Pectin and Nonstoichiometric

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Auto-template microcapsules of CaCO3/pectin and nonstoichiometric complexes as sustained tetracycline hydrochloride delivery carriers Marcela Mihai, Stefania Racovita, Ana-Lavinia Vasiliu, Florica Doroftei, Cristian Barbu-Mic, Simona Schwarz, Christine Steinbach, and Frank Simon ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09333 • Publication Date (Web): 03 Oct 2017 Downloaded from http://pubs.acs.org on October 4, 2017

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ACS Applied Materials & Interfaces

Auto-template microcapsules of CaCO3/pectin and nonstoichiometric complexes as sustained tetracycline hydrochloride delivery carriers Marcela Mihai,* Stefania Racovita, Ana-Lavinia Vasiliu, Florica Doroftei, Cristian Barbu-Mic. "Petru Poni" Institute of Macromolecular Chemistry of Romanian Academy, 41A Grigore Ghica Voda Alley, 700487 Iasi, Romania Simona Schwarz, Christine Steinbach, Frank Simon Leibniz-Institut für Polymerforschung Dresden e.V., Hohe Straße 6, D-01069 Dresden, Germany

KEYWORDS. microcapsule, CaCO3, pectins, nonstoichiometric polyelectrolyte complex, tetracycline hydrochloride.

ABSTRACT. New types of composites were obtained by an auto-template method for assembling hollow CaCO3 capsules by using pH sensitive polymers. Five pectin samples – which differ in the methylation degree and/or amide content – and some nonstoichiometric polyelectrolyte complex dispersions, prepared with the pectin samples and poly(allylamine

ACS Paragon Plus Environment

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hydrochloride), were used to control the crystal growth. The morphology of the composites was investigated by scanning electron microscopy and the polymorphs characteristics by FTIR spectroscopy. The presence of the polymer in the composite particles was evidenced by X-ray photoelectron spectroscopy, particles charge density and zeta-potential. The new CaCO3/pectin hollow capsules were tested as possible matrix for tetracycline hydrochloride carrier. The kinetic of the drug release mechanism was followed using Higuchi and Korsmeyer-Peppas mathematical models.

INTRODUCTION Recent advances in medicine had induced the development of advanced drug delivery systems able to encapsulate a wide variety of therapeutics such as labile drugs, proteins, chemotherapeutics and nucleic acids. Advanced drug delivery systems could improve the therapy by increasing the efficacy and duration of drug activity, decreasing dosing frequency, improving targeting for a specific site, reducing the unwanted side effects. Advanced drug delivery systems should be “smart”, which means that they are able to deliver the cargoes at a specific time, place and/or at a specific stimulus (pH, temperature, ionic strength). Among the large variety of advanced drug delivery systems that have been developed, drugloaded nano- and micro-particles attained high attention due to their facile administration by injectable and oral drug delivery.1,2 However, many current strategies yielding nano- and microparticles are limited by the formation of potentially toxic byproducts using organic solvents or mineral oils as reaction environments. Moreover, non-reacted monomers, initiators, and surfactants present safety issues in the final product. Microcapsules are materials with a unique core–shell structure and high surface/volume ratio. The inner hollow core can be loaded with


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various cargoes or can act as a confined space, whereas the outer semi-permeable wall can be endowed with versatile functionalities. The composite capsules combine the properties of organic and inorganic components and can integrate multiple functionalities, and form hierarchical structures. A large variety of methods, including molecular self-assembly,3 layer-bylayer assembly,4 and biomimetic mineralization,5 were developed for the design and synthesis of composite capsules. Many efforts to prepare composite capsules have been focused on using sacrificial “hard template” (silica spheres,6 porous carbon7) and “soft template” (emulsion droplets,8 surfactant vesicles,9 block copolymer micelles10). However, the main disadvantages of the template-based methods are that calcination at high temperatures is often required for template removal and some processes use chemical etching to remove the core which implies the use of dangerous chemicals leading to hazardous wastes. Alternatively, the formation of hollow capsules without a template was tested mainly based on localized Ostwald ripening (the change of an inhomogeneous structure over time, i.e., small crystals or sol particles dissolve, and redeposit onto larger crystals or sol particles)11 or differential diffusion (Kirkendall effect – the motion of the boundary layer between two metals that occurs as a consequence of the difference in diffusion rates of the metal atoms).12 Calcium carbonate (CaCO3) is a common mineral that exists as three anhydrous crystalline polymorphs (calcite, aragonite, vaterite), two hydrated metastable forms (monohydrocalcite and calcium carbonate hexahydrate), and an unstable amorphous phase. The vaterite is the least stable and recrystallizes in water to non-porous calcite (the most stable polymorph) within some hours via Ostwald ripening.13 Extensive attention has been paid to CaCO3 mineralization mechanism and biomimetic synthesis, due to its wide application in different industries. Due to


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its biocompatible and biodegradable nature, a strong interest for pharmaceutical and biomedical application has emerged in recent years.14,15 In previous studies, the synthesis of hollow CaCO3 structures under various organic compounds control has been followed.16-18 Our studies showed that in the presence of poly(2acrylamido-2-methylpropanesulphonic

acid-co-acrylic

acid),

both

microparticles19

and

microcapsules20 with almost spherical shape could be obtained by controlling the nucleation and crystal growth in certain crystallization conditions: concentration of calcium and carbonate ions in the initial solutions, reaction time, pH, concentration of the polymer. CaCO3/polymer composite microcapsules are of increased interest due to their ideal biocompatibility and biodegradability, of critical importance for the clinical application. The large specific surface area and the ability to load various drugs also make CaCO3/polymer composites ideal candidates as a drug carrier.21,22 Some pectin samples (which differ by the methylation degree and/or amide content) and different carbonate sources (sodium carbonate, diethylcarbonate, and ammonium carbonate) were used in a previous study as control for CaCO3 crystal growth.23 It was shown that the composites characteristics (pectin concentration in anionic groups or the amide group presence, and also the calcite/vaterite ratio) influence the sorption capacity against Cu(II) and Ni(II) ions. The sorption studies suggested that the CaCO3/pectin composites may act as matrix for pectin transport through the gastrointestinal tract, with sorption properties against heavy metal ions. Tetracycline is the most important broad-spectrum antibiotic produced by the Streptomyces genus of the Actinobacteria, used against many bacterial infections. Microencapsulation of tetracycline hydrochloride (TCH) prolongs its action, reduces the number of dosing, and also solves the problem of bitter taste and photosensitivity. The local delivery of TCH was proven to


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be effective in controlling localized periodontal infection without apparent side effects.24 A recent study encapsulated TCH in the CaCO3 microstructure by involving macromolecular assembly of anionic polypeptide with cationic peptide-oligomer.25 The results showed that pHdependent drug release enables selective cytotoxicity toward cancer cell lines as compared to the normal cells, thus having the potential for further development of therapeutic applications. In this context, this paper aims to develop an auto-templating method for assembling hollow CaCO3 capsules controlled/induced by pH sensitive polymers, as possible matrix for TCH carrier with prolonged action. Even if there are numerous studies on polyanions control of CaCO3 growth,26-30 according to our knowledge no studies concerning the use of nonstoichiometric polyelectrolyte complexes (NPEC) as templates for controlling CaCO3 crystals growth as autotemplate microcapsules have been reported up to now. Therefore, in this study, new composite microparticles/microcapsules were obtained by CaCO3 mineralization from supersaturated solutions under the control of some pectin samples and corresponding NPECs with different molar ratios (0.5, 0.9, and 1.2). The morphology, the polymer presence in the composites, particles mean size and the hollow structure of the composites were deeply investigated. The new obtained CaCO3/polymers microcapsules/microparticles were tested as advanced drug delivery systems using TCH as model drug, in vitro, as a function of composites properties, the kinetic of its release mechanism from microparticles being followed using Higuchi and Korsmeyer-Peppas mathematical models.

EXPERIMENTAL SECTION Materials


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CaCl2⋅2 H2O, Na2CO3, NaOH, and tetracycline hydrochloride from Sigma-Aldrich were used as received. Five pectin samples (Table 1) from Herbstreith &Fox KG, Neuenbürg, Germany were used. Poly(allylamine hydrochloride) (PAH) of low molar mass from Fluka was used without further purification. The chemical structures of the carboxylate pectin, PAH and TCH are shown in Scheme 1.

Preparation of polyelectrolyte complexes The pectin samples listed in Table 1, as polyanions, and PAH, as polycation were used for the preparation of NPECs. Aqueous solutions of 10-3 M pectins (taking into account each pectin charge density) and 10-2 M PAH were used in NPEC preparation. The dispersions of NPECs were prepared at room temperature, by mixing the solutions of oppositely charged polyelectrolytes in adequate proportions, according to the desired mixing molar ratio, n+/n- (0.5, 0.9 and 1.2). The quantity of pectin was maintained constant in one complex series, while the quantity of PAH varied depending on the n+/n- molar ratio. The PAH solution was added dropwise to the pectin solution, under magnetic stirring. After mixing, the formed dispersions were stirred 60 min and were characterized and used in composite preparation after 24 h.

Preparation of CaCO3/pectins and CaCO3/NPEC composite microcapsules The composite microcapsules were obtained through co-precipitation of Na2CO3 and CaCl2 solutions in the presence of pectins or NPEC dispersions. The 10-3 M pectins aqueous solutions were first prepared (24 hours prior use) and then Na2CO3 was dissolved aiming to obtain a concentration of the inorganic part of 0.05 M. For CaCO3/NPEC composites formation, 0.05 M Na2CO3 was dissolved into NPEC dispersion. The CaCO3 particles were formed under continuous stirring by adding equal volumes of CaCl2 (0.1 M) over the Na2CO3 (0.05 M)


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containing pectin solution (10-3 M) or NPEC dispersion. Immediately after stirring, the pH of the dispersions was adjusted to 10.5 ± 0.1 by dropwise addition of 1M NaOH. The obtained composite dispersions were stirred at 200 rpm for 8 hours at room temperature and then kept in static conditions for 60 minutes. The particles were separated by filtration, washed intensively with distilled water and then with acetone, dried in air for 24 hours and kept at room temperature in hermetically closed containers.

Characterization methods The morphologic characterization of the obtained particles was conducted with the Scanning Electron Microscope type Ultra plus (Carl Zeiss NTS) whereas samples after TCH loading/release were characterized with the Environmental Scanning Electron Microscope type Quanta 200, in high vacuum mode. To avoid electrostatic charging, the samples were coated with a 3 nm layer of platinum or gold. The energy dispersive X-ray (EDAX) system on Quanta 200 microscope was used for qualitative and quantitative analysis. The mean size of composite particles was determined from the overview SEM images and at least 20 particles using the ImageJ software. The qualitative and quantitative elemental surface compositions of the carbonate/polymer microparticles were carried out by means of an Axis Ultra X-ray photoelectron spectrometer (Kratos Analytical, Manchester, U.K.). The spectrometer was equipped with a monochromatic Al Kα (h⋅ν = 1486.6 eV) X-ray source of 300 W at 15 kV. The kinetic energy of the photoelectrons was determined with a hemispheric analyzer set to pass energy of 160 eV for wide scan spectra and 20 eV for high-resolution spectra. During all measurements, electrostatic charging of the sample was avoided by means of a low-energy electron source working in


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combination with a magnetic immersion lens. Later, all recorded peaks were shifted by the same amount which was necessary to set the C1s peak to 285.00 eV for saturated hydrocarbons.31 Quantitative elemental compositions were determined from peak areas using experimentally determined sensitivity factors and the spectrometer transmission function. Spectrum background was subtracted according to Shirley.32 The high-resolution spectra were deconvoluted by means of a computer routine (Kratos Analytical, Manchester, U.K.). Free parameters of component peaks were their binding energy (BE), height, full width at half maximum and the GaussianLorentzian ratio. The maximum information depth of the XPS method is not more than 8 nm for the C1s region.33 FTIR in the attenuated total reflection (FTIR-ATR) spectra of the CaCO3/polymer microparticles were recorded using a Vertex 70 Bruker FTIR spectrometer. The polymorph content has been determined from FTIR-ATR spectra following the method proposed by Vagenas et al.,34 taking into account the absorption peaks at 745 and 713 cm-1. The concentration of the crystal phases (in mg) has been calculated applying the following relations: Cv = A745/αv745

(1)

CC = A713/αC713

(2)

where subscripts C and V denoted calcite and vaterite phases, respectively, Axxx is the measured absorption, and α is the calculated absorptivity for each polymorph and absorption band:34 αv745= 21.8 mm2 mg-1, αC713= 63.4 mm2 mg-1. The percentile polymorph content was then calculated and discussed. The concentrations of the charged groups in the pectin solutions, NPEC dispersions, and composite microparticles were determined by titration using the particle charge detector Mütek PCD 03 (BTG Instruments GmbH, Herrsching, Germany). The particle charge detector includes


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a Teflon piston, which moves up and down in a cylindrical Teflon cell containing the polyelectrolyte solution in the slit between the cylinder and piston. Displacement of the ion cloud around the polymer chains which are stuck on the porous wall of Teflon cylinder creates the streaming potential (measured in mV) at the electrode pair located in upper and bottom parts of the cell. The measured streaming potential is linearly correlated to the charge density of polyelectrolytes and it becomes zero in case of charge neutrality. The concentration of the charged groups of each solution was evaluated by titration with a standard solution of a strong oppositely charged polyelectrolyte, poly(sodium ethylenesulfonate) or poly(diallyldimethylammonium chloride), with a concentration of 10-3 M. The concentration of the charged groups in the examined solution was calculated from the amount of standard solution needed to reach the zero value of the streaming potential, according to eq. 3: q=V*c/m

(3)

where: q - specific charge quantity [eq/g], V - consumed titrant volume [l], c - titrant concentration [eq/l], m - solids of the sample or its active substance [g]. All measurements were run at room temperature. The particle average hydrodynamic diameter, Dh, and the zeta-potential (ζapp) of NPEC particles was determined using a ZetaSizer Nano ZS (Malvern, UK) equipped with a 10 mW HeNe laser (633 nm) as light source. The equipment measured 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. The MPT-2 accessory was used for automatic determination of Dh and ζapp variation as a function of pH.

TCH loading experiments


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The stock solution of 1g/L TCH was prepared in double distilled water. Batch adsorption experiments were carried out in 20 cm3 vials using 10 mg microcapsules and 10 mL TCH aqueous solution, at pH=5.5. The vials were placed in a thermostat shaker bath (Memmert M00/M01, Germany) and shaken at 180 rpm and 25 °C for 4h. The concentration of TCH remained in solution was measured using a UV-Vis spectrophotometer (SPEKOL 1300, Analytik Jena) at the wavelength of 276 nm. The concentration of remained TCH, indirectly determined using the concentration/absorbance etalon curve, was situated in the range of 5 – 36 mg/L. For this interval, the calibration curve fits the Lambert and Beers’ law (eq. 4): A = 0.33349⋅C – 0.00566

(4)

where A is the absorbance at 276 nm and C is the concentration (mg/L). The correlation coefficient (R2) value was 0.999. The amount of TCH adsorbed per gram of adsorbent at any time, Qt, was calculated with eq. 5: Qt =

V (C0 − Ct ) W

(5)

where V is the volume of solution (L), C0 and Ct are the initial and instant concentration of TCH (mg/L) and W is the mass of adsorbent (g).

In vitro TCH release In vitro TCH release studies were conducted in 10 mL phosphate buffer solution (pH = 7.4) using 2 mg TCH-loaded composite particles. The release experiments were performed in thermostated shaker bath, at 37 °C. After predetermined intervals of time, a known volume (Vi, mL) of the supernatant was extracted and the content of released TCH was determined by UVVis spectrophotometer at 363 nm, using the calibration curve (eq. 6):


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A = 0.33431⋅C – 0.00568 (R2=0.999)

(6)

Subsequently, the same volume of fresh buffer solution was added into the release medium to top up to the initial volume. The cumulative TCH release (Q%) was calculated by the eq. 7,35 as follows: n −1

VxC n + Vi x ∑ Ci Q% =

i =1

M

(7)

x100

where M is the total mass of TCH absorbed into particles, Cn and Ci are TCH content released from particles in phosphate buffer solution determined for the different times, respectively. Release kinetic The kinetic of the release mechanism from microparticles loaded with TCH were examined using two mathematical models, namely Higuchi (eq. 5) and Korsmeyer-Peppas (eq. 8) models. Qt = k H t 1 / 2

(8)

where Qt is a fraction of drug released at time t, kH is the Higuchi constant, t is the time. F = kr t n

(9)

where F is a fraction of drug released, F= Mt/M∞, Mt and M∞ are the cumulative amounts of drug released at time t and at infinite time (the maximum released amount found at the plateau of the release curves), respectively, kr is the release rate constant that is characteristic to drugpolymer interactions, n is the diffusion coefficient that is characteristic to the release mechanism.

RESULTS AND DISCUSSION NPECs as colloidal dispersions


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Polyelectrolyte complexes, obtained by mixing aqueous solutions of oppositely charged polymers,36 are of interest in biomedical applications due to their facile preparation, responsiveness to environmental stimuli, and water usage as a solvent. The characteristics of the NPECs

are

determined

by

the

polyelectrolyte

components

(molecular

weight,

dissociation/protonation equilibria of ionic groups, charge density, and architecture) and the solvent (ionic strength, pH). NPECs are formed when complementary polyelectrolytes are mixed in such a ratio that there is an excess of one charge (either positive or negative), at relative low concentration stable colloidal dispersions are being obtained.37-41 When weak polyelectrolytes are involved in NPECs preparation, the molar ratio between charges when neutral complex particles were obtained, (n+/n-)n, is strongly influenced by the pH value of the solutions of the polyions, conferring an amphoteric character of NPEC aggregates.42 The amphoteric behavior of the NPEC particles, namely the sign of net surface charge, is determined by the positive or negative excess charges on the loops and chain ends on the particle surface, and depends on the molar ratio between the cationic and anionic charges, n+/n-. Table 2 summarizes the charge density (CD) and the hydrodynamic diameter (Dh) values of NPECs formed from the pectin samples (Table 1, Experimental section) and PAH, as a function of the charges molar ratio, n+/n-. NPECs were prepared starting with a 10-3 M pectin solution. Then, different amounts of a 10-2 M PAH solution were added to obtain the molar ratios of n+/n- = 0.5, 0.9 and 1.2. The NPEC stoichiometry, (n+/n-)n, i.e. the molar ratio between charges when neutral complex particles were obtained, was about n+/n- = 1.04 ± 0.04 irrespective of pectin structure, which suggest a facile intrinsic charge compensation between the complementary polyions charges. Due to the excess of the polyanion, NPEC nanoparticles having charge ratios of n+/n- = 0.5 and 0.9 were negatively charged, while the NPEC nanoparticles with a charge ratio of n+/n- = 1.2 were positively


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charged. NPECs having small charge excess, such as n+/n- of 0.9 and 1.2, tended to aggregation. Therefore, the NPEC dispersions were characterized and further used in composite microparticles formation no longer than 24 h after their formation. Before the stoichiometry, for the same n+/n- ratio, the CD values decreased with the decrease in the pectins charge density given by the carboxylate groups on the polymeric chains. After the stoichiometry, n+/n- = 1.2, the higher CD was obtained when amidated pectins were used, most probably due to the contribution of amino groups of pectin to the overall particles charge beside the PAH charges in excess. Before the stoichiometry, for the same n+/n- ratio (0.5 and 0.9), the hydrodynamic diameter of NPECs increased with the decrease of the pectins charge density, due to the larger amount of polymer needed to get the same charge compensation. After the stoichiometry, n+/n- = 1.2, the particle size sharply increases most probably because NPECs particles tend to agglomerate. As the NPECs are formed with pH-sensitive polymers, the modifications of Dh and apparent zeta-potential (ζapp) values as a function of pH were followed starting from the intrinsic pH of the NPECs (pH = 4 – 5) up to the pH of the crystallization medium (10.5) (Figure 1). The NPEC dispersions with n+/n- = 0.9 flocculated during titration and the results are not shown here. For NPECs formed by an excess of negatively charged polyelectrolytes (n+/n- = 0.5) the Dh and zeta-potential (ζapp) values slightly decrease with the increase of pH (Figure 1, a and b). The progressively dissociation of the pectins carboxylate groups with the pH increase resulted in an increase of negative charges which determines the slight decrease of the zeta-potential values with increasing the pH value. However, the majority of the separated protons would protonate the PAH’s amino groups in the immediate neighborhood of the formed carboxylates. The formed electrically neutral salt pairs (–COOϴ…H⊕–NH2 ↔ –COOϴ…H⊕…NH2) did not contribute to changing the zeta-potential. A different behavior was observed if amidated pectin samples were


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used to produce the NPECs (PCT59_A8 and PCT53_A21). With increasing the pH values the particles sizes were strongly increased and the zeta-potential values tended to zero and remained stable at ζapp ≈ -10 mV at pH > 8.5. The strong increase of the Dh values indicated swelling of NPEC particles in the moderate basic aqueous solution. Parts of the expanding NPEC’s macromolecules bring potential-determining charges into the electrokinetic shear plane or near them, which results in the reduction of the potential difference between the surface of the particle and the aqueous bulk phase. For NPECs consisting of an excess of positively charged polyelectrolyte molecules (n+/n- = 1.2) the functions ζapp = ζapp(pH) showed that the surface net charge was only slightly affected by the variation of the pH value (Figure 1d). The behavior seemed to be surprising because the increase of pH propagated the dissociation of the pectin’s carboxylate groups and the deprotonation of protonated PAH’s amino groups. Both processes also lead to the weakening of the electrostatic interactions between the PAH and pectin molecules. Water can penetrate into the NPECs and causes swelling of the particles as can be seen in the Figure 1c. As mentioned above, the swelling compresses the electrochemical double layer and lowers the apparent zeta-potential values. With increase of the pH, this process influences the ζapp = ζapp(pH) function in the opposite direction to the formation of carboxylate groups. Composite microcapsules based on CaCO3/pectins and NPECs The composite microcapsules were obtained by co-precipitation of Na2CO3 and CaCl2 aqueous solutions in the presence of pectin samples or NPECs of pectins, as polyanion, and PAH as polycation. To ensure the proper conditions for microparticles formation (by Ostwald ripening), an excess of Ca2+ ions (as compared to CO32-) in the starting solution, a pH higher than 10.5


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(CO32- becoming the dominant ionic species in the reaction mixture against HCO3-), and a prolonged reaction time of 8 hours were set (see Experimental part). The first approach to obtain capsules was to use as template the pectin series, the morphology of the composite particles being investigated by SEM (Figure 2) and the chemical structure evidenced by FTIR (Figure 1S). As small magnification micrographs in Figure 2 show, microparticles were obtained in the presence of pectins, with different shapes and sizes as a function of the pectins chemical structure. To evidence the microcapsule structure, high SEM magnification images of selected broken particles find in each sample are also presented. As previous studies showed,19,29,43,44 the presence of carboxylate and primary amine groups on the polymeric structures ensured CaCO3 mineralization, inducing mainly spherical shaped vaterite formation, characterized by crystallites of 25-35 nm.45 The gel matrix formed between the high content in carboxylic groups (95%) in PCT95 and the divalent calcium ions favor the vaterite polymorph growing inside the matrix, and stabilized the composite as microspheres, with a mean size of about 4.5 µm, and less as capsules. On the other hand, the very low content in carboxylic groups of 26% (PCT26) seemed not enough to form microcapsules with uniform shape, a mean size of 4.9 µm being obtained. The best results were obtained when 49% of functional groups were carboxylated, similar with the carboxylic group content in the synthetic polymer used in our previous study.20 Thus, PCT49 forms microcapsules with a mean size of ~6.6 µm, where the most probably formed polymorph is calcite which usually crystallizes in the form of mono-crystalline well-faceted rhombohedral particles.45 However, even at a similar content in carboxylic groups but different amino group content, the pectins PCT59_A8 and PCT53_A21 formed mixtures of calcite capsules and vaterite spheres,


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with a mean size of ~7.9 µm and ~6.2 µm, respectively. Taking into account that the amino groups also favor the vaterite formation and stabilization, and thus the total percent of groups which act as crystallization centers is about 67 and 74 %, respectively, we may assume that a similar mechanism as PCT95 takes place. Therefore, the stabilization of vaterite polymorph decreases the ability of uniform microcapsules formation by its dissolution and secondary nucleation of calcite on the external microparticles surface. The polymorph types and the pectins presence in the composites were evidenced by infrared spectroscopy. Figure 1S (supplementary information) shows the FTIR spectra of composites based on PCT 49 and PCT53_A21 in the domain 600 ≤ ν ≤ 1600 cm−1 where the most important absorption bands of calcium carbonate appear: calcite at 712 cm-1 (ν4) – the carbonate in-plane bending absorption and at 871-874 cm-1 (ν2) – carbonate out-of-plane bending absorption, whereas the band at 744 cm-1 (ν2), which is characteristic to vaterite, can be clearly observed only in the spectrum of CaCO3/PCT53_A21. The band at 1391-394 cm-1 (ν3) is ascribed in ATR spectra to asymmetric stretch of carbonate groups in calcite45 (in transmission spectra the characteristic band is at ~ 1490 cm-1),46 whereas the peak shift at 1407 cm-1 is determined by the pectin presence and its amide groups. The pectins carboxylate groups are evidenced by the band at 1650 cm-1 and the ester groups at 1796 cm-1.47 The bands at 1088 and 1013 cm-1 are characteristic for the vibration of C–O–C glyosidic bonds48 and therefore are characteristic also for pectin backbone. The second approach to obtain capsules was to use as templates the corresponding NPECs, with molar ratio between charges of 0.5, 0.9 and 1.2. The morphology of the composite particles was investigated by SEM (Figure 3) and the chemical structure was evidenced by FTIR (Figure 2S).


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As shown in Figure 3, the morphology of the composites obtained with pectin based NPECs depend both on pectin chemical structure and on the molar ratio between charges. Thus, taking into account the polycrystalline spherical shape observed in SEM images, the polymorph vaterite is the main one in the samples prepared with NPEC having n+/n- = 0.9, except those prepared with PCT95 and PCT49 based NPECs where a mixture of microcapsules and microspheres are obtained. A good ability to form capsules has been observed for NPECs with n+/n- = 0.5 and 1.2, irrespective of pectin structure. However, as the primary amino groups stabilize the vaterite polymorph, the composites prepared with a high content in PAH (n+/n- = 1.2) and those with amidated pectins (PCT59_A8/PAH and PCT53_A21/PAH) contain both almost spherical microcapsules covered with a calcite layer and also vaterite microspheres. The polymorph content in NPEC-based composites was also confirmed using ATR-FTIR, Figure 2S which include the 600 ≤ ν ≤ 1600 cm−1 region of the spectra of composites based on NPECs with PCT 49 and PCT53_A21, pectins with a similar content in carboxylic groups (49 and 53%) and which differ by amidation degree (0 and 21%). The FTIR spectra of composites formed with PCT49 and PAH confirm the calcite content by the characteristic bands at 713, 875, and 1394-1397 cm-1, as well as the vaterite content by characteristic peaks at 745 and 830-848 cm-1. The pectins characteristic bands are found at 1682-1688 cm-1 – carboxylate groups, 17951798 cm-1 – ester groups, and 1015 and 1089 cm-1 – C–O–C glyosidic bonds vibration. The shift of the absorption band from 1394 cm-1 (ν3, ascribed to the asymmetric stretch of carbonate group in calcite) up to 1403-1413 cm-1 for PCT49 based composites can be explained by the increase of PAH content. The amidated pectin PCT53_A21 based composite FTIR spectra contain similar characteristic bands as NPEC-PCT49-CaCO3 composites, but also a band at 1423 cm-1, which


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can be ascribed both to calcite and to amino group in pectin and in PAH. Table 1S summarized the FTIR characteristic bands for all investigated samples. FTIR-ATR spectra were also used to quantify the polymorph content, as described in the experimental part, taking into account the absorption peaks at 745 and 713 cm-1. As shown in Table 2S, the polymorph content is influenced by the pectin structure and NPEC molar ratio. Thus, when PCT49 was used during the microparticles formation, calcite is the main polymorph (~95%), whereas for amidated pectin PCT53_A21 based composite vaterite content increased up to ~36%, sustaining the previous SEM observations. The NPECs use in the composite formation increased the vaterite polymorph content in PCT49 based composites (ranging between 38 and 48%) the highest vaterite content being found in the composites with n+/n- of 0.9. On the other side, when PCT53_A21 was used in NPECs formation, the vaterite percentile amount increased up to ~53 % when anionic NPECs were used (n+/n- of 0.5 and 0.9) and up to ~64% when cationic NPECs were used, the higher content in amine groups favoring the vaterite formation. To evidence the particles composition, the amount of retained polymers has been indirectly determined by PCD titration, comparing the ionic concentration of starting polyanion solution or NPECs dispersion and that of the supernatant resulting after microparticles separation (% PAs) (Table 3S). As shown in Table 3S, the amount of the pectin remained in supernatant is about 1416% for pectin-based composites and decreased up to ~ 9%, 3-5% and ~2% with the polycation content increased, i.e. n+/n- increased from 0.5 to 0.9 and 1.2, respectively. The results suggest that almost all the polymeric amount used in composite preparation was included into microparticles, and the increase of polycation content favor the polymers retention. To further sustain the embedment of NPECs in the composites, the zeta-potential values of the CaCO3-NPEC composites were determined and compared with those of corresponding NPECs at


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pH = 10 (Table 3). As compared to the zeta-potential values of the NPEC dispersions at pH = 10, the zeta-potential values sharply decreased after composite formation close to that of bare CaCO3 particles values (7.6 mV), being influenced by the NPECs n+/n− ratio. This can be considered as a hint of the NPEC embedment into the composites irrespective of pectins or NPEC characteristics. The surface compositions of some CaCO3-NPEC samples were studied by means of XPS. Table 4 summarizes the surface composition of CaCO3 composite samples, which were obtained in the presence of PCT49 and NPECs dispersions with different ratios of PCT49 and PAH. For the CaCO3-PCT49 sample the [O]:[Ca]|spec ratio is higher ([O]:[Ca]|spec = 3.37) than the theoretical ratio for CaCO3 ([O]:[Ca]|stoi =3.0) indicating the presence of the pectin in composite particles. XPS spectra recorded from CaCO3 composites with NPECs clearly show the N1s peak resulting from photoelectrons escaped from the nitrogen atoms of the amino groups of the PAH. Compared to pristine CaCO3 and the CaCO3-PCT49 sample the corresponding [O]:[Ca]|spec ratios were slightly increased confirming increases of the polymer fractions retained on the surface of the composite particles. The highest amounts of oxygen and nitrogen were found for CaCO3-NPEC sample having a charge ratio of n+/n- = 0.9. As can be seen in Figure 3, for this sample a mixture of microcapsules and microparticles was obtained while the use of NPEC having other charge ratios (n+/n- = 0.5 and 1.2) drove the precipitation to the sole formation of capsules. According to former studies, the differences between the relative nitrogen and carbon contents in NPEC-based samples can be also explained by the NPECs distribution on the formed structures: a uniform distribution of NPECs on spheres50 whereas in capsules is expected NPECs to be placed on the inner part of the capsule shell.20 In order to get more insight into the chemical


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structure and the interaction mechanisms between the oppositely charged polyions (pectin and PAH) high-resolution element spectra were recorded (Figure 4). All C1s spectra showed a bimodal distribution of the C1s photoelectrons. The first maximum mainly resulted from photoelectrons of saturated hydrocarbons (component peak A at 285.00 eV). Since pectin (PCT49) does not contain saturated hydrocarbons in its structure, the C1s spectrum of sample CaCO3-PCT49 (Figure 4a) clarified that hydrocarbons contaminated the sample surfaces. Such surface contaminations are commonly observed on sample surfaces which are prepared or stored under ambient conditions. The second peak maximum mainly appeared from the carbonate ions (CO32-) of CaCO3 (component peak F at 289.4 eV). Component peaks C (286.5 eV), D (287.6 eV), and E (288.3 eV) are characteristic peaks for pectin (Scheme 1 in the Experimental part illustrates the assignment of the component peaks to the structure of the pectin polymer). The ratios of the component peaks resulting from C–O(H or C) (component peaks C), the hemiacetals (O–C–O, component peaks D), and the carbonyl carbon atoms of the carboxylic acid and/or carboxylate groups (component peaks E) were [C]:[D]:[F] = 9:2:2, which equalled the stoichiometric ratio of pectin. The addition of PAH (n+/n- = 0.5 or 1.2) was detected by the appearing of the N1s peaks in the wide-scan spectra but also in an additional component peak B at 285.7 eV showing the C–N bonds of the amino groups of the PAH polymer. The high-resolution N1s spectra (Figure 4, middle column) were deconvoluted into two component peaks (K and L). Component peaks K (399.5 eV) resulted from photoelectrons escaped from the nitrogen atoms of the non-charged primary amino groups (C–NH2). The binding energy values (> 401 eV) of the second component peak L were characteristic for protonated amino groups (C–N+H3). Normally, the occurrence of the protonated species reflects the C–NH2⋅HCl adduct formation in PAH applied for the


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production of CaCO3-NPEC composites. However, it can be assumed that the presence of CaCO3 neutralizes the hydronium ions (provided by the HCl) during the composite formation. Hence, the protonated amine species found in the N1s spectra mainly resulted from the salt formation, where the hydronium ions of the dissociating carboxylic acid groups (of pectin) protonated the electrically neutral amino groups (of PAH) in their immediate neighborhood. These findings supported the discussion of the electrokinetic behavior above. The electrostatic interactions between the intermolecularly formed ion pairs (–COOϴ…H⊕–NH2) can be considered as the fundament for remaining the NPECs stable during the composite formation. The formation of ion pairs strengthens the polarity of the NPECs. Thus, water can penetrate into the NPEC molecular aggregates, reduce the electrostatic interactions by solvation of the ions, and expand the particles structure, as observed in Figure 1. The Ca2p spectra are composed of the Ca2p3/2 and Ca2p1/2 peaks. The appearance of one component peak indicated the presence of only one binding state. The binding energy values for these component peaks found in the Ca2p3/2 spectra (346.81 eV) were characteristic for CaCO3.51,52 To further demonstrate the polymer presence mainly in the inner part of capsules walls, EDAX investigations were performed by determining the elemental composition on selected areas corresponding particles surfaces and capsules inner wall. The results obtained for the samples based on PCT49 and corresponding NPECs are included in Table 5. The theoretical elemental composition was calculated assuming a uniform distribution of the inorganic and organic components of the composites and taking into account their initial concentration. As results in Table 5 shows, the values of C/Ca and O/Ca atomic ratios obtained at the outside wall of the microcapsules were lower than the calculated ones, taking into account the amount of


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used polymers in samples preparation. Moreover, the values are closer to the corresponding atomic ratios in CaCO3, namely C/Ca = 1 and O/Ca = 3, which sustain the mainly CaCO3 composition of the particles surface as was also indirectly observed by zeta potential measurements. By contrary, inside the microcapsules the values of C/Ca, N/Ca and O/Ca atomic ratios are higher than the calculated ones, results explained by a lower content in CaCO3 and an excess of polymers on the microcapsules inner walls. Taking into account all the above results and how the Ostwald ripening process takes place for CaCO3 polymorphs, as described in the literature,53-55 the formation process of hollow CaCO3 capsules in the presence of pectins and NPECs is schematically represented in Scheme 2. As the mechanism seems to be similar for pectins and NPECs as well, the scheme combines both structures. The formation process of hollow CaCO3 capsules in the presence of pectins and NPECs is expected to take place in the following steps (Scheme 2): (1) interaction of calcium ions with ionic or ionizable groups along the polymeric chain or on the free loops and end chains on NPECs nanoparticles with the formation of gel microparticles and calcium carbonate crystallization promoted by the calcium rich sites; (2) calcium carbonate crystallization grow with formation of microparticles (in the first minute of crystallization), (3) dissolution of less stable CaCO3 fractions (amorphous and vaterite) mainly by dissolution of the middle core; (4) secondary nucleation of the crystalline polymorph (mainly calcite) on the external surface; (5) progressively increase in the thickness of crystalline shell as the core becomes depleted and produce hollow microspheres.

Tetracycline loading


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CaCO3 composite samples prepared with PCT49 and PCT53_A21, pectins with a similar content in carboxylic groups (49 and 53%) and different amidation degree (0 and 21%), and the corresponding NPECs with different molar ratio between charges were explored as drug delivery vehicles using TCH as a model drug. The morphology of the microparticles after TCH loading is shown in Figure 3S and the mean particles size, as compared to that of the starting microparticles/microcapsules, are summarized in (Table 4S). As shown in Figure 3S, and as compared to the SEM images in Figures 2 and 3, there are no significant changes in the microparticles shape after the TCH sorption. Moreover, the mean particle diameter (Table 4S) was close to that of initial particles sustaining a good stability of the samples to the loading protocol. The highest percentile mean diameter variation after TCH loading of about 11% was obtained when the molar ratio was n+/n- = 0.9, which can be assigned to the low crosslinking of Ca2+ ions with the small amount of anionic chains remained uncompensated by the cationic polymer, which probably conduct to a loosed network matrix which could less protect the CaCO3 nanocrystals against dissolution. The maximum loading capacity was determined taking into account the initial TCH concentration in aqueous solutions and the residual concentration of the supernatant after sorption, by measuring the absorbance at 276 nm (Table 6). As Table 6 shows, the sorption capacity of the tested samples depends on both pectin chemical structure and on the NPECs molar ratio between charges. Thus, both CaCO3 based microparticles prepared with PCT49 and PCT53_A21 have a good loading capacity, more than 220 mg/g. The NPEC-based composites capacity to load TCH depends on the ratio between charges, the microparticles shape and polymorph content. It is important to know that tetracycline acid salts have three acidity constants in aqueous solution (pKa1=3.3, pKa2=7.7 and pKa3=9.7) due to three protonation sites


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(Scheme 1c), and at pH = 3.3 ~ 7.7 exists as a zwitterion. Therefore, both the negative charges on the pectin carboxylate backbone and also the positive charges of amino group on PAH and amidated pectins can interact with TCH, and contribute to the drug loading, which can explain the highest loading capacity of the sample prepared with NPEC based on PCT53_A21 with the highest content in PAH (n+/n- = 0.9). Also, CaCO3 polymorphs influence the loading capacity, as each polymorph is characterized by different porosity (calcite porosity is lower than that of vaterite) and different surface charge (under the used experimental conditions – the Ca2+ concentration of 0.1 M – the surface of calcite is negatively charged whereas vaterite is slightly positively charged56). Also, increasing the calcite content conducts to an increased surface area and thus increased availability of more adsorption sites. As a consequence, the TCH retention in the microparticles/microspheres can be explained by the synergic contribution of organic/inorganic component materials while interacting with TCH: (1) the polymeric matrix capacity for TCH sorption by electrostatic interactions or H-bonds, (2) the availability of functional groups after CaCO3 growth, (3) the distribution of CaCO3 crystals on/onto microparticles/microcapsules, (4) the capacity of crystals to interact with TCH as a function of their arrangement on/onto microparticles/microcapsules.

TCH in-vitro release In vitro release studies were performed at pH 7.4 (in phosphate buffer) and the results were summarized in Figure 5. As shown in Figure 5, the release profiles indicated that about 22% and 27% of TCH was released after 10 h from TCH-loaded microspheres of PCT49-CaCO3 and PCT53_A21-CaCO3, respectively. The TCH release from NPEC based microparticles can be related to the CaCO3 polymorphs. Thus, the vaterite polymorph observed in the samples


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prepared with NPEC having n+/n- = 0.9, conduct to a similar cumulative release percent after 10 h as the corresponding pectin-based composites. The cumulative release percent of calcite-shelled capsules obtained with NPECs having n+/n- = 0.5 and 1.2 varied as a function of different factors. Thus, as compared to the corresponding pectin-based composites, the TCH-loaded NPEC-PCT49-CaCO3 microcapsules present an increased release amount after 10 h when n+/n- = 0.5, namely when carboxylic groups are in excess, whereas the n+/n- = 1.2 the PCT53_A21-CaCO3 microcapsules had a higher release amount in the same condition. In the first case, the relaxation of pectin free anionic end chain conducts most probably to the increase of TCH release whereas in the second case the vaterite polymorph stabilized by the primary amino groups (from PAH and amidated pectin PCT53_A21/PAH) would increase the shell permeability. However, the release of a drug from a matrix (as full or empty particles) generally involves both pore diffusion and matrix erosion. The structure of the PCT53_A21-CaCO3 and corresponding NPEC samples after TCH release is shown in Figure 6. Thus, as micrographs in Figure 6 show, the surface of the microparticles/microcapsules differ significantly from that of TCH loaded composites. The formed hexagonal plate-like new structures can be assigned to vaterite polymorph as was also previously observed for vaterite polymorph formed in the presence of NH4+ ions.57 It seems that the sample PCT53_A21/PAH=1.2, which already contains vaterite as the main polymorph preserves the spherical shape of the microparticles, whereas the samples containing calcite were transformed into vaterite plates, without spherical reorganization. Similar abnormal polymorph transformation from calcite to vaterite was found in different systems58,59 in the present case being most probably ascribed to the influence of TCH presence in the system as interfering in the crystal growth of the new materials. Thus, the incomplete drug release can be assigned probably


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to the entrapment of a certain fraction of TCH by the new formed material, which is most probably dissolved in acidic conditions, at pH lower than 5.

Release kinetic The kinetic of the release mechanism from microparticles loaded with TCH was examined using two mathematical models, Higuchi (Figure 7) and Korsmeyer-Peppas (Figure 8). The release constants were calculated from the slope of the appropriate plots, and regression coefficient (R2) by linear regression analysis (Table 7). Thus, the release profiles of samples could be explained by Higuchi model (Figure 7), as the plots showed a good linearity, with correlation coefficient (R2) values ranged in the domain 0.946–0.995 (Table 7). Accordingly, the drug release was proportional to the square root of time, indicating diffusion controlled-release mechanism for all tested composites. The diffusion mechanism of drug release was further confirmed by Korsmeyer-Peppas plots (Figure 8) that showed high linearity (R2 values between 0.983 and 0.999). In spherical matrices the dominant drug release mechanism can be assumed taking into account the n values: n < 0.4, Fick’s diffusion (case-I); 0.4 < n < 0.85, anomalous or non-Fick’s transport; n > 0.85, case-II transport (zero order). As shown in Table 7, n values are greater than 0.85 for all investigated samples, indicating a case II transport mechanism of TCH diffusion. Also, values of n > 1 have been observed, which are regarded as super case II kinetics. Case-II transport is described by a characteristic relaxation constant and reflects the influence of polymer relaxation on molecules’ movement

in

the

matrix.

This

behavior

can

also

be

ascribed

to

the

recrystallization/reorganization of CaCO3 polymorphs on the composites. Also, the dynamic relaxation/swelling behavior of micro-gels formed by pectins or NPECs and Ca2+ ion depend on


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the relative contribution of diffusion and polymer relaxation. In the studied composites, the polymer relaxation is affected by the ionization of functional groups of polymers and also by the charge density of the polymer backbone or net charge of NPEC nanoparticles. An increase in the ionic group content results in the electrostatic repulsion between ionized functional groups, leading to chain expansion, which in turn affects macromolecular chain relaxation. Thus, the drug release mechanism becomes more relaxation-controlled when the ionization of complex microparticles increases. For the studied composites the release pH of 7.4 favors a decrease of protonation of amine groups on PAH and amidated pectins and simultaneously deprotonation of carboxylic acid groups on pectins. Therefore, the recrystallization/reorganization mechanism becomes more relaxation-controlled as ionization of constitutes polymers becomes prominent, and thus n values increase.

CONCLUSIONS New types of composites were obtained by an auto-templating method for assembling hollow CaCO3 capsules, by using five pectin samples – which differ in the methylation degree and/or amide content – and some nonstoichiometric polyelectrolyte complex dispersions. The best results were obtained when 49% of functional groups were carboxylated and for NPECs with n+/n- = 0.5 and 1.2, irrespective of pectin structure. The composites prepared with a high content in PAH (n+/n- = 1.2) and those with amidated pectins (PCT59_A8/PAH and PCT53_A21/PAH) contain both almost spherical microcapsules covered with a calcite layer and also vaterite microspheres. As a hint of the good NPEC embedment into the composites, irrespective of pectins or NPEC characteristics, is the sharply decreased zeta-potential values after composite formation, close to that of bare CaCO3 particles values (7.6 mV). XPS high-resolution N1s


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spectra sustained the salt formation between the protonated amine species of PAH and the dissociated carboxylic acid groups of pectin, the electrostatic interactions between the intermolecular formed ion pairs being considered as the fundament for NPECs stability during the composite formation. A formation mechanism of microcapsules was proposed, starting with the interaction of calcium ions with ionic or ionizable groups along the pectin or NPECs nanoparticles, calcium carbonate crystallization growth with formation of microparticles, dissolution of amorphous and vaterite and secondary calcite nucleation on the external surface with progressively increased thickness of crystalline shell. CaCO3 based microparticles had a good loading capacity, more than 220 mg/g. The diffusion controlled-release mechanism for all tested composites was sustained by Higuchi model and further confirmed by Korsmeyer-Peppas plots. The n values greater than 0.85, for all investigated samples, indicates a case II transport mechanism of TCH diffusion.

ASSOCIATED CONTENT Supporting Information. The following file is available free of charge: Figure 1S. FTIR spectra of composites based on CaCO3 and the pectins PCT 49 and PCT53_A21; Figure 2S. FTIR spectra of composite microparticles of CaCO3 and NPECs based on PAH and (a) PCT49 and (b) PCT53_A21 and different molar ratios; Figure 3S. SEM micrograph of pectin-CaCO3 and NPEC-CaCO3 composite microparticles after TCH retention; Table 1S. FTIR characteristic bands of investigated samples; Table 2S. The percentile polymorph content of investigated samples; Table 3S. The percentile of polyanion remained in the supernatant (% P) after composite microparticle separation, comparative with starting polyanion solution; Table 4S. Mean diameter of the CaCO3-polymer samples before and after TCH loading. (file type, PDF)


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AUTHOR INFORMATION Corresponding Author *Marcela Mihai, Telephone number: +40.2322217454; Fax number: +40.232211299, email address: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This work was supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CNCS – UEFISCDI, project number PN-II-RU-TE-2014-4-1433.

ABBREVIATIONS CD, charge density; Dh, hydrodynamic diameter; NPEC, nonstoichiometric polyelectrolyte complexes; PAH, poly(allylamine hydrochloride; PCT, pectin; SEM, scanning electron microscopy; TCH, tetracycline hydrochloride; XPS, X-ray photoelectron spectroscopy; ζapp,, apparent zeta-potential. REFERENCES (1)

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(10) Fu, J.; Wang, J.; Li, Q.; Kim, D. H.; Knol, W. 3D Hierarchically Ordered Composite Block Copolymer Hollow Sphere Arrays by Solution Wetting. Langmuir 2010, 26, 12336–12341. (11) Baldan, A. Progress in Ostwald Ripening Theories and Their Applications to Nickel-Base Superalloys. J. Mater. Sci. 2002, 37, 2171 – 2202. (12) Tang, Y.; Chen, S.; Mu, S.; Chen, T.; Qiao, Y.; Yu, S.; Gao, F. Synthesis of Capsule-like Porous Hollow Nanonickel Cobalt Sulfides via Cation Exchange Based on the Kirkendall Effect for High-Performance Supercapacitors. ACS Appl. Mater. Interfaces 2016, 8, 9721– 9732.= (13) Volodkin, D. CaCO3 Templated Micro-Beads and -Capsules for Bioapplications. Adv. Colloid Interface Sci. 2014, 207, 306–324. (14) Render, D.; Samuel, T.; King, H.; Vig, M.; Jeelani, S.; Babu, R. J.; Rangari, V. Biomaterial-Derived Calcium Carbonate Nanoparticles for Enteric Drug Delivery. J. Nanomat. 2016, Article ID 3170248, 8 pag. (15) Kong, F.; Zhang, H.; Zhang, X.; Liu, D.; Chen, D.; Zhang, W.; Zhang, L.; Santos, H. A.; Hai, M. Biodegradable Photothermal and pH Responsive Calcium Carbonate@Phospholipid@Acetalated Dextran Hybrid Platform for Advancing Biomedical Applications. Adv. Funct. Mater. 2016, 26, 6158–6169. (16) Suzuki, M.; Nagasawa, H.; Kogure, T. Synthesis and Structure of Hollow Calcite Particles. Cryst. Growth Des. 2006, 6, 2004–2006.


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(25) Begum, G.; Reddy, T. N.; Kumar, K. P.; Dhevendar, K.; Singh, S.; Amarnath, M.; Misra, S.; Rangari, V. K.; Rana, R. K. In Situ Strategy to Encapsulate Antibiotics in a Bioinspired CaCO3 Structure Enabling pH-Sensitive Drug Release Apt for Therapeutic and Imaging Applications. ACS Appl. Mater. Interfaces 2016, 8, 22056–22063. (26) Ouhenia, S.; Chateigner, D.; Belkhira, M. A.; Guilmeaub, E.; Krauss, C. Synthesis of Calcium Carbonate Polymorphs in the Presence of Polyacrylic Acid. J. Cryst. Growth 2008, 310, 2832–2841. (27) McKenna, B. J.; Waite, J. H.; Stucky, G. D. Biomimetic Control of Calcite Morphology with Homopolyanions. Cryst. Growth Des. 2009, 9, 4335–4343. (28) Matahwa, H.; Ramiah, V.; Sanderson, R. D. Calcium Carbonate Crystallization in the Presence of Modified Polysaccharides and Linear Polymeric Additives. J. Cryst. Growth 2008, 310, 4561–4569. (29) Mihai, M.; Mountrichas, G.; Pispas, S.; Stoica, I.; Aflori, M.; Auf der Landwehr, M.; Neda, I.; Schwarz, S. Calcium Carbonate Microparticle Templates Using a PHOS-b-PMAA Double Hydrophilic Copolymer. J. Appl. Cryst. 2013, 46, 1455–1466. (30) Mihai, M.; Socoliuc, V.; Doroftei, F.; Ursu, E.-L.; Aflori, M.; Vekas, L.; Simionescu, B. C. Calcium Carbonate−Magnetite−Chondroitin Sulfate Composite Microparticles with Enhanced pH Stability and Superparamagnetic Properties. Cryst. Growth Des. 2013, 13, 3535−3545. (31) Beamson, G.; Briggs, D. High Resolution XPS of Organic Polymers; Wiley: Chichester, NY, 1992. (32) Shirley, D. A. High-resolution X-ray Photoemission Spectrum of the Valence Bands of Gold. Phys. Rev. B 1972, 5, 4709-4714.


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(41) Dragan, E. S.; Mihai, M.; Schwarz, S. Complex Nanoparticles Based on Chitosan and Ionic/Nonionic Strong Polyanions: Formation, Stability, and Application. ACS Appl. Mater. Interfaces 2009, 1, 1231-1240. (42) Kötzl, J.; Philippi, B.; Sigitov, V.; Kudaibergenov, S.; Bekturov, E. A. Amphoteric Character of Polyelectrolyte Complex Particles as Revealed by Isotachophoresis and Viscometry. Colloid Polym. Sci. 1988, 266, 906-912. (43) Barhoum, A.; Rahier, H.; Abou-Zaied, R. E.; Rehan, M.; Dufour, T.; Hill, G.; Dufresne, A. Effect of Cationic and Anionic Surfactants on the Application of Calcium Carbonate Nanoparticles in Paper Coating. ACS Appl. Mater. Interfaces 2014, 6, 2734–2744. (44) Schenk, A. S.; Cantaert, B.; Kim, Y.-Y.; Li, Y.; Read, E. S.; Semsarilar, M.; Armes, S. P.; Meldrum, F. C. Systematic Study of the Effects of Polyamines on Calcium Carbonate Precipitation. Chem. Mater. 2014, 26, 2703−2711. (45) Brecevic, L.; Nothig-Laslo, V.; Kralj, D.; Popovic, S. Effect of Divalent Cations on the Formation and Structure of Calcium Carbonate Polymorphs. J. Chem. Soc. Faraday Trans. 1996, 92 (6), 1017-1022. (46) Shimadzu application news. Spectrophotometric Analysis, Application Note No.a395 (47) Andersen, F. A.; Brecevic, L. Infrared Spectra of Amorphous and Crystalline Calcium Carbonate. Acta Chem. Scand. 1991, 45, 1018-1024. (48) Urias-Orona, V.; Rascón-Chu, A.; Lizardi-Mendoza, J.; Carvajal-Millán, E.; Gardea, A. A.; Ramirez-Wong, B. A Novel Pectin Material: Extraction, Characterization and Gelling Properties. Int. J. Molec. Sci. 2010, 11, 3686-3695. (49) Baum, A.; Dominiak, M.; Vidal-Melgosa, S.; Willats, W. G. T.; Søndergaard, K. M.; Hansen, P. W.; Meyer, A. S.; Mikkelsen, J. D. Prediction of Pectin Yield and Quality by


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(58) Wu, Q.-S.; Sun, D.-M.; Liu, H.-J.; Ding, Y.-P. Abnormal Polymorph Conversion of Calcium Carbonate and Nano-Self-Assembly of Vaterite by a Supported Liquid Membrane System. Cryst. Growth Des. 2004, 4, 717-720. (59) Yang, B.; Nan, Z. Abnormal polymorph conversion of calcium carbonate from calcite to vaterite. Mater. Res. Bull. 2012, 47, 521–526.


37


650

a

550

-10

PCT95 PCT49 PCT26 PCT59_A8 PCT53_A21

-15 -20

450

ζapp(mV)

Dh [nm]

500

-5

b


600

400 350

-25 -30

300 -35 250 -40

200 150

-45 4

5

6

7

8

9

10

11

4

5

6

7

pH

d


9

10

11

10

11

70 60 50

ζapp(mV)

2000

8

pH

2500

c

Dh [nm]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 55

1500

1000


40 30 20 10

500 0 3

4

5

6

7

8

9

10

11

3

4

pH

5

6

7

8

9

pH

Figure 1. The hydrodynamic diameter (Dh, a and c) and the apparent zeta-potential (ζapp, b and d) values as a function of pH of NPECs based on different pectin samples and PAH at n+/n- = 0.5 (a and b) and n+/n- = 1.2 (c and d)


38

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PCT95

PCT49

PCT59_A8

PCT26

PCT53_A21

Figure 2. SEM micrograph of pectine-CaCO3 composite microparticles


39


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 55

PCT95/PAH = 0.5

PCT95/PAH = 0.9

PCT95/PAH = 1.2

PCT49/PAH = 0.5

PCT49/PAH = 0.9

PCT49/PAH = 1.2

PCT26/PAH = 0.5

PCT26/PAH = 0.9

PCT26/PAH = 1.2

PCT59_A8/PAH = 0.5

PCT59_A8/PAH = 0.9

PCT59_A8/PAH = 1.2

PCT53_A21/PAH = 0.5

PCT53_A21/PAH = 0.9

PCT53_A21/PAH = 1.2

Figure 3. SEM micrograph of NPEC-CaCO3 composite microparticles


40

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


C 1s

A F

D

E

N 1s

K

Ca 2p Ca 2p 1/2

K

Ca 2p 1/2

L

Ca 2p 3/2

C B

c) Ca 2p 3/2

A F

D L

E

C B

b) Ca 2p 3/2

A F E

Ca 2p 1/2

D C

a) 298 294 290 286 282 410 405 400 395 390 Binding Energy [eV] Binding Energy [eV]

356 351 346 341 Binding Energy [eV]

Figure 4. High-resolution C1s (left column), N1s (middle column), and Ca2p (right column) element spectra recorded from the CaCO3-PCT49 (a), CaCO3-PCT49/PAH = 0.5 (b), and CaCO3-PCT49/PAH = 1.2 (c) composite samples.


41


30

Cumulative Release (%)

25

20

15

10

PCT49 loaded TCH PCT49/PAH=0.5 loaded TCH PCT49/PAH=0.9 loaded TCH PCT49/PAH=1.2 loaded TCH

5

0 0

10

20

30

40

50

60 500

600

Time (min)

35 30

Cumulative Release (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 55

25 20 15

PCT53_A21 loaded TCH PCT53_A21/PAH=0.5 loaded TCH PCT53_A21/PAH=0.9 loaded TCH PCT53_A21/PAH=1.2 loaded TCH

10 5 0 0

10

20

30

40

50

60 500

600

Time (min)

Figure 5. Tetracycline in-vitro release


42

Page 43 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PCT53_A21

PCT53_A21/PAH=0.5

PCT53_A21/PAH=0.9

PCT53_A21/PAH=1.2

Figure 6. SEM micrograph of PCT53_A21-CaCO3 and corresponding NPEC-CaCO3 composite microparticles after TCH release


43


20

Qt

15

10


5

0 1

2

3

4 1/2

5

6

7

1/2

t (min )

30

25

20

Qt

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 55

15

10


5

0 1

2

3

4 1/2

5

6

7

1/2

t (min )

Figure 7. Graphical representation of Higuchi equation


44

Page 45 of 55

log t 0.0 0.6

0.7

0.8

0.9

1.0

-0.2

log F

-0.4

-0.6


-0.8

-1.0

log t 0.0 0.6

0.7

0.8

0.9

1.0

-0.2

-0.4

log F

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-0.6

-0.8


-1.0

Figure 8. Graphical representation of Korsmeyer-Peppas equation


45


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(c)

(a) O O EC C HO

OR C O C C OH

Page 46 of 55

R = H; CCH3

(b) A

A

CH2 CH

D

BCH2 KNH

2

O

Scheme 1. Chemical structure of (a) the carboxylate pectins, (b) poly(allylamine) (both structures with assignment of the different component peaks for XPS discussion), and (c) tetracycline hydrochloride


46

Page 47 of 55

+ + + + + +

+ + +

+ + +

+ + +

+ + +

+ + +

+ + +

+ + +

+ + +

+ + +

+ + +

(1)

+ + +

+ + +

+ + +

+ + +

+ + + + + +

(3)

(2)

- PCT + + +

- vaterite

+ + +

- calcite

+ + +

(5)

+ + +

+ + +

- Ca2+

+ + +

- NPEC

+ + +

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(4)

Scheme 2. The schematically representation of the hollow CaCO3 capsules formation process in the presence of pectins and/or NPECs: (1) interaction of calcium ions with ionic or ionizable groups along the polymeric chain or on the free loops and end chains on NPECs nanoparticles with the formation of gel microparticles and calcium carbonate crystallization promoted by the calcium rich sites; (2) calcium carbonate crystallization grow with formation of microparticles, (3) dissolution of less stable CaCO3 fractions (amorphous and vaterite) mainly by dissolution of the middle core; (4) secondary nucleation of the crystalline polymorph (mainly calcite) on the external surface; (5) progressively increase in the thickness of crystalline shell as the core becomes depleted and produce hollow microspheres.


47


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 55

Table 1. Chemical structure of pectin samples

X = O-, OMe, NH2

% O-

% OMe

% NH2

Sample Code

95

5

0

PCT95

49

51

0

PCT49

26

74

0

PCT26

59

33

8

PCT59_A8

53

26

21

PCT53_A21


48

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2. Charge density (CD) and hydrodynamic diameter (Dh) values of NPECs formed from the studied polyelectrolyte pairs. Sample

n+/n-

CD, meq/g

Dh, nm

PCT95

0

-18.22

-

PCT95/PAH

0.5

-17.37

257.8

PCT95/PAH

0.9

-4.51

296.0

PCT95/PAH

1.2

+5.02

660.5

PCT49

0

-18.87

-

PCT49/PAH

0.5

-16.45

337.9

PCT49/PAH

0.9

-3.48

531.5

PCT49/PAH

1.2

+4.55

871.5

PCT26

0

-17.75

-

PCT26/PAH

0.5

-12.01

444.2

PCT26/PAH

0.9

-1.41

981.8

PCT26/PAH

1.2

+3.13

1255.9

PCT59_A8

0

-17.09

-

PCT59_A8/PAH

0.5

-13.00

303.6

PCT59_A8/PAH

0.9

-2.21

380.7

PCT59_A8/PAH

1.2

+6.22

968.9

PCT53_A21

0

-18.37

-

PCT53_A21/PAH

0.5

-12.51

332.9

PCT53_A21/PAH

0.9

-2.02

438.5

PCT53_A21/PAH

1.2

+6.87

1040.5


49


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 50 of 55

Table 3. The apparent zeta-potential (ζapp) values of the CaCO3-NPEC composites compared with those of corresponding NPECs at pH = 10 Sample

n+/n-

ζapp (NPEC), mV

ζapp (CaCO3-NPEC), mV

PCT95/PAH

0.5

-41.2

-5.2

PCT95/PAH

0.9

-22.4

+3.6

PCT95/PAH

1.2

+32.7

+10.1

PCT49/PAH

0.5

-37.4

-3.7

PCT49/PAH

0.9

-17.3

+4.4

PCT49/PAH

1.2

+26.6

+10.9

PCT26/PAH

0.5

-31.9

-7.9

PCT26/PAH

0.9

-15.8

+2.7

PCT26/PAH

1.2

+11.8

+4.5

PCT59_A8/PAH

0.5

-12.1

-5.1

PCT59_A8/PAH

0.9

+29.8

+3.7

PCT59_A8/PAH

1.2

+59.1

+4.5

PCT53_A21/PAH

0.5

-8.8

-9.0

PCT53_A21/PAH

0.9

+34.4

+4.5

PCT53_A21/PAH

1.2

+70.3

+7.6


50

Page 51 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 4. Relative element composition of CaCO3 composites prepared from PCT49 and NPECs with different molar ratios, n+/n-. n+/n- = 0.5

n+/n- = 0.9

n+/n- = 1.2

[O]:[Ca]|spec 3.367

4.130

4.219

4.131

[N]:[Ca]|spec –

0.193

0.319

0.237

0.878

0.789

0.854

0. 791

[Ca]:[C]|spec 0.261

0.191

0.202

0.192

PCT49

[O]:[C]|spec


51


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 52 of 55

Table 5. Atomic ratios of the elements characteristic for polyanion (C and O), polycation (C and N) versus Ca from CaCO3 in composite samples based on PCT49 and its corresponding NPECs, as compared to the calculated ratios.

Atomic ratio

CaCO3/PCT49 calculated

CaCO3/PCT49/PAH = 0.5

inside

outside

calculated

inside

outside

C/Ca

3.2681

3.7304

1.8383

3.439

3.9271

1.6172

N/Ca

0

0

0

0.0571

0.0873

0.0033

O/Ca

5.2685

4.7004

3.2081

5.2681

3.8412

3.1681

CaCO3/PCT49/PAH = 0.9 calculated

inside

outside

CaCO3/PCT49/PAH = 1.2 calculated

inside

outside

C/Ca

3.6111

4.9403

1.2961

3.7813

6.6414

1.4081

N/Ca

0.1142

0.1301

0.0091

0.1714

0.2163

0.0087

O/Ca

5.268

4.0404

3.1844

5.268

4.9745

3.3571


52

Page 53 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 6. The maximum TCH loading capacity of pectin-CaCO3 and NPEC-CaCO3 composite microparticles Sample

n+/n-

TCH loading, (mg/g)

PCT49

0

254.72

PCT49/PAH

0.5

152.78

PCT49/PAH

0.9

188.68

PCT49/PAH

1.2

171.43

PCT53_A21

0

222.77

PCT53_A21/PAH

0.5

187.50

PCT53_A21/PAH

0.9

200.00

PCT53_A21/PAH

1.2

399.04


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Table 7. The maximum release percent after 10 h, and the release constants (kH – the Higuchi constant, kr – the release rate constant), the diffusion coefficient (n), and regression coefficients (R2) calculated using the Higuchi and Korsmeyer-Peppas mathematical models

Sample

n+/n-

Maximum release, %

Higuchi model

Korsmeyer-Peppas model

kH, min1/2

R2

nr

kr, min-n

R2

PCT49

0

22.89

9.066

0.995

1.072

0.052

0.999

PCT49/PAH

0.5

25.32

4.268

0.980

0.850

0.078

0.998

PCT49/PAH

0.9

17.57

6.168

0.984

1.023

0.060

0.985

PCT49/PAH

1.2

15.8

6.351

0.997

1.536

0.018

0.983

PCT_A21

0

28.31

9.652

0.980

0.892

0.072

0.986

PCT_A21/PAH

0.5

22.89

4.548

0.946

1.029

0.053

0.992

PCT_A21/PAH

0.9

26.88

4.503

0.948

1.133

0.030

0.987

PCT_A21/PAH

1.2

31.84

11.678

0.983

0.905

0.075

0.993


54

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Table of Contents

SYNOPSIS. New types of composites were obtained by auto-template assembling CaCO3 capsules by using five pectin samples, which differ in the methylation degree and/or amide content, and some nonstoichiometric polyelectrolyte complex dispersions. The new CaCO3/pectin hollow capsules were tested as possible matrix for tetracycline hydrochloride carrier. The kinetic of the drug release mechanism was followed using Higuchi and KorsmeyerPeppas mathematical models.


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Pectin and Nonstoichiometric

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