Accepted Manuscript Composite Microparticles of Halloysite Clay Nanotubes Bound by Calcium Carbonate Yi Jin, Raghuvara Yendluri, Bin Chen, Jingbo Wang, Yuri Lvov PII: DOI: Reference:

S0021-9797(15)30415-X http://dx.doi.org/10.1016/j.jcis.2015.12.031 YJCIS 20956

To appear in:

Journal of Colloid and Interface Science

Received Date: Revised Date: Accepted Date:

3 November 2015 11 December 2015 17 December 2015

Please cite this article as: Y. Jin, R. Yendluri, B. Chen, J. Wang, Y. Lvov, Composite Microparticles of Halloysite Clay Nanotubes Bound by Calcium Carbonate, Journal of Colloid and Interface Science (2015), doi: http:// dx.doi.org/10.1016/j.jcis.2015.12.031

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Composite Microparticles of Halloysite Clay Nanotubes Bound by Calcium Carbonate Yi Jina*, Raghuvara Yendlurib, Bin Chena, Jingbo Wanga, Yuri Lvovb,c*

a

Institute of Chemical Engineering, Ningbo University of Technology, Ningbo 315016, China

b

Institute for Micromanufacturing, Louisiana Tech University, Ruston, Louisiana 71272, USA c

Ural Federal University, Ekaterinburg, 620002 Russia

ABSTRACT: Natural halloysite clay nanotubes with 15 nm inner and 75nm outer diameters have been used as vehicles for sustained release of drugs in composite hollow microparticles “glued” with CaCO3. We used a layer-by layer assembly accomplished alginate binding with Ca2+ followed by CO2 bubbling to prepare the composite microspheres of CaCO3 and polyelectrolytes (PE) modified halloysite nanotubes (HNTs-PE2/CaCO3) with the diameter of about 5-10 µm. These microparticles have empty spherical structure and abundant pore distributions with maxima at 2.5, 3.9, 6.0 and 13.3 nm, and higher surface area of 82.3 m2g-1 as characterized by SEM and BET test. We loaded drugs in these micro-nano carriers of tight piles of halloysite nanotube with end clogged with CaCO3. The sustained release of Nifedipine drug from HNTs-PE2/CaCO3 composite microspheres was slower than for pristine halloysite nanotubes. Keywords: Halloysite, LbL self-assembly, calcium carbonate, drug sustained release a* Corresponding author. Tel.: +86 138 0587 4816; fax: +86 0574 8708 1240. E-mail address: [email protected] (Y. Jin). b* Corresponding author. Tel.: +1 318 257 5144; fax: +1 318 257 5104. E-mail address: [email protected] (Y. Lvov).

1. Introduction Halloysite nanotubes (HNTs) are a naturally available clay mineral with external diameter of 50-75 nm, lumen of 10-15 nm and length of about 1000 nm [1-3]. Its inner surface consists of a gibbsite octahedral sheet (Al-OH) groups with positive electrical potential, whereas the external surface is composed of siloxane groups (Si-O-Si) with negative surface charge [4-10]. Halloysite has been proved to be environmental friendly and biocompatible which enables it to be a promising nanomaterial for developing organic/inorganic composites for control release in the biomedicine field including drugs, enzymes and DNA delivery [11-16]. With 15 nm lumen consisting ca 15 vol. % of the nanotubes, usage of halloysite nanocontainers for loading and sustained release of functional chemicals seems to be the most remarkable for applications. The in vitro release characteristics of different drugs from halloysite show sustained release with typical time period of 5-20 hours [3]. The drug-halloysite release curves give a bust release 50-60 % of the load in the first 1-2 hrs with slowing release in the following time. We assume that clogging the nanotube ends (making a kind of end-stoppers) would allow further slowing down the release kinetics. Therefore, a complexation of clay nanotube with biocompatible calcium carbonate looks as a promising strategy for developing nano-micro vehicles for controlled drug delivery. Vaterite CaCO3 spherical particles with the diameter of 3-5 µm are composed of nanocrystals bound to each other as fibrous aggregates thus forming channel-like structure with a pore size in the range of 20-40 nm [17, 18]. They have been useful decomposable cores for templating bioactive molecules ether by formulation of microbeads with the biomolecules such as proteins or by encapsulation of the biomolecules into the layer-by-layer formed LbL-multilayer capsules [19-24]. For its nontoxic, highly surface area and mild decomposition conditions the vaterite CaCO3 particles were used for drug release systems [25-28]. Recently, B. Parakhonskiy et al. prepared CaCO3 particles whose geometry was controlled by varying optimizing speed, time, and pH value of the reaction solution, and ratio of the interacting salts. These microparticles were used for studying uptake by living cell and appeared to be an attractive drug delivery platform [29]. Considering high potential of these two of biocompatible nano-micro materials for controlled release, new HNTs-PE2/CaCO3 composite microspheres were designed. We used a layer by layer assembly accomplished with alginate binding with Ca2+ followed by CO2 bubbling to prepare the

hollow microspheres of CaCO3 and halloysite nanotubes (HNTs/CaCO3) with the diameter of about 5-10 µm. This nano-micro composite has several advantages: both of the components are none or low toxic, and are highly porous but with different pore size and internal pore chemistry (positive and negative), which can be loaded with drugs. Nifedipine is a dihydropyridine calcium channel blocker that primarily blocks L-type calcium channels [30]. Its main uses are as an antianginal and antihypertensive. It is also commonly used for the small subset of pulmonary hypertension patients whose symptoms respond to calcium channel blockers. We loaded nifedipine in the pristine halloysite nanotubes and porous HNTs/CaCO3 nano-micro composites to optimize the drug releasing profile. 2. Experiment Sections 2.1. Materials The raw halloysite was supplied from Henan Province, China. All other chemicals and drugs were purchased from Sigma-Aldrich and used without further purification. Chitosan (CH) (Mw ~ 100 kDa) and sodium polyalginic acid (PLGA) were used as polycation and polyanion for LbL assembly with their sequential adsorption. Calcium chloride dehydrate (CaCl2), sodium carbonate (Na2CO3) were used for synthesis of calcium chloride microspheres. Nifedipine was used for loading and releasing study. The water used was purified with a three-stage Millipore Milli-Q Plus 185 system. 2.2. Formation of nanoshells on halloysite tubes with layer-by-layer (LbL) method. The negatively charged halloysite (60 mg) was incubated with 20 mL chitosan-CH solution containing 1 wt. % acetic acid and 0.5 Mol/L NaCl for 30 min, followed by three centrifugation (5000 rpm, 5 min)

washing cycles with 1wt % acetic acid solution. At the second step, the

CH-coated halloysite was added into 20 mL alginate - PLGA solution containing 0.5 Mol/L NaCl; 30 min was allowed for adsorption, and three centrifugation/washing cycles were performed. The cationic chitosan and anionic alginate adsorption steps were repeated to build two bilayer shell on the clay nanotubes. The assembly process was monitored with zeta-potential measurements using Zeta Plus, Brookhaven Instruments Corp. 2.3. Synthesis of HNT-PE2/CaCO3 microspheres

Co-precipitation reaction of two salts is a typical method to prepare vaterite CaCO3 microcores, and introducing protein or other chemicals into these particles [31-34]. This method was used to prepare

the

HNTs-PE2/CaCO3

composite

microspheres

(Scheme

1).

PE2-means

two

polycation/polyanion bilayer shell. First, halloysite was coated with LbL chitosan/alginate shell, then CO2 was bubbled 20 min into this dispersion, followed by addition of 5 mL 0.5M CaCl2 and bubbling for another 30 min. The final composite nano-micro spheres were washed in water three times and dried at 60ºC. To compare with the nanoshelled halloysite composite HNT-PE2/CaCO3, we also prepared the bare pristine halloysite HNT/CaCO3 composites via co-precipitation. The HNT was dispersed in 10 mL 0.5M CaCl2 solution, then CO2 was bubbled into the halloysite dispersion for 30 min. The obtained composite microspheres were washed by water and dried at 60ºC. 2.4. The loading and releasing experiments Saturated solution of nifedipine was prepared by dissolving 20 mg of the drug in 1 mL of ethanol. 50 mg of HNTs or 50 mg of HNTs-PE2/CaCO3 composites was added to the above solution. The mixture is dispersed and sonicated for 5-10 minutes to form a homogeneous dispersion. The dispersion is placed in a vacuum chamber for 30 minutes for three cycles followed by overnight vacuuming. The samples are washed twice with ethanol by centrifuging at 8000 rpm for 3 minutes and dried in a vacuum dessicator. The loading efficiency was determined using TGA. For release control the drug-loaded halloysite and composite nano-micro spheres were placed into 10 mL of phosphate buffers (pH 7.4) at 37 °C with stirring. At suitable intervals, 1 mL of the dissolution medium was taken for testing. The equivalent volumes of fresh medium were added. The concentration of nifedipine was determined at 236 nm using UV/VIS Agilent Techn. Spectrophotometer. 2.5. Characterization Hitachi scanning electron microscopy (SEM, HITACS S-4800) and transmission electron microscopy (Tecnai G2 F30 Twin, USA) were used to characterize the morphology of halloysite and composite particles. X-ray studies were performed with Bruker-D8 Discover XRD instrument, the scan rate was 0.5º (2θ) min-1 with a step size of 0.02º. Thermogravimetric analysis (TGA) was

performed under nitrogen flows from room temperature to 1000 °C at a heating rate of 20°C/min using a DuPont 1090B Thermal Analyzer. BET was conducted with nitrogen porosity meter NOVA 2200e, Quatachrome Instrument Inc. 3. Results and Discussion 3.1. Halloysite LbL nanocoating with chitosan and alginate. Halloysite has negatively charged outermost with the surface ξ-potential at normal aqueous conditions of -25 ± 2 mV.

So, the LbL assembly may be started with cationic chitosan adsorption

followed with anionic sodium polyalginic acid (CH/PLGA). The sequential polycation/polyanion assembly was followed by alternating nanotube ξ-potentials which was an indicator of LbL-multilayer shell formation [35]. With the assembly of CH/PLGA multilayer, the ξ -potential has increased from -25 to +36 ± 2 mV after deposition of the first chitosan layer and then went down to -40 mV after deposition of anionic polyalginic acid; further the adsorption cycle was repeated to make total two-bilayer coating (Fig.1 a).

a

b

c

Fig. 1. ξ-potential versus layer number for the LbL assembly of CH and PLGA on clay nanotube (a) and TEM of pristine (b) and LbL coated tubed decorated with additional layer of 5 nm silica for visualization (c) (error ±5 %).

The alternating ξ-potential is a driving force of sequential CH/PLGA electrostatic binding [36-39]. To visualize the coating on the surface of the nanotubes, an additional layer of silica nanoparticles was assembled above HNTs-PE2. From the TEM images in Fig. 1b-c, silica decorated HNTs-PE2 was visualized showing difference with bare pristine halloysite. The PLGA coating plays

the key role on the forming of the composites because carboxyl groups of PLGA can be cross linked by Ca2+, Scheme 1.

Chitosan

PLGA

Introduce Ca2+ and CO3 2

－

Co-precipitation

Scheme 1. Preparation of HNTs-PE2/CaCO3 composite nano-micro spheres

3.2 Three dimensional structures of the HNTs-PE2/CaCO3 microparticles The polyalginic acid with carboxyl group was crosslinked by Ca2+, which induced assembly of halloysite modified by PLGA forming composite microparticles. Then, we bubbled CO2 through HNT-PE2 dispersion to introduce the carbonate ions CO32- for calcium carbonate production. The SEM images of the resulted nano-micro composite particles are shown in Fig. 2. These images illustrate that after introducing the Ca2+ and CO32- into the halloysite dispersion resulted CaCO3 formation (this was also confirmed with superposition of crystalline X-ray diffraction patterns from halloysite showed in Fig. 3 a and its composition with CaCO3 showed in Fig. 3 b). SEM images in Fig. 2 a-b demonstrate the particles formed by pristine halloysite bound with CaCO3. One can see that CaCO3 cores are loosely coated by the unmodified halloysite tubes and the morphology is different as compared with the more perfect microcomposites formed by chitosan/alginate modified nanotubes (Fig. 2 c-e). The obtained nano-micro composites prepared by with alginate coated and CaCO3 bound halloysite narrower the particle diameter distribution to 5-10 µm. Also the hollow structure with wall thickness of ca. 3 µm can be seen from the broken particles in Fig. 2 d-e. The mechanism of formation such hollow spheres is unclear; one of the assumptions

is that a micro-template may be the CO2 microbubbles (the gas bubbling was a conditions of empty particle formation). Besides, the chitosan has hydrophobic areas which may induce formation of spherical micelles. Similar hollow spheres were observed with chitosan-halloysite shell formation on 5-10 µm droplets oleic oil emulsion [40].

b

a

c

d

e

Fig. 2. SEM images of co-precipitation CaCO3 with bare halloysite (a, b), and co-precipitation CaCO3 with LbL-modified halloysite, HNT-PE2 (c, d, e)

The X-ray patterns of pristine halloysite and HNT/PE2-CaCO3 nano-micro spheres are presented in Fig. 3. Compared with the X-ray pattern of pristine halloysite (Fig. 3 a) the pattern of composite appeared five major vaterite CaCO3 particle reflections (Fig. 3 b, marked with diamonds) , which illustrated that the CaCO3 was embedded between halloysite tubes while reflections of halloysite (multilayer 0.72 nm packing maximum at 2Ɵ = 12.3˚) in the composite

sample still appeared but its strength has reduced, indicating that CaCO3 bulk and halloysite nanotubes are composited physically and no calcites penetrate between alumosilicate layers.

Fig. 3. XRD patterns of HNT/PE2 (a) and HNT-PE2/CaCO3 (b).

The fraction quantification of the halloysite, polyelectrolytes and CaCO3 in the composite particles was obtained with thermogravimetric analysis. Fig. 4 gives the TGA curve of pristine halloysite-HNT,

pure

CaCO3 microspheres,

HNT/PE2

and

HNT-PE2/CaCO3

composite

microspheres recorded at a heating rate of 20 ºC/min in nitrogen. The weight loss temperature of PE2, HNT and CaCO3 were ca. 220, 430 and 570 ºC, respectively. The polyelectrolyte shell PE2 is 5.1 ± 0.2 wt. % of total HNT/PE2. The TGA curve of HNT-PE2/CaCO3 composite microspheres shows that there are three stages of weight loss corresponding to decomposition of polyelectrolytes - PE2, dehydration of halloysite - HNT and decomposition of CaCO3, respectively, with fractions’ weight loss of 3.7, 9.4 and 10.7 ± 0.3 wt. %. This confirms that polyelectrolytes are presenting at 3.7 wt. % and CaCO3 consists 10.7 x 2 = ca. 21 wt. % of total composite sample mass because only 55 % is decomposed at 570 ºC (Fig. 4a). Therefore, in nano-micro composite spheres there are ca. 75 wt. % of halloysite nanotubes and 25 wt. % of binding polyelectrolytes and calcium carbonate.

a

b

c

Fig. 4. The TGA curves of four different samples: a) pristine halloysite and pure CaCO3; b) halloysite coated with chitosan/alginate shell, and c) HNT-PE2/CaCO3 nano-micro composite

With BET analysis we characterized the samples pore size distribution. The surface area of the HNT/CaCO3 nano-micro spheres prepared with CO2 was 82.3 ± 0.2 m2/g, Table 1. Compared with the surface area of 45.0 ± 0.2 m2/g for pristine HNT and 40.0 ± 0.2 m2/g for CaCO3, the HNT-PE2/CaCO3 microspheres has much higher surface area and more abundant pore distributions with characteristic sizes of 2.5, 3.9, 6.0 and 13.3 ± 0.3 nm. It is closer to maxima in the pore distribution of pristine halloysite of 3, 5 and 8.5 ± 0.3 nm. The pore distribution picks of pure CaCO3 microspheres are at 18 and 23 nm. These pores with different diameter can load more medicine or biomolecules with different molecular weight and the amine group in the chitosan can link better proteins. Recently it was reported that a three dimensional flowerlike microstructures of halloysite and layered double hydroxide- HNT@LDH as a carrier to load enzyme [41]. It was constructed by in situ growth of hydroxide nano-platelets on the surface of HNTs via a layer-by-layer deposition process. The results showed that the prepared HNT@LDH exhibited well-defined 3D dimensional architecture and large surface area that rendered it as candidates for hosting for biomolecules. We compared the pore characteristics of halloysite, calcium carbonate, their composite and HNT@LDH which were listed in Table 1. Comparison of HNT@LDH particles with our HNTs-PE2/CaCO3 composites, the HNT@LDH particles had the biggest surface area of 154.6

m2g-1 but no pores, while the HNTs-PE2/CaCO3 composites had smaller sizes and possess more abundant pore distribution for loading different size chemicals. Table 1. Pore characteristics of halloysite, calcium carbonate, their composite and HNT@LDH Samples

Surface area (m2g-1)

Pore volume (cc/g)

Pore diameter (nm)

Particle size (µm) 0.05∼1

HNT

45.1

0.24

3.0, 5.0, 8.5

CaCO3

40.0

0.30

18.0, 23.0

3∼5

HNT-PE2/CaCO3 HNT@LDH

82.3 154.6

0.38 -

2.5, 3.9, 6.0,13.3 -

5∼10 20∼50

3.3. In vitro release study of Nifedipine Nifedipine was used to study the drug loading/releasing behavior of halloysite and its nano-micro composites. From TGA analysis (Fig. 5) the loading efficiency of the drug in the nanotubes was 10.1 ± 0.2 wt. %. Release profiles of Nifedipine from these two nano-micro particles are presented in Fig. 6, which exhibited a two-stage release behavior including a fast release at the initial bust phase in the first half hours and a subsequent prolonged release. The release data was fitted to first order kinetics model given by the equation, R = Mஶ (1 − eି୩୲ ) with an R-squared of 0.84, where Mஶ = amount of active agent released at infinite time and k = release rate constant [42]. For drug in the HNT-PE2/CaCO3 composite the release time was slowed down to 9 hours for 70 wt. % drug released (complete release needs ca. 23 hours) whereas from pure pristine halloysite larger 94 wt. % fraction of the drug was released at the same time. A slower release rate of the composite is due to polymers clogging ends of the nanotubes and the pile up structure of the nanotubes.

Fig. 5. TGA curve of the drug-loaded HNTs-PE2/CaCO3 composite and as compared with drug-loaded pristine halloysite

Fig. 6. Nifedipine releasing curve of the halloysite-CaCO3 composite and as compared with pristine halloysite

The different decomposition conditions of halloysite and CaCO3 may also facilitate the different release kinetics due to decomposition of calcium carbonate at low pH. In stomach pH of 1.2, CaCO3 can be easily dissolved, and the drug loaded in CaCO3 should be released firstly, followed with the second stage release of the drug loaded in halloysite tubes in intestines. 4. Conclusions Calcium carbonate is used in the drug delivery formulations due to its mesoporous structure and biocompatibility, such as the decomposable cores templates by encapsulation of the biomolecules into the layer-by-layer formed LbL-multilayer capsules. Halloysite also has been proved to be environmental friendly and biocompatible for control release field. We combined CaCO3 with biocompatible natural clay nanotubes to produce nano-micro composites with synergetic properties. The HNT-PE2/CaCO3 composite microspheres with the diameter of 5-10 µm were prepared by co-precipitation method using alginate coated clay nanotubes. The prepared composites have empty spherical shape with halloysite as the main component, and remaining mass

is CaCO3 binder of ca. 21 wt. % and polyelectrolytes ca. 4 wt. %.

These microspheres had larger

pores of 2.5, 3.9, 6.0 and 13.3 nm and had higher surface area of 82.3 m2/g than that of pristine halloysite with 3.0, 5.0, 8.5 nm and 45.1 m2/g [43], and CaCO3 particles with 18, 23 nm and 40.0 m2/g [44]. The porous structure and high surface area make it a promising material for the drug delivery. Ca2+ binding of alginate was an important feature of the microparticles formation. The loading of nifedipine drug in the nanotube composites was 10.1 ± 0.2 wt. %.

Release profiles of nifedipine

from halloysite-composites exhibit an exponential behavior including the initial bust and a subsequent prolonged release. The 70 % drug release time from HNT-PE2/CaCO3 composite was 9 hours (complete 95 % releasing needed about 20 hours), it is much slower than nifedipine release from a pristine halloysite. A sustained nifedipine release illustrated kinetics which was retarded with the composite nanoformulation of these clay nanotubes bound through calcium carbonate. This two natural mineral composites with different pore size and structures have great potential to loading different kinds and size drugs, proteins and nanoparticles to carry out the control-release or catalytic reaction.

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Graphic Abstract A layer-by layer assembly technique was used to accomplish halloysite clay nanotube alginate binding with Ca2+ followed by CO2 bubbling to prepare nanotube-calcium carbonate composite spheres. These composite particles have abundant pore distribution range of 2.5 to 13 nm and surface area of 82 m2g-1.

Highlights


Halloysite nanotubes were modified with chitosan and alginate by LbL self-assemble technique.




Composite microspheres of CaCO3 (HNTs-PE2/CaCO3) were prepared by alginate binding with Ca2+ followed by CO2 bubbling.




The composite particles were non toxic and biocompatible.




The composite particles have empty spherical structure, abundant pore distributions and higher surface area.


The sustained release of Nifedipine drug from HNTs-PE2/CaCO3 composite microspheres was slower than that from pristine halloysite nanotube.