Article pubs.acs.org/Langmuir
The Amine Content of PEGylated Chitosan Bombyx mori Nanoparticles Acts as a Trigger for Protein Delivery Daniela Vasquez,† Rakiya Milusheva,‡ Patric Baumann,† Doru Constantin,§ Mohamed Chami,∥ and Cornelia G. Palivan*,† †
Department of Physical Chemistry, Basel University, Klingelbergstrasse 80. 4056, Basel, Switzerland Institute of Polymer Chemistry and Physics of the Academy of Science of Uzbekistan, Tashkent, Uzbekistan § Laboratoire de Physique des Solides, Univ. Paris-Sud, CNRS, UMR 8502, F-91405 Orsay Cedex, France ∥ Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Mattenstrasse 26, CH-4058 Basel, Switzerland ‡
S Supporting Information *
ABSTRACT: In modern medicine, effective protein therapy is a major challenge to which a significant contribution can be expected from nanoscience through the development of novel delivery systems. Here we present the effect of the amine content of nanoparticles based on PEGylated chitosan Bombyx mori (PEG-OChsBm) copolymers on the entrapment of molecules in a search for highly efficient nanocarriers. PEG-O-ChsBm copolymers were synthesized with amine contents from 1.12% to 0.70%, and nanoparticles were generated by self-assembly in dilute aqueous solutions. These nanoparticles successfully entrapped molecules with a wide range of sizes, the efficiency of which was dependent on their amine contents. While hydrophobic molecules were entrapped with high efficiency in all types of nanoparticle, hydrophilic molecules were entrapped only in those with low amine content. Bovine serum albumin, selected as a model protein, was entrapped in nanoparticles and efficiently released in acidic conditions. The triggered entrapment of molecules in PEG-O-ChsBm nanoparticles by selection of the appropriate amine content represents a straightforward way to modulate their delivery by fine changes in the properties of nanocarriers.
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INTRODUCTION There is a significant need in medicine today to improve therapeutic strategies, such as protein or gene therapy. The objective of protein therapy is to increase levels that are insufficient or to replace dysfunctional proteins related to pathological situations by protein delivery into cells.1 This approach has been already proposed for various pathologies, including cancer,2 diabetes,3 and brain diseases,4 but direct protein administration is in many cases impossible either because of the inactivation of sensitive proteins or low bioavailability.1 In this respect, the development of nanocarriers for proteins serves both to protect them from proteolytic attack and to deliver them in high amounts due to their localization in the carrier. The concept of a protein delivery nanocarrier involves 3D assemblies with sizes in the nanometer scale (particles, capsules, micelles, or vesicles) in which proteins are encapsulated/ inserted and then released upon changes in the overall architecture (degradation or stimuli-induced changes).5 Polymer 3D assemblies represent more efficient nanocarriers than lipidic ones, because of higher mechanical stability combined with the possibility to modulate their properties by chemical modifications.6 Natural polymers are of particular interest for generating nanocarriers because of their biocompatibility, © 2014 American Chemical Society
biodegradability, and nontoxicity, and products derived from dextran, cellulose, chitosan, or gelatin have been proposed as nanocarriers for protein delivery.7−9 In particular, chitosan, the N-deacetylated form of the linear polysaccharide chitin and the second most abundant natural polymer, with properties depending on the nature and quality of chitin, has been used to produce nanocarriers.10 However, chitosan has drawbacks for biomedical applications, namely poor water solubility11 and a high positive charge in acidic media (pH < 6.5), which induces blood-contact problems, such as hemolysis or thrombosis.12 To overcome these problems, chitosan was chemically modified by grafting poly(ethylene glycol) (PEG) chains at different points in the chitosan. PEGylation of chitosan has mainly been achieved by PEG grafting on amino groups of glucosamide units (N-modifications), but this approach induced changes in the chitosan skeleton and a loss of physicochemical properties.13 Grafting PEG onto OH groups (O-modifications) represents a better approach,14 because such PEG-O-chitosan derivatives are water-soluble over a broad pH range, and the O-modifications do not affect Received: July 30, 2013 Revised: January 10, 2014 Published: January 14, 2014 965
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purchased from Sigma Aldrich or Fluka. Bodipy (Invitrogen; Mw: 660.5 Da; Emax = 630 nm), sodium fluorescein (Fluka Chemika; Mw: 376.3 Da; Emax = 514 nm), isothiocyanate conjugate of bovine albumin (BSA−FITC) (Mw = 66 kDa), and all the solvents were used without further purification. Preparation of MPEG Iodide (MPEGI). Monomethyl ether poly(ethylene glycol) iodide (MPEGI) was obtained by iodation of the monomethyl ether of PEG with iodomethane (CH3I) in the presence of triphenylphosphate at 120 °C for 6 h under continuous stirring. After 6 h, the reaction was stopped by cooling to room temperature, and the reaction product was purified by redissolving in toluene and precipitating in diethyl ether. The precipitate was separated by filtration, washed twice with ether, and dried under vacuum. The final product was a light yellow color, and the reaction yield was calculated as 98.5%. N-Phthaloylation of Chitosan. Modifications of chitosan Bombyx mori are necessary to convert it to polymerizable organosoluble precursors. N-Phthaloylchitosan was synthesized by an etherification reaction and then used as a reaction intermediate with its free amine groups protected.36 A mixture of chitosan Bombyx mori (2.5 g; 15.5 mmol) (ChsBm) and phthalic anhydride (PHA) (6.9 g; 46.55 mmol) in dimethylformamide (50 mL) was heated under stirring at 130 °C in N2. After 5−7 h, the mixture was precipitated in ice−water and filtered. Further, the precipitate was extracted with ethanol in a Soxhlet apparatus and dried in air. The resulting N-phthaloylchitosan was obtained in a yield of 82−100%. Synthesis of PEG-O-Chitosan Graft Copolymers. PEG monomethyl ether iodide (MPEGI) was reacted with N-phthaloylchitosan at a molar ratio of 1:2 in DMF for 16 h under continuous stirring. To unprotect the amine groups protected by Nphthaloylation, an aqueous solution of hydrazine monohydrate was added and the reaction was continued for an additional 15 h at 90 °C under stirring. At the end of the reaction, excess hydrazine monohydrate was removed by distillation at reduced pressure with a Rotavapor R-215 (Büchi, Switzerland 230v 50/60 Hz). The solution was then dialyzed against water for 96 h (dialysis tubes Spectra/Por, Biotech, cellulose ester dialysis membrane MWCO 3500−5000), concentrated by distillation at reduced pressure, and finally freezedried with a Maxi_dry-Lyo/Plus system (Jonan Nordic A/S, Germany). The degree of O-substitution (DS) of chitosan was determined according to the content of initial amine groups of copolymers,37 which was obtained by potentiometric titration (eqs 2 and 3 of the Supporting Information). PEG-O-chitosan Bombyx mori copolymers were synthesized with different amine contents in the copolymers. We use the following nomenclature for the copolymers: PEG-O-ChsBm1.12; PEG-OChsBm0.84; PEG-O-ChsBm0.70 for copolymers with 1.12%, 0.84%, and 0.70% amine content, respectively. Methods. X-ray diffraction (XRD) was performed with a DRON-3 M X-ray diffractometer using monochromatic Cu K α-radiation, at a 22 kV voltage and 16 mA current intensity. The degree of crystallization of PEG-O-ChsBm samples was determined according to eq 1:
the free amino groups of the chitosan backbone. PEGylated chitosan self-assembled and formed 3D supramolecular assemblies (nanoparticles and micelles) with PEG as the hydrophilic outer shell, and this was then able to protect them from the reticuloendothelial system,15 prevent bacterial surface growth, decrease the plasma protein binding, and improve blood compatibility.16,17 Three-dimensional assemblies based on grafted copolymers of PEGylated chitosan have been intensively studied as drug delivery carriers,18 both for small molecular mass drugs19 and for larger biomolecules, such as insulin,20 heparin,21 DNA22 and siRNA,23,24 and albumin.25 A considerable number of studies have explored the entrapment and release behavior of drugs as a function of properties of 3D assemblies of PEGylated chitosan, such as the PEGylation degree26,27 and molecular mass of chitosan and PEG.28,29 In addition, PEGylated chitosan derivatives used to produce nanoparticles were able to increase the stability of insulin but did not affect its transport across cell membranes at physiological pH.30,31However, to the best of our knowledge, the effect of the total amine content of PEGylated chitosan nanoparticles on the entrapment and release behavior of drugs has not previously been evaluated. In this study, we aimed to establish whether the amine content of PEGylated-O-chitosan Bombyx mori-based nanoparticles affects the insertion efficiency of active compounds with a wide range of molecular masses and whether it can be used as a trigger to improve the effectiveness of delivery, especially for hydrophobic molecules and proteins. We synthesized poly(ethylene glycol)-O-chitosan Bombyx mori (PEG-O-ChsBm) graft copolymers with different amine contents (1.12%, 0.84%, 0.70%) and prepared nanoparticles by self-assembly in dilute aqueous solutions. First, we tested the ability of nanoparticles to entrap small molecular mass molecules (hydrophobic and hydrophilic dyes). Then we extended the entrapment study to high molecular mass biomolecules using a protein example. As a model protein we selected bovine serum albumin (BSA), a multifunctional nonglycosylated plasma protein responsible for maintaining the colloidal osmotic pressure of blood and which has been used as a plasma expander and for dialysis therapy in patients with liver diseases.32 By entrapment of BSA inside PEG-OChsBm nanoparticles we expected to trigger its delivery under specific conditions (a specific domain of pH) and overcome the conflicting results reported on risks and benefits of direct BSA administration to patients.33,34 The uptake/release of molecules inside/from PEG-O-ChsBm polymer nanoparticles was then assessed as a function of the amine content of nanoparticles. On the basis of entrapment differences, such nanoparticles support a straightforward approach for protein delivery in which intrinsic fine differences in properties of the nanoparticles determine the specificity and efficiency of the delivery. Our study represents a step further in understanding and using fine molecules’ properties/factors, such as the amine content of PEG-O-ChsBm nanoparticles, to modulate the entrapment and release of molecules (ranging from small mass molecules up to proteins).
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CK (%) = (IK − Ia)/IK × 100
(1)
where IK and Ia are the intensities of crystal reflex and amorphous dispersion, respectively. 1 H NMR was used to determine the molecular mass of copolymers and the presence of specific groups after each step of their synthesis. 1 H NMR spectra were recorded with a Bruker DRX-400 spectrometer in D2O for MPEG and CDCl3 for MPEGI, chitosan, and copolymers, respectively. Fourier transform infrared (FTIR) spectra of all reaction products were recorded on a Shimadzu 84005 spectrometer as KBr pellets. Self-Assembly of Chitosan-O-PEG Graft Copolymers. Aqueous solutions of chitosan-O-PEG graft copolymers were prepared in Millipore water at a concentration of 10 mg mL−1, and the water was evaporated under reduced pressure until a polymer film was formed. Water was added dropwise to the polymer film to achieve a final sample volume of 1 mL. The average size of the self-assembled
EXPERIMENTAL SECTION
Materials. Chitosan Bombyx mori with a degree of deacetylation of 88−96% (eq 1 of Supporting Information) and a molecular mass Mw of 151.8−418.0 kDa (Table S1 of Supporting Information) was synthesized from chitin Bombyx mori by heterogenic deacetylation.35 Poly(ethylene glycol) monomethyl ether (MPEG) (Mw 2000) was 966
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Scheme 1. O-PEGylation of Chitosan Bombyx mori (where R = CH2CH2(OCH2CH2)mOCH3 and PHA = phthalic anhydride)
Fluorescence Correlation Spectroscopy (FCS). FCS was performed with a Zeiss LSM 510-META/Confcor2 laser-scanning microscope equipped with a HeNe laser (633 nm) for hydrophobic dye, an Ar laser (488 nm) for hydrophilic dye, and a 40× water-immersion objective (Zeiss C/Apochromat 40X, NA 1.2), with the pinhole adjusted to 90 and 70 μm for hydrophobic and hydrophilic dyes, respectively. Solutions of polymer nanoparticles (5 mg/mL) with entrapped dyes (bodipy, λexcitation 630 nm; sodium fluorescein, λexcitation 488 nm) were measured at room temperature in special chambered quartz-glass holders (Lab-Tek; eight-well, NUNC A/S) that provide optimal conditions for imaging while reducing evaporation of the aqueous solutions. Intensity fluctuations were analyzed using an autocorrelation function with the LSM 510/Confocor software package (Zeiss, AG). Spectra were recorded over 100 s, and each measurement was repeated ten times; results are reported as the average of three independent experiments. Adsorption and bleaching effects were reduced by exchanging the sample droplet after 2 min of measurement. The excitation power of the HeNe laser was PL = 15 mW, and the excitation transmission at 633 nm was 30%. The excitation power of the Ar laser was PL = 200 mW, and the excitation transmission at 488 nm was 25%. To reduce the number of free fitting parameters, the diffusion times for free dye (τD bodipy = 56 μs; τD Nafluorescein = 41 μs) as well as the labeled albumin (τD BSA−FITC = 82 μs) were independently determined and fixed in the fitting procedure. The release of protein from nanoparticles was measured by FCS. The pH of solutions of loaded nanoparticles was decreased to acidic conditions and stabilized for equilibrium for 20 min; a drop (15 μL) was then placed on a quartz-glass holder and measured immediately. Before each experiment, a dye solution was measured under similar conditions. Cell Toxicity Assay. HeLa cells (2 ×104 per well) were cultured in Dulbecco’s Modified Eagle’s Medium (DEME) containing 10% fetal calf serum (FCS) for 24 h in a 96-well plate and then incubated with different concentrations of nanoobjects (50, 100, 150, 200, 300, 500 μg/mL) and 20 μg/mL doxorubicin for 24 h at 37 °C in 5% CO2. THP-1 cells were cultured in RPMI medium with10% FCS at a density of 2 × 104 cells per well. Nanoobjects with different concentrations (50, 100, 150, 200, 300, 500 μg/mL) and 20 μg/mL doxorubicin were added immediately and incubated for 24 h. Cell viability for both cell lines was tested using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay (Promega, Madison, WI). After 1 h incubation with MTS, the absorbance of each well was measured at 490 nm with a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA).
nanoobjects was reduced by repeated extrusions (11 times) through filters (0.4 μm pore diameter) using a miniextruder from Avanti-Polar Lipidics Inc. The resulting solutions were characterized by static and dynamic light scattering (SLS and DLS) and observed by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and cryo-TEM. Solutions of PEG-chitosan nanoparticles containing active molecules were prepared by the same method as described above for selfassembly but using PBS as solvent instead of water. Dyes and protein solutions in PBS were added dropwise to the copolymer film to entrap them in the self-assembled supramolecular structures (a final sample volume of 1 mLwas obtained). The concentration of bodipy was fixed at 1 μM. Bodipy was dissolved in ethanol and PBS (20:80%, respectively), and an aliquot (1 mL) was added to the chitosan-PEG solution. For hydrophilic dye, the powder was dissolved in an aliquot (1 mL) of PBS to reach the desired concentration of 100 μM. For protein, the powder was dissolved in 1 mL of PBS to reach a concentration of 10 μM. The final solution was stirred overnight and the sample then dialyzed for 48 h to remove excess dye. The fluorescent particles used in FCS are small and thus experience thermal motion in solution. Light Scattering. DLS and SLS were performed simultaneously to calculate the average molar mass (Mw), radius of gyration (Rg) and hydrodynamic radius (Rh). DLS and SLS were measured using an ALV laser goniometer with a linearly polarized He−Ne laser operating at a wavelength of 632.8 nm (JDS Uniphase). Copolymer solutions were maintained at a constant temperature of 20 ± 0.1 °C. Measurements were carried out by varying the scattering angle (θ) from 30° to 150° with a 10° stepwise increase. The viscosity of the solutions was assumed equal to that of pure water at 20 °C (η = 0.1 cP). The time correlation function G(t) was determined with an ALV/LSE-5004 correlator. Diffusion coefficients at zero deviation (D0) were evaluated from G(t) using both a nonlinear decay-time analysis and the Laplace inversion method (CONTIN algorithm). Small Angle Neutron Scattering (SANS). SANS data were collected at the D22 beamline of the Laue-Langevin Institute (ILL) in Grenoble, France. The average wavelength of the neutrons was λ = 6 Å and the polydispersity Δλ/λ = 10%. Two experimental configurations were used, with a sample−detector distance of 1.5 and 11 m (corresponding to high and low scattering vector q, respectively). The samples were contained in 1 mm thick quartz cells, at a temperature of 25 °C. Radial regrouping was done with GRASP software, developed at the ILL. The signal of pure D2O, measured in the same conditions, was subtracted as background correction. Transmission Electron Microscopy (TEM). TEM images were obtained with a Phillips EM400 electron microscope operating at 100 kV. Nanoobjects were negatively stained by adding 5 μL of 2% uranyl acetate solution and deposited on a carbon-coated copper grid. Excess uranyl acetate was removed under vacuum. Scanning Electron Microscopy (SEM). SEM images were recorded with a FEI Quanta-200 equipped with a Gatan 3View camera. All samples were prepared at a concentration of 1 mg/mL in water. An aliquot of solution was placed on a sheet of muscovite mica and dried at room temperature.
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RESULTS AND DISCUSSION Synthesis and Characterization of Grafted PEG-OChsBm. The synthesis of grafted copolymers of PEG-OChsBm was carried out in four steps: (i) synthesis of MPEGI based on MPEG activation by iodomethane using triphenyl phosphate to avoid hydrolysis of the product; (ii) protection of chitosan amine groups by 3-fold excess of phthalic anhydride under N2; (iii) reaction of N-phthaloylchitosan and MPEGI, 967
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Table 1. Physicochemical Parameters of PEG-O-ChsBm Nanoparticlesa
a
sample
NNH2 %
Mw (kDa)
Mw/Mn
DS (%)
ζ potential (mV)
DH (nm)
Dg (nm)
DTEM (nm)
ρ = Rg/RH
PEG-O-ChsBm PEG-O-ChsBm PEG-O-ChsBm
1.12 0.84 0.70
4.9 5.9 4.3
1.23 1.14 −
54 75 94
18.8 ± 1.3 14.9 ± 1.2 9.4 ± 1.8
126 ± 19 139 ± 14 143 ± 21
160 ± 40 155 ± 16 150 ± 19
190 140 210
1.20 1.11 1.05
DH: hydrodynamic diameter; Dg: diameter of gyration; DTEM: diameter by TEM; DS: degree of O substitution
Figure 1. (A) Small angle neutron scattering of PEG-O-ChsBm nanoparticles: PEG-O-ChsBm1.12 (solid symbols); PEG-O-ChsBm0.84 (empty symbols). Inset: Guinier plot approximation. (B) SAXS results (dots) and models: polydisperse solid spheres (dashed line), and porous spheres (solid line), described as a binary sphere model (see text for details).
and (iv) removal of N-phthaloyl groups by hydrazine monohydrate (Scheme 1). The characteristic FTIR bands of chitosan (Figure S1A, Supporting Information) are 3345 cm−1 for O−H and 2879 cm−1 for C−H groups, 1648 and 1572 cm−1 correspond to C O (amide I) and N−H (amide II) groups, respectively, and 1022 cm−1 for the C−O group of pyranose. The FTIR spectrum of the PEG-O-ChsBm copolymer (Figure S1B, Supporting Information) shows, in addition, the characteristic bands for the CH group at 2867 cm−1 and for CO groups at 1110 cm−1 (CO oscillation of PEG). The band at 3400 cm−1 indicates substitution of hydroxyl groups by the PEG chain. Efficient blocking of amine groups by deacetylation is indicated by the presence of the characteristic chitosan band at 1650 cm−1 (amide I) in the PEG-O-ChsBm copolymer spectrum. The 1H NMR spectrum of chitosan (Figure S2A, Supporting Information) is characterized by a peak at δ = 3.0 ppm, corresponding to H-2 from the glucosamine ring, and two weak peaks in the region from δ = 3.5 to δ = 3.8, characteristic of H-3 and H-6 of the glucosamine ring of chitosan. In the 1H NMR spectrum of PEG-O-ChsBm copolymer (Figure S2B, Supporting Information), peaks from the PEG oxymethyl group (the region from δ = 3.4 to δ = 3.8) overlap those of the chitosan proton signals. The degree of crystallization of ChsBm samples was determined as 53−60% ± 1%. Self-Assembly of PEG-O-ChsBm. Amphiphilic PEG-OChsBm grafted copolymers spontaneously self-assemble in aqueous medium and generate 3D supramolecular assemblies through the formation of hydrogen bonds between nonmodified glycosamine units of chitosan. Various interactions contribute to the self-assembly process, namely electrostatic
interactions from chitosan amine groups, hydrophobic interactions from CH2−CH2 and acetyl groups, and H-bonding interactions involving OH groups of chitosan.19 The solubilization effect of PEG chains on chitosan is based on a decrease in the number of hydrogen bonds between chitosan moieties induced by the presence of the PEG chains.20 Supramolecular assemblies generated by different PEG-O-ChsBm copolymers at a molar ratio PEG:chitosan of 1:2 and different amine contents were characterized by light scattering and a combination of TEM, SEM, and cryo-TEM. SLS analysis provided the values of the molar mass, Mw, and radius of gyration, Rg, while DLS provided the values of the hydrodynamic radius RH (Table 1). The ratios between Rg and RH, ρ = Rg/RH of PEG-O-ChsBm assemblies have values between 1.05 and 1.2 for all PEG-O-ChsBm copolymer assemblies. Theoretical values of the ρ ratio that characterize the architecture of supramolecular assemblies are 1.0 for thin vesicles, 0.779 for homogeneous hard spheres, and >1, up to 2 or 3, for polymers in extended conformations.38 The experimental values of ρ for PEG-O-ChsBm assemblies suggest that these nanobjects behave as porous spheres. The poroussphere model predicts a Rg/RH ratio ∼1.03, and the value increases as the particle size increases.39,40 The hydrodynamic diameter of particles decreased (from 143 to 126 nm) when the amine contents of copolymers increased (from 0.7% to 1.12%). This small effect is attributed to Hbonds between the amine groups of chitosan moieties and water. In contrast, the radius of gyration remained almost constant (Rg ∼ 75−80 nm) when the amine content was changed. The polydispersities (PDI) of copolymer particles were determined by DLS with the cumulant method using the 968
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Figure 2. Nanopaticles generated by self-assembly of PEG-O-ChsBm1.12 examined by (A) transmission electron microscopy, scale bar: 200 nm, (B) scanning electron microscopy, scale bar: 50 nm, and (C) cryotransmission microscopy, scale bar: 200 nm.
Figure 3. (A) FCS autocorrelation curves (continuous lines) and their fit (dotted lines) of free bodipy (a) and bodipy-PEG-O-ChsBm1.12 nanoparticles (b). (B) FCS autocorrelation curves (continuous lines) and their fit (dotted lines) of free sodium fluorescein (a) and sodiumfluorescein-PEG-O-ChsBm0.70 nanoparticles (b). Curves normalized to 1 to facilitate comparison.
improve the fit of the experimental curve and investigate the possible presence of porosity, we used a second model: Itotal(q) = Isph(q) + Ipores(q), where the pores are described as monodisperse spheres and their contribution is added incoherently (we neglected the positional correlation between pores or between pores and particles). The model based on a pore radius of 10 nm describes fairly well the excess scattering with respect to the first model between 0.01 and 0.03 Å−1, as illustrated by the solid line in Figure 1B. The volume fraction of the pores is more difficult to determine, because this parameter is perfectly correlated with the pore contrast (difference in electron density with respect to the particle density). If we consider that the pores are full of solvent, we can estimate their volume fraction at about half that of particles: Vpores/Vsph = 0.5. We used this value for the model shown in Figure 1B. In addition, SAXS data are consistent with SANS results and indicate the presence of spherical objects with a radius around 100 nm. However, the data quality is lower than for neutron scattering, mainly due to the soft nature of nanoparticles, which induces a reduced electron density contrast between particles and solvent. We emphasize that the measurements were done in state-of-the-art conditions: at a synchrotron facility, using a setup adapted to low-signal samples (both the sample and the solvent are injected into the same capillary, for optimal background subtraction). The morphology of the nanoobjects was estimated by TEM using negative staining (Figure 2A) and SEM (Figure 2B). The nanoobjects were nanoparticles, with mean diameter in the range 140 nm < D < 210 nm, dependent on the amine content
best fit for the angle range 30−90°. PDI values for all PEG-OChsBm copolymer particles after successive extrusions were 0.4−0.5, but we did not reduce further the size distribution of the supramolecular assemblies by additional extrusions to preserve the amount of copolymer. PEG-O-ChsBm nanoparticles are positively charged due to the amino groups of the chitosan backbone and thus should associate easily with negatively charged biomolecules, an important factor for entrapment of active compounds. To confirm the particle morphology, a combination of SANS and SAXS was performed. SANS analysis (Figure 1A) for nanoobjects based on PEG-O-ChsBm1.12 (solid symbols) and PEG-O-ChsBm0.84 (open symbols) shows that the intensity decayed markedly above q = 0.1 Å−1, which indicates the presence of small objects with a gyration radius estimated at Rg = 1.4 nm by a Guinier fit (Figure 1A inset). In addition, the PEG-O-ChsBm1.12 assemblies also contained large objects (typical size ≥100 nm), in good agreement with SLS and DLS results (Rg = 80 nm). SAXS data (Figure 1B) provides information on the overall size of the particles. The presence of several oscillations shows that the objects are relatively monodisperse. We plotted (as dashed line) the prediction of a sphere model, Isph(q), with mean radius ⟨R⟩ = 36 nm and polydispersity p = σ/⟨R⟩ = 0.03 (where the radius distribution is described by a Schulz function). The agreement is only qualitative, probably due to other types of objects (in particular, the increase of I(q) at small angles indicates the presence of larger aggregates), in agreement with the PDI values we obtained by DLS. To 969
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(Table 1). The difference between the Rh values obtained from DLS and those from TEM was due mainly to the presence of small aggregates, which were also confirmed by SEM (Figure S3 of the Supporting Information). To get more insight into the morphology of the spherical objects of ChBM-O-PEG, we used Cryo-TEM, which confirmed a porous spherical morphology (Figure 2C). While the size of pores of about 10 nm prevented a more detailed view into their internal structure, this value is in agreement with that obtained by SAXS analysis. The combination of methods we used for characterization of PEG-O-ChsBm nanoparticles did not indicate a difference in nanoparticle porosity as a function of the amine content, due to the small size of the pores and the soft nature of the nanoparticles (a reduced electron density contrast between particles and solvent). While we cannot exclude this, in the limit of experimental errors and with the specific resolution of the methods we used, the porosity of nanoparticles did not change upon variation of the amine content. Insertion of Small Molecular Mass Molecules into ChBm-O-PEG Nanoparticles. Insertion of various molecules ranging from small molecular mass molecules up to proteins represents an essential step in developing medical applications of PEG-O-ChsBm nanoparticles. As models for small molecular mass molecules, we investigated the entrapment of both hydrophobic (bodipy) and hydrophilic (sodium fluorescein) dyes and used fluorescence correlation spectroscopy to characterize their insertion inside nanoparticles. The change in diffusion time from τD = 56 μs for free bodipy to τD = 6273 μs for bodipy-containing bodipy-ChsBm-OPEG1.12 (Figure 3A) indicates that the hydrophobic molecule was inserted in polymer nanoparticles. The entrapment efficiency (EE) of the hydrophobic dye was 98.6% (eqs 4−7 of the Supporting Information), and all PEG-O-ChsBm nanoparticles allowed successful insertion of a hydrophobic dye (Figure S2 of the Supporting Information and Table 2), with EE decreasing as the amine content of the copolymer nanoparticles decreased. The change of the diffusion time τD = 41 μs for the free hydrophilic dye fluorescein to τD = 3603 μs for fluoresceinChsBm-O-PEG0.70 (Figure 3B) indicates a successful insertion of the hydrophilic small molecular mass molecule. However, the EE was significantly lower for the hydrophilic dye in
ChsBm-O-PEG0.70 than for the hydrophobic dye (Table 2). In addition, the hydrophilic dye was only inserted into nanoparticles with low amine content (PEG-O-ChsBm0.84; PEG-OChsBm0.70); for nanoparticles with higher amine contents, the insertion did not take place. To estimate the number of dye molecules per nanoparticle, we determined the molecular brightness as count rate per dye molecule (CPM in kHz) and compared it to the count rate per dye containing nanoparticle (eq 4 of the Supporting Information). The number of hydrophobic molecules present in nanoparticles was three times higher than that of hydrophilic molecules under the same entrapment conditions. The diffusion time allows the hydrodynamic radius of diffusing particles to be calculated using the Einstein−Stokes equation. When hydrophobic molecules were entrapped, the RH of nanoparticles decreased as the amine content decreased (Table 2). Nanoparticles with less amine content present a decreased hindrance effect (due to the H bonds of amino groups and water), which favors the entrapment of molecules (both hydrophobic and hydrophilic molecules were successfully entrapped inside). In addition, hydrophobic forces induce shrinkage of particles upon entrapment of hydrophobic molecules. A different dependence of the particle size as a function of amine content was observed in the case of entrapment of the hydrophilic dye: as the amine content decreased, the RH of nanoparticles increased (as the insertion was not associated with an increase of the hydrophobic forces). Thus, upon entrapment, the final size of the particles is affected by the nature of the entrapped molecules (hydrophobic/ hydrophilic). We observed two scenarios for the entrapment of small mass molecules: (i) a hydrophobic molecule can be entrapped in all PEG-O-ChsBm nanoparticles, whereas (ii) a hydrophilic molecule is inserted only in nanoparticles with low amine contents. PEG-O-ChsBm nanoparticles are positively charged (ζ potential, Table 1). We can explain the favored entrapment of the hydrophobic dye bodipy, which has a micellar conformation, by the presence of negative charges exposed to the aqueous phase. Together with hydrophobic interactions the electrostatic interactions support its entrapment inside PEG-OChsBm copolymer nanoparticles. The hydrophilic dye sodium fluorescein, at pH 7.2, has a pKa of 6.4, which means that under these conditions it is negatively charged. Therefore, the entrapment is favored by electrostatic interactions. However, the particles produce an intrinsic screening effect due both to the H-bonds of primary amines in chitosan and water and to a hindrance effect of the PEG shell, preventing the insertion of molecules.19,20 In the case of entrapment of hydrophilic molecules, the screening effect of nanoparticles becomes dominant and can block the entrapment for high amine contents. On the contrary, in the case of hydrophobic molecules, the electrostatic and hydrophobic forces predominate and support their entrapment irrespective of the amine content of nanoparticles. While we cannot exclude a difference, as the porosity did not change as a function of the amine content (in the limit of experimental errors and with the specific resolution of the methods we used), we consider that it does not play a role in the entrapment process. The values of EE obtained with our PEG-O-ChsBm nanoparticles are in the same range as those previously reported for entrapment of other hydrophobic compounds, such as calcitonin (EE of 52−44%),41 and methotrexate (EE of 95−88%)19 or hydrophilic compounds, such as ammonium
Table 2. Dye-Entrapment inside PEG-O-ChsBm Nanoparticles nanoparticle (NP) PEG-OChsBm1.12 PEG-OChsBm0.84 PEG-OChsBm0.70 PEG-OChsBm1.12 PEG-OChsBm0.84 PEG-OChsBm0.70
τ D, dye (μs)
τD, dye− NP (μs)
56a
DH (nm)
free dye fraction (%)
dye− NP fraction (%)
no. of dye/ NP
EE (%)
6273
185
27
73
3
98.6
56a
4832
143
39
61
3
64.2
56a
3966
117
43
57
4
44.3
41b
−
−
−
−
−
−
b
41
3391
230
38
62
1
26.1
41b
3603
244
33
67
1
21.8
Hydrophobic dye. bHydrophilic dye; EE: entrapment efficiency; τD: diffussion time a
970
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Figure 4. (A) FCS autocorrelation curves (continuous lines) and their fit (dotted lines) of free protein (a) and protein-PEG-O-ChsBm1.12 nanoparticles (b). (B) FCS autocorrelation curves (continuous lines) and their fit (dotted lines) of free protein (a) and protein-PEG-O-ChsBm0.70 nanoparticles (b). Curves normalized to 1 to facilitate comparison.
Table 3. Protein Release from PEG-O-ChsBm Nanoparticlesa
a
nanoparticle (NP)
τD, free protein (μs)
PEG-O-ChsBm1.12 PEG-O-ChsBm0.84 PEG-O-ChsBm0.70
82 82 82
τD, protein−NP (μs) DH (nm) 6456 5982 5577
free protein fraction (%)
protein−NP fraction (%)
no. of protein/NP
EE (%)
15 12 26
85 88 74
1 2 2
3.8 9.5 11.7
219 203 189
τD: diffusion time; EE: entrapment efficiency.
Figure 5. Protein release from PEG-O-ChsBm nanoparticles as a function of time: PEG-O-ChsBm1.12 (squares), PEG-O-ChsBm0.84 (triangles), and PEG-O-ChsBm0.70 (circles) at pH 6.5 (A) and pH 5.8 (B).
glycyrrhizinate (EE of 63−35%).42 To the best of our knowledge, this is the first study, which uses PEG-O-ChsBm nanoparticles with different amine content to entrap both hydrophobic and hydrophilic compounds and examines the specific conditions in each case. The entrapment process did not modify the morphology of nanoparticles, as indicated by a combination of TEM and LS (Supporting Information, Figures S5 and S6). Changes in the size of nanoparticles upon entrapment of the hydrophobic dye were observed, in agreement with the FCS results (see above). Entrapment and Release of a Protein in/from PEG-OChsBm Nanoparticles. A step further toward the development of medical applications was to study whether high molecular mass molecules, such as proteins, could be inserted into the PEG-chitosan nanoparticles, and we investigated the influence of amine content on EE. We selected BSA (Mw = 66 kDa) as a model protein and have used its negatively charged
backbone for pH > PI (isoelectric point PI = 4.7 at 25°) to support an efficient entrapment. The changes in diffusion time from τD = 82 μs for the free protein to τD = 6456 μs for the protein-containing PEG-OChsBm1.12 or to τD = 5577 μs for FITC labeled BSAcontaining PEG-O-ChsBm0.70 indicate that the protein was entrapped in the nanoparticles (Figure 4). At physiological pH (pH = 7.2), nanoparticles with an amine content of 1.12− 0.84% entrapped less protein than nanoparticles with a lower amine content (Table 3). As was explained above, RH decreased as the amine content decreased (see Table 3). Also, the RH values for protein-containing nanoparticles were slightly higher than those with small mass molecules, as expected from the large differences between the masses of the entrapped molecules (Mw dyes ∼300−600 Da; Mw protein ∼66 kDa). The EE increased from 3.8% to 11.7% as the amine content of the nanoparticles decreased. Thus, the screening effect produced by H-bonds of chitosan amine groups and water in 971
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coefficient R2. The kinetic release data for all protein-loaded PEG-O-ChsBm nanoparticles required a model for pH = 5.8 different from that for pH = 6.5 (Table 4). The release
the case of nanoparticles with high amine content (PEG-OChsBm1.12; PEG-O-ChsBm0.84) is stronger than the electrostatic interaction, which controls the entrapment in particles with lower amine contents (PEG-O-ChsBm0.70). We consider two factors which play a role in the analysis of proteinentrapment behavior: (i) Mw of the entrapped molecule, and of PEG, respectively, and (ii) the amine content of copolymers. Low Mw biomolecules, such as insulin Mw 5.7 kDa (EE 20− 29%)43 were entrapped easier than molecules with high Mw, such as BSA (with Mw 66.4 kDa), for which we obtained EE values of 3.8−11.7%. In addition, PEG with small molecular mass (M w PEG: 350 and 750 Da) 12,43 reduced the encapsulation efficiency less than PEG with high molecular mass, as is the case in our copolymers (Mw PEG: 2000 Da). Taken together with the effect of the amine content, these results indicate that fine details in the molecular properties of nanoparticles are important factors in the entrapment of bioactive compounds. Protein entrapment did not modify the morphology of the nanoparticles, exactly as was observed for the entrapment of small molecules (Supporting Information, Figure S7-A). The release of protein from nanoparticles was monitored as a function of time and pH by FCS (Figure 5). Nanoparticles successfully released the protein at pH 6.5 and 5.8. We note that the initial values of released protein were obtained at 20 min after the decrease of pH to allow complete pH equilibrium of the protein-containing nanoparticle solution (Figure 5). By decreasing the pH from 7.2 up to 6.5, nanoparticles with higher amine content (1.12% and 0.84%) released 80% of the protein after 500 min, whereas those with lower amine content (0.70%) released slightly less protein. This behavior was different at pH 5.8 where nanoparticles with the lowest amine content (0.70%) released 92% of the protein, while only 65% was released from the nanoparticles with higher amine contents. As the pI value of protein is 4.7, for the interval of pH we studied the release process, the protein is negatively charged, which means that the key factor for the release process is the overall charge variation associated with the decrease of pH. It is known that PEGchitosan particles tend to dissociate in acidic conditions due to intermolecular electrostatic repulsion of protonated amino groups of the chitosan moieties.20 At pH = 5.8, the overall positive charge in the environment of nanoparticles induces a dissociation process of particles, which depends on the amine content: particles with low amine contents tend to dissociate to a higher proportion than those with high amine content. This explains why particles with high amine content (1.12%) released only 65% of their protein after 500 min, compared with those with 0.7% amine, which released 92% of the protein. The difference in protein release at higher pH (6.5) is probably due to a lower intermolecular electrostatic repulsion of protonated amino groups of the chitosan moieties, which does not induce the dissociation of particles and therefore the complete release of protein. Interestingly, the particles with medium amine content had a dramatic change in their release profile after 250 min, when a burst of protein was observed. Ongoing experiments intend to clarify the factors, which influence the protein release in the case of medium amine content of particles. To analyze the mechanism of protein release, we used different release kinetic models, some of them already reported for chitosan nanospheres44 (Figure S8, Supporting Information). The criterion applied to determine the best model for the systems under investigation was the value of the correlation
Table 4. Protein Release Kinetic Parameters of PEG-ChsBM Nanoparticlesa first-order model, pH 5.8
Korsmeyer−Peppas model, pH 6.5
BSA−NP
K × l0−3 (min−1)
R2
K (min−n)
n
R2
PEG-ChsBM1.12 PEG-ChsBM0.84 PEG-ChsBM0.70
3.73 4.35 6.05
0.9674 0.9440 0.9818
0.1109 0.6589b 0.8939
0.45 − 0.45
0.9845 0.8683b 0.9206
a 2
R : correlation coefficient; K: release rate constant; n: release exponent. bPossible combination of mechanisms.
mechanism at pH 5.8 follows a first-order model, as the release is directly proportional to the protein concentration.45 Similar release kinetics has been already reported for PEG-Chs nanoparticles.46 At pH 5.8, the release rate constant increases with decreasing amine content of PEG-O-ChsBm nanoparticles, thereby promoting a fast protein release due to the content of H+ at this pH. At pH 6.5, the protein release kinetic was modeled with a Korsmeyer−Peppas model45 in which the release exponent, n, describes the drug release mechanism. A release exponent n = 0.45 indicates a Fickian diffusion process based on a molecular diffusion of the drug along a chemical potential gradient.47 Note that for PEG-ChsBM0.84 nanoparticles a different behavior was obtained (as mentioned above), presumably due to a combination of release mechanisms (footnote b in Table 4). Because the amine content of nanoparticles influences the release profiles of proteins by changing the pH, it can trigger a specific and efficient protein delivery. Depending on the biological conditions of the desired application, PEG-O-ChsBm nanoparticles with specific amine contents can be selected for protein entrapment and release “on demand” when the required conditions are met. Cell Toxicity of PEG-O-ChsBm Nanoparticles. The toxicity of nanoparticles was assessed in HeLa and THP-1 cell lines. The toxicity of chitosan depends on its physicochemical properties and the concentration of polymer,48 but this has been reported to be reduced by 10 and 20 times as a result of PEG-grafting on the chitosan backbone.26 The MTS assay with HeLa cells in the presence of chitosan-PEG nanoparticle concentrations up to 500 μg/mL resulted in >80% viability after 24 h incubation for all copolymer concentrations (Figure 6). Similar results were obtained when THP-1 cells were incubated with chitosan-PEG nanoparticles (up to a concentration of nanoparticles of 500 μg/mL), while a positive control using 20 μg/mL of doxorubicin significantly decreased the viability of both cell lines. The MTS assay demonstrates the nontoxicity of our PEG-O-ChsBm nanoparticles up to high concentrations on two different cell lines. The lack of toxicity together with the specific release profiles support the potential medical use of PEG-O-ChsBm nanoparticles for protein delivery. In addition, depending on the properties of the protein (charge, molar mass, and solubility), the amine content of PEG-O-ChsBm nanoparticles can be tailored to maximize the delivery efficiency. 972
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Figure 6. HeLa and THP-1 cell viability after 24 h incubation of (A) PEG-O-ChsBm1.12 and (B) PEG-O-ChsBm0.70 nanoparticles. A positive control based on incubation of cells with 20 μg/mL doxorubicin is included.
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CONCLUSIONS To develop efficient protein delivery systems, nanoparticles based on poly(ethylene glycol)-O-chitosan Bombyx mori copolymers with different amine contents (1.12%, 0.84%, and 0.70%) were generated by self-assembly in dilute aqueous solutions. Entrapment of both small mass molecules and proteins was modulated by the amine content of PEG-OChsBm nanoparticles in a specific way, dependent on the hydrophobic and hydrophilic nature of the molecules. Entrapped BSA−FITC could be released from the nanoparticles by decreasing the pH. Specific release kinetics was obtained for different pH values, due to the triggered effect of the amine content of nanoparticles, which provides conditions that can be adapted for a desired application. The fact that PEG-O-ChsBm nanoparticles entrap selectively hydrophobic or hydrophilic molecules suggests that they represent ideal candidates to deliver drugs “on demand”, by a straightforward selection of their properties that will support a wide range of biomedical applications. To the best of our knowledge, this is the first report indicating that the amine content of PEG-OChsBm nanoparticles acts as a trigger for the delivery of molecules and will serve to orient the strategy of entrapment in a more efficient way.
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performed the cell toxicity assay, and M.C. performed cryoTEM experiments. C.P. conceived the project. All authors have approved the final version of the manuscript. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS Financial support was provided by the Swiss National Science Foundation and the National Center of Competence in Nanoscale Science and is gratefully acknowledged. C.P. thanks Prof. W. Meier from the University of Basel for fruitful discussions. R.M. thanks the Swiss National Foundation for the International Short Visit fellowship, which served for the synthesis of the copolymers. D.V. acknowledges Gabriele Persy (University of Basel) for TEM measurements, and Patrick Judeinstein from Laboratoire Léon Brillouin (LLB) of the CEA-Saclay, for SANS experiments. D.V. thanks Adrian Najer (University of Basel) for the analysis of FCS experimental data. Authors thank Dr. B.A. Goodman for editing the manuscript.
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ASSOCIATED CONTENT
S Supporting Information *
Determination of the degree of deacetylation (DD), degree of O-substitution (DS), FTIR and 1NMR spectra of chitosan and copolymers, SEM micrograph of chitosan assemblies, determination of entrapment efficiency (EE), insertion of hydrophobic molecules in chitosan nanoparticles (TEM and DLS), and insertion and release of protein from chitosan nanoparticles (TEM). This material is available free of charge via the Internet at http://pubs-acs.org.
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REFERENCES
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AUTHOR INFORMATION
Corresponding Author
*Tel: +41-61-2673839. E-mail:
[email protected]. Author Contributions
The manuscript was written through contributions from all authors. R.M. performed the synthesis of copolymers, D.V. prepared the nanoparticles with different entrapped compounds and characterized them, D.C. performed SANS and SAXS, P.B. 973
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