Article pubs.acs.org/Biomac
Chitosan Nanogels by Template Chemical Cross-Linking in Polyion Complex Micelle Nanoreactors Flavia Maggi,† Serena Ciccarelli,† Marco Diociaiuti,‡ Stefano Casciardi,§ and Giancarlo Masci*,† †
Department of Chemistry, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy Dipartimento di Tecnologie e Salute, Istituto Superiore di Sanita, Viale R. Elena 299, 00161 Rome, Italy § Dipartimento di Igiene del Lavoro, INAIL ex-ISPESL, 00040 Monte Porzio Catone, Italy ‡
S Supporting Information *
ABSTRACT: Chitosan covalent nanogels cross-linked with genipin were prepared by template chemical cross-linking of chitosan in polyion complex micelle (PIC) nanoreactors. By using this method, we were able to prepare chitosan nanogels using only biocompatible materials without organic solvents. PIC were prepared by interaction between chitosan (Xn = 23, 44, and 130) and block copolymer poly(ethylene oxide)-blockpoly[sodium 2-(acrylamido)-2-methylpropanesulfonate] (PEO-b -PAMPS) synthesized by single-electron transfer− living radical polymerization (SET-LRP). PIC with small size (diameter about 50 nm) and low polydispersity were obtained up to 5 mg/mL. After cross-linking of chitosan with genipin, the nanoreactors were dissociated by adding NaCl. The dissociation of the nanoreactors and the formation of the nanogels were confirmed by 1H NMR, DLS, and TEM. The size of the smallest nanogels was about 50 nm in the swollen state and 20 nm in the dry state. The amount of genipin used during reticulation was an important parameter to modulate the size of the nanogels in solution.
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shape, and composition, some new technologies have been introduced such as liposomal template,13−16 temperatureinduced association,17 or inverse miniemulsion18 controlled radical polymerization (CRP) and photolithographic fabrication.19−21 Chitosan (CS) is a weak cationic linear biopolymer soluble in acidic solution (pH < 6.4) containing β-(1−4)-linked 2-amino2-deoxy-D-glucopyranose units partly acetylated mainly obtained by deacetylation of chitin extracted from the skeletal materials of crustaceans.22 The positive charge enable chitosan to be exploited for gene delivery by interaction with DNA and RNA or with other pharmaceutically active agents forming nanocarriers that can be internalized mostly through an endocytotic mechanism upon interaction with the cellular membrane.23 Furthermore, chitosan has the special ability of adhering to the mucosal surfaces24 and to open tight junctions between epithelial cells.25 For this reason, preparation of chitosan nanogels gained a lot of attention in recent years and has been extensively reviewed.5,6 The methods most frequently used to prepare chitosan nanogels are water-in-oil heterogeneous gelation by the inverse microemulsion method26,27 and aqueous homogeneous gelation based on chemical and physical cross-linking.28−34
INTRODUCTION Hydrogels are hydrophilic three-dimensional polymer networks that are able to take up large amounts of water or physiological fluid maintaining their internal network structure. Their high water content and low surface tension contribute to their biocompatibility. Nanogels, which vary in size from a few nanometers to 1000 nm, have added values such as the deployment in areas of the body not easily accessible after intravenous injection, intracellular drug delivery, and large surface area that allow easy multivalent conjugations and very rapidly response to environmental stimuli.1−7 Rigorous control over the size of nanocolloids is essential for their in vivo biodistribution. For instance, for intravenous administration where nanoparticles need to extravasate through the capillary endothelium to reach the target tissue, “leaky” vasculature can be found in liver, spleen, and bone marrow, but also in inflammation sites and tumors which allows extravasation of particles even up to 400 nm in diameter.8,9 Nanogels with small size can be particularly useful because they evade the reticuloendothelial system (RES) but, at the same time, should be cleared from the bloodstream by renal filtration, reducing risk of accumulation.10 To prevent dissolution of the nanogel in the aqueous environment, chemical cross-links involving the formation of covalent bonds are preferred as compared to physical crosslinks. This is mostly obtained by chemical cross-linking in (inverse) emulsion polymerization11 and precipitation polymerization.12 Recently, to obtain nanogels of well-controlled size, © 2011 American Chemical Society
Received: May 13, 2011 Revised: August 23, 2011 Published: August 25, 2011 3499
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= 85%) was used as received. Genipin was purchased from Wako GmbH. Chitosan Hydrolysis by Nitrous Acid. Low molecular weight chitosan samples were prepared by nitrous acid hydrolysis. 47 Commercial chitosan (2 g) was dissolved (10 mg/mL) in 60 mM HCl. The hydrolysis was started by adding the proper amount of a 0.1 M NaNO2 solution in order to obtain the desired degree of polymerization (1.11 × 10−4 mol to obtain Xn = 100). The reaction was carried out at 25 °C. At the end of the depolymerization, chitosan was precipitated by neutralizing with 1 M NaOH and then washed three times with distilled water. This product was finally dialyzed with distilled water and isolated by lyophilization. The molecular weight was determined by GPC. The samples will be named C26, C44, and C130, where the number represents the number-average degree of polymerization (Xn) of chitosan determined by GPC (Figure S1 of the Supporting Information). The degree of deacetylation after hydrolysis, determined by 1H NMR, was the same as the starting chitosan. Preparation of PEO-b-PAMPS. PEO114-Br macroinitiator was synthesized as previously described.48 Block polymerization of AMPS on PEO macroinitiator was carried out by SET-LRP using a method for AMPS polymerization already reported by us in a preceding paper.49 Briefly, PEO114-b-PAMPS20 was prepared in DMF:water 50:50 (v/v), T = 20 °C, [AMPS]:[PEO-Br]:[CuCl]:[CuCl2]:[Me6TREN] = 50:1:1:1:2 and [AMPS] = 1 M. A solution of AMPS (0.60 g, 2.61 mmol), PEO114-Br (0.267 g, 0.052 mmol of initiating groups), 0.65 mL of water, and 1.3 mL of DMF was prepared in a Schlenk tube and degassed purging with argon. In a 25 mL, two-necked, roundbottomed flask previously degassed by three vacuum−argon cycles, a Cu(I)−Cu(II)−Me6TREN water stock solution was prepared by adding 0.65 mL of degassed water to 5.12 mg (0.052 mmol) of CuCl, 6.96 mg (0.052 mmol) of CuCl2 and 26 μL (0.10 mmol) of Me6TREN. After taking from the Schlenk tube an initial sample to measure the monomer concentration at t = 0, the freshly prepared Cu(I)−Cu(II)−Me6TREN water stock solution was added. The polymerization was carried out at 20 °C. To measure the monomer conversion by HPLC, samples were withdrawn and diluted up in the eluent. At the same time, a given amount of reaction mixture was withdrawn and diluted in the GPC eluent for the molecular weight determination. The polymerization was stopped after 70 min at 40% conversion (Xn = 20) by diluting with water and bubbling with air for 5 min. The polymers were purified by extensive dialysis against distilled water. Then the solution was treated with a strong acid ion-exchange resin Dowex Marathon C (Na+ form) to completely remove Cu2+ ions, and the solid product was obtained by lyophilization. PEO114-bPAMPS40 was prepared with the same method starting from [AMPS]: [PEO-Br] = 100:1 and stopping the polymerization at 40% conversion. Molecular weight of the final polymers was confirmed by GPC and 1H NMR. Preparation of Polyion Complex (PIC) Micelles. To prepare 1 mg/mL PEO114-b-PAMPSx/chitosany PIC (AxCy, where x and y are the Xn of PAMPS and chitosan, respectively) in 0.1 M acetate buffer pH 4.3, PEO114-b-PAMPSx and Cy were separately dissolved in the solvent at a concentration of 1 mg/mL and left to stand overnight at room temperature to achieve complete dissolution. PIC micelles were obtained by quickly adding the proper amount of chitosan solution to 1.5 mL of PEO-b-PAMPS solution at room temperature with vigorous stirring. The mixture was left to equilibrate until a stable scattered intensity was obtained. Obviously, since kinetic control of the complexation is reasonably possible, the results obtained in this work have to take into account the technique of mixing used. If not otherwise stated, the mixing fraction f+ = n+/(n+ + n−), with n+ and n− being the moles of positive and negative charges in solution, respectively, was fixed to 0.5 in order to obtain complete charge neutralization. PIC micelles will be named AxCy, with Ax and Cy being the PEO114-b-PAMPSx and chitosany polymers, respectively. Dissociation of the PIC Micelles by Addition of NaCl. PIC micelles were dissociated by adding a 2 M NaCl solution. After each addition of NaCl we waited 10 min before measurements. This time was sufficient to obtain a stable measure. PIC dissociation as a function
Using the first method, tumor targeted chitosan-based nanogels cross-linked with glutaraldehyde and containing doxorubicin-modified dextran with a diameter of 100 nm were prepared for drug delivery applications.26 Ohya et al. was the first to present data about chitosan nanospheres for drug delivery applications of an anticancer drug using a water-in-oil emulsion method followed by glutaraldehyde cross-linking.27 For the approach based on chemical and physical crosslinking in water, the gelation is generally carried out in diluted solutions in order to prevent macroscopic gelation. Chitosanbased nanogels (60−120 nm) were prepared by covalent chemical cross-linking based on a carbodiimide coupling of chitosan with an oligo(ethylene glycol) dicarboxylic acid in water.29 Stable pH-responsive chitosan nanogels with a diameter of 70−80 nm were prepared by reaction of chitosan with ethylenediaminetetraacetic dianhydride in water.30 Chitosan nanogels with diameter ranging from 200 and 400 nm were prepared by physical cross-linking with tripolyphosphate (TPP).28,31 Chitosan physical nanogels were also prepared by electrostatic interaction with various polyelectrolytes. 32−34 This type of nanoparticle is usually named polyion complex or interpolyelectrolyte complex. Aiming to the preparation of covalently cross-linked chitosan nanogels with size below 100 nm, in this paper we report the use of polyion complex micelles (PIC) as nanoreactor for template cross-linking of chitosan. PIC are formed by selfassembly of oppositely charged polyelectrolytes, driven by ionic interactions between the ionogenic groups of the polymer partners.35−43 To avoid undesired aggregation, double hydrophilic block copolymers are frequently used, consisting of neutral and ionogenic blocks, in which the neutral block will form the shell of PIC micelles. Poly(ethylene oxide) (PEO) is widely used as a neutral block because of its biocompatibility and low toxicity. Numerous studies have shown that the size and shape of the formed PIC nanoparticles significantly depend on the polymerization degree of PEO block, medium conditions, and molar ratio between the polyelectrolyte blocks. By controlling these parameters, small and highly uniform in size PIC can be obtained with diameter of 20−50 nm and a coacervate core of 5−15 nm.35−37 In this paper we will report the preparation of PIC micelles between chitosan and the block copolymer poly(ethylene oxide)-block-poly[sodium 2-(acrylamido)-2-methylpropanesulfonate] (PEO-b-PAMPS) synthesized by SET-LRP. This procedure allows the preparation of small chitosan nanogels using only biocompatible materials without organic solvents. Formation of PIC micelles will be studied as a function of polyelectrolyte chain length, mixing ratio, polymer concentration, and ionic strength. PIC micelles will be used as nanoreactors for cross-linking of chitosan with genipine, a naturally occurring cross-linker.44,45 The nanoreactors will be dissociated by increasing the ionic strength in order to obtain the free nanogels.
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EXPERIMENTAL SECTION
Materials. Tris(2-dimethylaminoethyl)amine (Me6TREN) was synthesized as previously described.46 Poly(ethylene oxide) methyl ether with number-average molecular weight 5000 (PEO114-OH, Mw/ Mn = 1.03) from Aldrich were used as received without purification. CuCl from Fluka was washed with acetic acid followed by methanol to remove impurities. CuCl2 from Fluka was used as received. AMPS acid form (Aldrich) was converted in the sodium form by neutralizing an aqueous solution up to pH 7.5. The salt was isolated by freeze-drying. Chitosan (low viscous, Fluka, Mn = 1.5 × 105, degree of deacetylation 3500
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GATAN GIF energy filter and a 794 IF Peltier cooled slow scan charge-coupled device multiscan camera. To enhance image contrast and resolution, chromatic aberrations were eliminated by collecting only elastic electrons (ΔE = 0). Briefly, a droplet of a PIC or nanogel solution (0.1 mg/mL) in 0.05 M acetic acid or 0.1 M acetate buffer pH 4.3 was deposited onto 400 mesh copper grids covered with a very thin (about 20 nm) amorphous carbon film. The excess of liquid was removed by placing the grid onto a piece of filter paper. Finally, the grid was dried at room temperature. Samples were observed staining with 2% w/v phosphotungstic acid (PTA) buffered at pH 7.3.
of NaCl concentration was followed by DLS, measuring the scattering intensity and the hydrodynamic diameter, and by 1H NMR. Cross-Linking of PIC Micelles with Genipin. PIC micelles (1 mg/mL in 0.1 M acetate buffer pH 4.3) were cross-linked by adding the proper amount of a 10 mg/mL solution of genipin prepared in the same solvent. Different molar ratios between genipin and the total amount of glucosamine group present in the PIC micelles were used (1:1, 2:1, 4:1, 10:1). Cross-linking was carried out at 25 °C under gentle stirring in a shaking water bath for 4 days. After this period, a stable pale blue coloration as a result of the reticulation was observed and a stable DLS scattered intensity was measured. Dissociation of the Cross-Linked Nanoreactors by Addition of NaCl. The chitosan nanogels obtained after genipin cross-linking but still inside the nanoreactors were released by adding a concentrated solution (2 M) of NaCl. After each addition of NaCl we waited 10 min before measurements. This time was sufficient to obtain a stable measure. The release of the nanogels as a function of NaCl concentration was followed by DLS and 1H NMR. After addition of NaCl, the solution containing the nanogel and free PEO-b-PAMPS chains has been dialyzed first against 0.1 M acetate buffer + 0.6 M NaCl and then against distilled water in dialisys tube with 50 000 Da cutoff (Float-A-Lyzer G2, Spectrum). The residual solution in the dialysis tube was lyophilized. The lyophilized materials was dissolved in CD3COOD (0.05 M) in D2O for 1H NMR and 0.1 M acetate buffer for DLS. Instrumentation. HPLC. HPLC experiments for the determination of monomer conversion were carried out using a LabFlow 4000 HPLC pump (LabService Analytica, Bologna, Italy) equipped with a Knauer K-2501 UV detector and a C18 (Phenomenex Luna, 5 μm) column. AMPS was analyzed eluting with tetramethylammonium chloride (0.01 M)/acetonitrile 98/2 (v/v). The injection volume was 20 μL. DMF was used as internal standard. GPC. Molecular weight distributions were obtained using a GPC system equipped with a LabFlow 4000 HPLC pump, TSK-GEL α3000 (30 cm × 7.8 mm i.d., 7 μm) column and a Shimadzu RID-10A refractive index detector (Shimadzu, Kyoto, Japan). The columns were thermostated at 25 °C. PEO-b-PAMPS was analyzed by eluting at a flow rate of 0.8 mL/min with 0.2 M NaCl and using monodisperse PEO standards. Relative molecular weights were obtained in this case. Lactose was used as volume marker. The concentration of the polymeric solutions was 1 mg/mL. The injection volume was 50 μL Chitosan was analyzed by eluting with 0.25 M acetate buffer (pH 4.7). Monodisperse pullulan standards were used for calibration. Molecular weights were obtained using the universal calibration method. 1 H NMR. 1H NMR spectra were recorded at 298 K on a Varian XL300 spectrometer operating at 300 MHz. 1H NMR spectra of PIC micelles and nanogels were obtained preparing 5 mg/mL solutions in CD3COOD (0.05 M) in D2O. Dissociation of PIC and nanoreators was carried out by adding a 2 M NaCl solution prepared in the same solvent. Dynamic Light Scattering (DLS). DLS data were obtained with a Brookhaven Instruments Corp. BI-200SM goniometer equipped with a BI-9000AT digital correlator using a solid-state laser (125 mW, λ = 532 nm). If not otherwise stated, measurements of scattered light were made at a scattering angle θ of 90°. The temperature of the copolymer solution was controlled with accuracy of 0.1 °C. All samples were prepared by filtering PIC solutions with a 0.45 μm Millipore filter (Durapore) into a clean scintillation vial. Experiments were carried out at 25 °C. The solution was allowed to equilibrate until stable scattered intensity was obtained. Experiment duration was in the range of 5−20 min. Cumulant analysis or CONTIN was used to fit the data to obtain the z-average hydrodynamic diameter (Dh). The data reported were calculated as the average of three different measures carried out preparing three different PIC and nanogels batches for each sample. The error reported in the tables was calculated as standard deviation of these three measurements. Energy-Filtered Transmission Electron Microscopy (EF-TEM). The samples were observed with a FEI TECNAI 12 G2 Twin (120 KeV) transmission electron microscope equipped with a “postcolumn”
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RESULTS AND DISCUSSION Preparation of PEO-b-PAMPS. Two block copolymers with different length of PAMPS block (Xn = 20 and 40, as calculated by the conversion of AMPS measured by HPLC) were prepared (PEO114-b-PAMPS20 and PEO114-b-PAMPS40) by polymerizing AMPS starting from a PEO-Br macroinitiator using SET-LRP. The polymerization of AMPS with this method has been previously reported in a preceding paper. 49 It has to be underlined that at the time the polymerization was though to be an atom transfer radical polymerization (ATRP) or “waterborne” ATRP. However, this polymerization was carried out using CuCl/Me6-TREN as a catalyst in binary mixtures of organic solvents and water. Under these conditions, extensive disproportionation has been demonstrated, and therefore, SET-LRP is the right mechanism. 50−57 This polymerization proceeds using “nascent” Cu(0), generated in situ by the disproportionation of Cu(I) precursor, as a catalyst. Cu(0) acts as electron donor, and the initiator and dormant propagating species act as electron acceptors. The length of the PAMPS block was confirmed by 1H NMR and GPC (GPC chromatograms in Figure S2 of the Supporting Information). The polydispersity of PEO114-b-PAMPS20 and PEO114-b-PAMPS40 was 1.17 and 1.16, respectively. Preparation of Polyion Complex Micelles. Effect of the Polyelectrolytes Chain Length. PIC micelle formation should proceed as reported in Figure 1. The PIC micelles
Figure 1. Schematic representation of PIC micelle formation, crosslinking, and dissociation of the nanoreactor.
prepared in this work are expected to behave as lyophobic colloids,58 which means that kinetic aspects like exchange or addition reactions and structural rearrangements could be important. Therefore, to avoid inaccurate particle characterization, all size and shape data were determined 48 h after complexation. In any case, size and shape were measured at different time 3501
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after preparation. We did not observe significant differences between measures carried out after 1 h, 48 h, and 2 months after PIC preparation. After 48 h the size of the PIC micelles was similar to that observed after 1 h within 10%. Only a slight decrease of the polydispersity index was observed. The size was the same also after 2 months, indicating great stability of the micelles. Three samples of chitosan having different number-average degree of polymerization Xn (C26, C44, and C130) and two block copolymers (PEO114-b-PAMPS20 and PEO114-b-PAMPS40) were used to prepare the PIC micelles (that will be named AxCy, where x and y are Xn of PAMPS and chitosan, respectively) in 0.1 M acetate buffer (pH 4.3) at a polymer concentration of 1 mg/mL. The final concentration of the PIC micelles (CPIC) was 1 mg/mL. The use of a pH 4.3 buffer allowed us to have almost all chitosan amino groups ionized.59 Because of the complete ionization of the strong acid PAMPS independently on pH, we have been able to work maximizing the interpolyelectrolyte interactions. Chitosan, PEO, and PAMPS lengths were chosen taking into account the important role of these parameters in PIC micelles stabilization, prevention of the formation of macroscopic aggregates, and precipitation.38−41 Cohen Stuart et al. demonstrated that the molar ratio between the neutral and the polyelectrolyte block had to be equal to or higher than 3 in order to prevent precipitation of PIC nanoparticles and obtain coacervate PIC core near-stoichiometric ratio.41 As the aim of our work is to obtain small nanogels (
