Article pubs.acs.org/crystal
Calcium Carbonate Microparticles Growth Templated by an Oxadiazole-Functionalized Maleic Anhydride-co‑N‑vinyl-pyrrolidone Copolymer, with Enhanced pH Stability and Variable Loading Capabilities Marcela Mihai,*,† Mariana-Dana Damaceanu,† Magdalena Aflori,† and Simona Schwarz‡ †
“Petru Poni” Institute of Macromolecular Chemistry, Grigore Ghica Voda Alley 41 A, 700487 Iasi, Romania Leibniz-Institut für Polymerforschung Dresden e. V., Hohe Strasse 6, 01069 Dresden, Germany
‡
ABSTRACT: The crystallization characteristics of CaCO3 microparticles from supersaturate aqueous solutions in the presence of a conjugate drugcopolymer has been investigated, comparative with particles prepared in similar conditions but without polymer. The polymer conjugate, P(NVPMA-Ox), is based on poly(N-vinylpyrrolidone-co-maleic anhydride) as support and 2-amino-5-(4-methoxy-phenyl)-1,3,4-oxadiazole. The influence of the polymer/CaCO3 ratio on the microparticles' characteristics and the particles' pH stability was deeply investigated by scanning electron microscopy, X-ray diffraction, flow particle image analysis, particles charge density, and electrophoresis. The presence of P(NVPMA-Ox) as a template in the CaCO3 crystallization process induced the particles stability increase up to the polymer isoelectric point located at pH = 3.4, irrespective of carbonate content in composite particles. The adsorption capacity of the microparticles as a function of their characteristics was tested using methylene blue. The sorption capacity of composite materials increased with the increase of polymer content in the composites, suggesting that the sorption process takes place mainly by electrostatic interactions.
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INTRODUCTION A variety of self-organized and hierarchical microscopic skeletal structures composed of inorganic and organic components are produced in nature. Such complex, three-dimensional structures, if fabricated synthetically, could have numerous important applications. For example, porous inorganic microspheres with controlled meso- and macroporosity might find uses in catalysis, separation technology, and biomedical engineering.1−6 Calcium carbonate, one of the most abundant inorganic biominerals, exists as three anhydrous crystalline polymorphs (calcite, aragonite, vaterite), two hydrated metastable forms (monohydrocalcite and calcium carbonate hexahydrate), and an unstable amorphous phase. CaCO3 is pHsensitive and the large specific surface area and the capability to load various small molecules make CaCO3 an ideal candidate as a drug carrier.7,8 The application of CaCO3 particles is determined by a number of strictly defined parameters, such as morphology, structure, size, specific surface area, oil adsorption, chemical purity, and so on. 9−11 Among these factors, particle morphology is the most important one. As a consequence, controllable synthesis of calcium carbonate crystals with uniform size and morphology has been attracting considerable attention due to their fascinating mechanical and optical properties. A promising approach is to use organic additives and/or templates to control the nucleation, growth, and © 2012 American Chemical Society
alignment of inorganic materials. Such exploration of bioinspired morphosynthesis strategies using self-assembled organic superstructures, organic additives, and/or templates with complex functionalization patterns to template inorganic materials with controlled morphologies and textures has received much attention.3−6,8,11−13 pH-sensitive polymers, which are polyelectrolytes that bear in their structure weak acidic or basic groups that either accept or release protons in response to changes in environmental pH, can be incorporated into inorganic/organic hybrid materials that show both the advantages of inorganic materials (high mechanical stability) and conventional weak polyelectrolyte based materials (the controlled release/uptake properties of the composite microparticles resulting from changes in, among others, pH values and ionic strength).6,14−16 Besides the common applications of polyelectrolytes in the controlled release, the inorganic/organic materials can be also applied as mechanically stable microreactors for enzymatic reactions and synthesis employing gas phase reagents, in the form of hollow catalytically microcontainers. The inorganic part can also find medical application that, together with encapsulated drug material, can provide synergistic curing effects, i.e., the application of hydroxyapatitecontaining capsules in bone repair. Received: May 8, 2012 Published: August 8, 2012 4479
dx.doi.org/10.1021/cg301011c | Cryst. Growth Des. 2012, 12, 4479−4486
Crystal Growth & Design
Article
In this study, we prepared composite CaCO3 microparticles by crystallization from supersaturate aqueous solutions in the presence of a conjugate drug-copolymer. The conjugate molecule, synthesized by our group17 and denoted as P(NVP-MA-Ox), is based on poly(N-vinylpyrrolidone-comaleic anhydride) P(NVP-MA) as a support macromolecule and 2-amino-5-(4-methoxy-phenyl)-1,3,4-oxadiazole. Homoand copolymers of N-vinyl-2-pyrrolidone (NVP) are of considerable academic and industrial interest due to their unique properties allowing the use of these polymeric systems in lithography as light sensitive thin coatings for printing plates, for preparation of separation membranes for ultrafiltration, biocompatible polymers with low toxicity and carriers of biologically active compounds, sorbents, coagulants, and flocculants.18,19 The characterization of the obtained microparticles was performed by scanning electron microscopy, X-ray diffraction, flow particle image analysis, particles charge density, and electrophoresis. The influence of the polymer/CaCO3 ratio on the microparticles characteristics and on the particles' pH stability was thoroughly investigated. The adsorption capacity of the microparticles as a function of their composition and characteristics was tested using methylene blue (MB), known as dye for biological use, monoamine oxidase inhibitor,20 antimalarial drug21 or drugs component used in cancer therapy.22 To the best of our knowledge, this is the first investigation on the formation of stable CaCO3 particles by using this anionic copolymer, with advantageous properties concerning small cationic molecule uptake. These new composite materials may find useful application in the biomedical field, taking into account their biocompatibility properties and their enhanced pH stability and loading capabilities.
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Scheme 1. Chemical Structure of the Conjugate Polymer P(NVP-MA-Ox) and of Methylene Blue
composites was carried out in glass beakers, at 25 °C. For composite particles, first was prepared an 0.05 wt % P(NVP-MA-Ox) aqueous solution and then specific amounts of Na2CO3 were solved into it. Equal volumes of as prepared solutions (Na2CO3 with or without P(NVP-MA-Ox), and CaCl2), with Na2CO3 and CaCl2 having equal concentrations, were rapidly mixed; the pH of the mixtures was adjusted to 8.5. The mixtures were stirred for 10 min on a magnetic stirrer, at room temperature, and then the dispersions were kept under static conditions for 60 min. The obtained microparticles were separated by filtration, intensively washed with water, and finally washed with acetone and dried in an oven at 40 °C, for 1.5 h. The resultant microparticles were kept in hermetically closed tubes, at room temperature. In the following, the samples were coded CxPy, where x is the molar concentration of Na2CO3 and CaCl2 aqueous solutions and y is the polymer concentration in wt %. The concentration of the Na2CO3 and CaCl2 in the crystallization medium was varied between 0.1 and 0.3 M and the conjugate polymer concentration was zero or 0.05 wt %. Characterization of CaCO3/P(NVP-MA-Ox) Composite Microparticles. The particles' shape and surface were examined by using a FEI Phenom Desktop scanning electron microscope, in high vacuum mode. The size, size distribution, and the circularity of the carbonate particles were evaluated using the Sysmex Dynamic Flow Particle Image Analyzer 2100. To obtain correct values, the Sysmex FPIA 2100 image analyzer was checked before starting the main experiment by using certified size standards. The characterization of the samples by X-ray diffraction was carried out using a D8 Advance Bruker AXS device. The X-rays were generated using a Cu Kα source with an emission current of 36 mA and a voltage of 30 kV. Scans were collected over the 2θ = 20− 60° range using a step size of 0.01° and a count time of 0.5 s/step. The semiquantitative analysis was performed with an EVA soft from DiffracPlus package and an ICDD-PDF2 database, based on the patterns’ relative heights. The criteria used to compare the simulated and the measured scan is the R/R0 ratio, where R is the weighted reliability and R0 represents the inevitable discrepancy due to the statistics of the X-ray diffraction (noise modeled by the Poisson’s law). For an ideal fit R/R0 value is 1. Electrokinetic potential of carbonate samples was measured by means of ZetaSizer Nano ZS (Malvern, UK) operating at the wavelength 633 nm, using an MPT-2 automated titrator. The equipment measures the electrophoretic mobility of the particles and converts it into the zeta potential, using the von Smoluchowski equation. The results were expressed as the average of at least three independent measurements performed on 0.5 mg/mL aqueous dispersions, for each sample. The concentration of the charged groups in the examined solutions and microparticles dispersion, determined with the particle charge detector Mütek PCD 03 (BTG Instruments GmbH, Herrsching, Germany) was calculated from the amount of standard solution [poly(sodium ethylenesulfonate) or poly(diallyldimethyl-ammonium chloride), with a concentration of 10−3 M] needed to reach the zero
EXPERIMENTAL SECTION
Materials. CaCl2·2 H2O and Na2CO3 from Sigma-Aldrich were used as received. Methylene blue (MB) from Aldrich was used without further purification. The drug-copolymer conjugate P(NVP-MA-Ox) was synthesized and purified as was described in ref 17. Briefly, the reaction between maleic anhydride and N-vinylpyrrolidone takes place under stirring, on a water bath for 7 h, at 70−80 °C. The resulting P(NVP-MA) copolymer was separated by precipitation and washing three times in diethyl ether and dried under a vacuum. The molecular weights of P(NVP-MA) were measured by gel permeation chromatography, by using polystyrene standards (Mw = 7500 g/mol, Mn = 6700 g/mol, Mw/Mn = 1.12) and by viscometric measurements (Mv = 7600 g/mol). Then, the polymer was kept in a humidity atmosphere in order to obtain the acid form and dissolved in Nmethylpyrrolidone at 40 °C. The reaction with 2-amino-5-(4-methoxyphenyl)-1,3,4-oxadiazole takes place at 105 °C for 9 h using triphenylphosphite and pyridine as catalyst. The copolymer−drug conjugate was precipitated into a large amount of distilled water with constant stirring, washed thoroughly with distilled water and hot water, and dried in an oven at 80 °C under a vacuum for 6 h. The copolymer derivative was transformed in sodium salt by alkaline hydrolysis with 0.1 M NaOH aqueous solution, at 40 °C, and finally purified by diafiltration and recovered by freeze-drying. The oxadiazole derivative contents relative to maleic comonomer units (conversion degree), determined by both 1H NMR analysis and UV−vis spectroscopy, was estimated to be 29%. The molecular weights of oxadiazole-functionalized polymer are Mw = 6000 g/mol, Mn = 5700 g/mol, and polydispersity Mw/Mn = 1.05, as was measured by gel permeation chromatography. The chemical structures of P(NVP-MA-Ox) and MB are shown in Scheme 1. Preparation of CaCO3/P(NVP-MA-Ox) Composite Microparticles. The formation of CaCO3 and CaCO3/P(NVP-MA-Ox) 4480
dx.doi.org/10.1021/cg301011c | Cryst. Growth Des. 2012, 12, 4479−4486
Crystal Growth & Design
Article
value of the streaming potential. All measurements were run at room temperature. Methylene Blue Sorption on CaCO3/P(NVP-MA-Ox) Composites. The study of the sorption properties of the carbonate composites versus a cationic small molecule (MB) was carried out using a batch equilibrium procedure. Thus, 20 mg of dry composite material was placed in a flask and contacted with 5 mL of aqueous solution of MB with different concentrations, under gentle shaking, at room temperature, for 8 h. The residual concentration of MB remained in the supernatant was determined using a UV−vis spectrophotometer (UV−vis SPEKOL 1300, Analytik Jena), by measuring the absorbance in the supernatant at 665 nm, A665. The sorption efficiency, Cso, of the composite materials was calculated according to eq 1:
Cso = [(C0 − C)MV ]/(w· 1000), mg / g
different CaCO3 content, comparative with particles prepared without polymer. SEM images included in Figure 1 shows that the precipitate is made by more or less spherical aggregates having either spherulitic structure (CaCO3/P(NVP-MA-Ox) particles) or cauliflower shape (CaCO3 particles), each aggregate being composed by nanocrystallites. Calcite usually crystallizes as monocrystalline well-faceted particles. Vaterite particles, on the other hand, are polycrystalline, exhibit a spherical shape, and are built up by 25−35 nm crystallites.32 CaCO3/P(NVP-MAOx) particles of about 4 μm in diameter have been obtained when the initial Na2CO3 and CaCl2 aqueous solutions had the concentration 0.1 M (sample C03P05). Smaller particles, of about 2 μm in diameter, were obtained for the highest carbonate content (sample C03P05), whereas the sizes of C02P05 particles were between the values of the other two. For the same carbonate content, the presence of P(NVP-MA-Ox) has a significant role in the particles' formation, easily observed comparing samples CxP05 and CxP0. Thus, the particles' size decreases when CaCO3 crystallization takes place in the presence of P(NVP-MA-Ox), smoother particles with a lower surface porosity being evident comparative with particles in sample CxP0. The electrostatic complexation of carboxylic groups from copolymer and Ca2+ ions is also expected; therefore, by increasing the inorganic ratio the decrease of particle size occurred. Similar behavior was observed for other calcium carbonate systems, template on poly(acrylic acids).15 SEM images of some broken particles, shown as insets in Figure 2, showed that highly porous particles were obtained when CaCO3 crystallization takes place in the absence of the polymer, irrespective of initial solution supersaturation (Table 1). The composite particles inner morphology (insets in Figure 2) was strongly influenced by the polymer presence and less by the calcite/vaterite fraction. Almost the same structure were noticed in the insets in Figure 2a,c,e comparative with insets in Figure 2b,d,f, irrespective of carbonate content, probably due to the strong interaction between the carboxylic acid groups of P(NVP-MA-Ox) and the crystallizing CaCO3. The particle size distribution and the average values of circularity analysis of CaCO3 and CaCO3/P(NVP-MA-Ox) particles, obtained by FPIA measurements, are summarized in Figure 2 and Table 2. When comparing the FPIA data (Figure 2, Table 2) with SEM micrographs (Figure 1) a close agreement can be observed. Thus, the majority of the CaCO3/P(NVP-MA-Ox) particles have the particle size lower than 5 μm, the lowest particles size being obtained for the sample with the highest carbonate content (sample C03P05). The percent of counted particles with high values of particles diameter (>10 μm) can be ascribed to particle agglomeration during samples investigation, irrespective of studied samples. Moreover, the particles' mean diameter has significantly decreased when P(NVP-MA-Ox) was used in particle formation. The circularity is a measure to specify the shape of particles; the more spherical particle, the closer its circularity to unity, and the more elongated particle, the lower its circularity is observed. The value of circularity is defined as perimeter of equivalent circle vs perimeter of particle. As Table 2 shows, the circularity values suggest that the shape of almost all investigated samples is close to spherical, with a relatively small circularity distribution. However, the lowest value of mean circularity obtained for C03P05 sample (∼0.8) seems to be an artifact caused by the particles' tendency of agglomeration.
(1)
C0 and C are the concentrations of the dyes in aqueous solution (mol/L) before and after the interaction with the composite material, respectively; M is the molar mass of dyes (320 g/mol); V is the volume of the aqueous phase (mL); w is the amount of the dry composite material (g). C0 and C were indirectely determinated using the C/A665 etalon curve.
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RESULTS AND DISCUSSION The spontaneous precipitation of calcium carbonate from aqueous supersaturated solutions is triggered as a rule by a combination of factors including supersaturation and the presence of foreign particulate matter, which may act as a nucleation initiation point.23 The supersaturation ratio with respect to the polymorph (c-calcite or v-vaterite), Ωx, is given by eq 2: Ω x = (Ca 2 +)(CO32 −)/K sp ,x
(2)
In eq 2, parentheses denote the ion activity coefficients and Ksp,x is the thermodynamic solubility product of the polymorph (Ksp,c =3.313 × 10−9 for calcite, and Ksp,v =1.221 × 10−8 for vaterite).24 The activity coefficients were calculated from the extended form of the Debye−Hü c kel equation using SequentiX-WinIAP software.25 Table 1 summarized the values Table 1. Ion Activity Product (IAP) and the Solution Supersaturation with Respect of Calcite (Ωc) and Vaterite (Ωv) sample
[Ca2+] = [CO32−]
IAP, × 10−08
Ωc
Ωv
C01P0 C02P0 C03P0
0.1 0.2 0.3
1.522451 4.618999 8.909055
4.595385 13.94204 26.89120
1.246889 3.782964 7.296523
for ion activity product (IAP) and the initial solution supersaturation with respect of calcite (Ωc) and vaterite (Ωv), taking into account the initial molar concentration of solutions. As Table 1 shows, the preparation concentration of inorganic part used in this study ensures the solutions' supersaturation with respect of calcite (Ωc) and vaterite (Ωv). Thus, it is expected that by mixing the reactant solutions, first the thermodynamically unstable amorphous CaCO3 (ACC) is formed.26 Further, at low temperatures (
