hela cells can be hardly observed in 1 h (Figure 11). Considering that the long PEG chain cannot shield the cationic micellar surface completely, as described above, the diminished cell entry of G8mE5000 could be associated with the brush conformation of PEG corona (Figure 4). Further study is needed to better understand this result. 20


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3.6 Phagocytosis of PU micelles by mouse macrophages raw264.7 cells Phagocytosis is an important pathway for the clearance of exogenous nanomaterials from the blood48-49. A large quantity of nanoparticles in circulation are eventually cleared by the mononuclear phagocytic system50-51. Therefore, it is of great importance to investigate the interactions between cationic polyurethane micelles and macrophages. To this end, the macrophages uptake of PU micelles was studied using raw264.7 cell line. It was found that G8mE0 and G8mE500 exhibit remarkable green fluorescence signal in macrophages in 0.5 h (Figure S5), while the fluorescent intensity in G8mE1900-treated cells is much weaker (Figure 12). Importantly, the fluorescence signal G8mE5000 micelles could be hardly observed within 1 h. These results demonstrate that cationic polyurethane micelles without PEG corona may expose their GQA shell on the micellar surface and be rapidly recognized by the mouse macrophages. Moreover, the short PEG chain (Mn 500, 1900) grafted onto the micellar surface have no significant effect on the clearance of nanocarriers by macrophages, while PEG segments with higher molecular weight (Mn 5000) could reduce the internalization efficiency of nanoparticles in macrophages, and thus holding great promise to evade the interception of nanomedicines by the immune system.

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4. Conclusion In summary, taking polyurethanes bearing cationic GQA groups and different molecular weight PEG segments as models, we systematically investigate the distribution and interaction of PEG chains and GQA groups on the micellar surface. It was found that the distribution of the two hydrophilic groups and the molecular weights of PEG have great impacts on the physicochemical and biological properties of polyurethane micelles. In particular, the long PEG chains (Mn 5000) with brush conformation can improve the stability, drug loading capacity and sustained release properties of polyurethane micelles. Moreover, they could reduce the cytotoxicity and inhibit the protein absorption and macrophage uptake to some extent, even though they cannot completely cover the micellar surface due to the uneven distribution of GQA and PEG chains. It is known that the surface properties of nanocarriers is crucial to the fate of drug formulations in vivo. This point is especially important for multifunctional nanovehicles containing various functional moieties on the particle surface52. Therefore, it is of great significance to develop well-designed molecules to optimize the interaction and distribution of multiple groups and to maximize the efficacy of drug delivery systems. Our work presents a new understanding on the interaction of cationic and hydrophilic groups on the micellar surface and their effects on the biophysicochemical characters of nanocarriers, which can provide a guidance for the design of effective and stealthy nanovehicles for controlled delivery applications.

22


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Acknowledgment This work was supported by the National Natural Science Foundation of China (51173118, 51273126 and 51273124), the National Science Fund for Distinguished Young Scholars of China (51425305).

Supporting Information: The FTIR spectrum and thermal properties of polyurethanes with varying molecular weights of mPEG chains. The 1H NMR spectrum of polyurethane micelles dispersed in heavy water. The DPD simulated structure of polyurethane micelles. CLSM images of Hela cells and Raw264.7 cells incubated with polyurethane micelles for 0.5 h.

Conflict of Interest: The authors declare no competing financial interest.

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Table 1. Theoretical Composition and Molecular Weights of three types of polyurethane molecular weightsa

molar ratio samples

LDI

PCL

mPEG

G8mE0

4

2

G8mE500

4

G8mE1900 G8mE5000

Chain Extender

Mn

Mw

Mw/Mn

0.6

20162

33190

1.65

1.4

0.6

11118

16380

1.47

0.8

1.4

0.6

19093

23078

1.21

0.8

1.4

0.6

18310

24481

1.34

PDO

EG8

0

1.4

1.6

0.8

4

1.6

4

1.6

a

Measured by GPC analysis

24


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Table 2. The size, size distribution, zeta potential and CMC value of polyurethane micelles. Sample

size (d.nm)

zeta potential

CMC

(mV)

(µg/ml)

PdI

G8mE0

87.1±0.5

0.392

40.8±3.0

1.07

G8mE500

88.4±0.8

0.235

41.2±1.1

3.35

G8mE1900

74.8±0.3

0.245

30.6±1.4

0.93

G8mE5000

58.0±0.1

0.257

24.5±1.2

3.31

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29



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Figure 1. A) Schematic structure of polyurethane G8mEX. B) Schematic illustration of G8mEX micelles with different PEG chain lengths. 123x109mm (300 x 300 DPI)


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Figure 2. 1H NMR spectrum of (A) G8mE0, (B) G8mE500, (C) G8mE1900 and (D) G8mE5000 in DMSO-d6. 149x160mm (300 x 300 DPI)



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Figure 3. TEM images of (A) G8mE0, (B) G8mE500, (C) G8mE1900 and (D) G8mE5000 polyurethanes micelles. 90x100mm (300 x 300 DPI)


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Figure 4. The computer simulated structures of polyurethane micelles with different molecular weights of PEG. The blue, green and red particles represent PCL, PEG and GQA, respectively. 57x23mm (300 x 300 DPI)



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Figure 5. Change of size (A) and size distribution (B) of polyurethane micelles with different molecular weights of PEG for 24 h of incubation at 37 °C in PBS buffer solution (pH 7.4). 216x331mm (300 x 300 DPI)


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Figure 6. Change of size (A) and zeta potential (B) of polyurethane micelles with different molecular weights of PEG for 48 h of incubation at 37 °C in 10% FBS medium. The bold numbers in (B) show the decrease in zeta potential value (mV) of the micelles before and after incubation with 10% FBS medium. 214x328mm (300 x 300 DPI)



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Figure 7. Drug loading content (%) and encapsulation efficiency (%) of PTX in polyurethanes micelles: (A) G8mE0, (B) G8mE500, (C) G8mE1900 and (D) G8mE5000. 99x70mm (300 x 300 DPI)


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Figure 8. The release profile of PTX from polyurethane micelles in PBS (pH 7.4). 109x85mm (300 x 300 DPI)



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Figure 9. Cell viability of L929 mouse fibroblasts (A, B) and HeLa cells (C, D) after incubation with drug free polyurethane micelles for 24 (A, C) and 72 h (B, D). 115x95mm (300 x 300 DPI)


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Figure 10. Cytotoxicity of polyurethane micelles with different PEG lengths against HeLa tumor cells after 24 h and 72 h of incubation. Insets show the IC50 values of various PTX formulations toward HeLa cells for 72 h of incubation. 239x409mm (300 x 300 DPI)



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Figure 11. CLSM images of Hela cells incubated with polyurethane micelles with different PEG chain lengths for 1 h. 136x132mm (300 x 300 DPI)


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Figure 12. CLSM images of Raw264.7 cells incubated with polyurethane micelles with different lengths of PEG chains for 1 h. 138x136mm (300 x 300 DPI)



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Table of Content 83x35mm (300 x 300 DPI)


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Surface Distribution and Biophysicochemical Properties of Polymeric

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