- Email: [email protected]
Chitosan nanoparticles affect polymorph selection in crystallization of calcium carbonate
Accepted Manuscript Title: Chitosan nanoparticles affect polymorph selection in crystallization of calcium carbonate Authors: Matthew A. Hood, Katharina Landfester, Rafael Mu˜noz-Esp´ı PII: DOI: Reference:
S0927-7757(17)31145-7 https://doi.org/10.1016/j.colsurfa.2017.12.048 COLSUA 22172
To appear in:
Colloids and Surfaces A: Physicochem. Eng. Aspects
Received date: Revised date: Accepted date:
13-10-2017 16-12-2017 18-12-2017
Please cite this article as: Hood MA, Landfester K, Mu˜noz-Esp´ı R, Chitosan nanoparticles affect polymorph selection in crystallization of calcium carbonate, Colloids and Surfaces A: Physicochemical and Engineering Aspects (2010), https://doi.org/10.1016/j.colsurfa.2017.12.048 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chitosan nanoparticles affect polymorph selection in crystallization of calcium carbonate Matthew A. Hood1, Katharina Landfester1, and Rafael Muñoz-Espí1,2*
2
Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany
SC RI PT
1
Institute of Materials Science (ICMUV), Universitat de València, c/ Catedràtic José Beltrán 2, 46980 Paterna, València, Spain * E-mail: [email protected]
+ CO32− Ca2+
Ca2+ Ca2+
Ca2+
CaCO3
Ca2+
A
CC
EP
TE
D
M
A
N
U
Graphical abstract
Abstract. Stable suspensions of chitosan nanoparticles were produced by the inverse miniemulsion technique and used as substrates for the crystallization of various polymorphs of calcium carbonate (CaCO3). These bioinspired chitosan nanoparticles were cross-linked with 1,4-butanediol diglycidyl ether, had an average particle diameter of 100–350 nm, and displayed 1
pH sensitive swelling behavior. X-ray diffraction demonstrated that vaterite and calcite were obtained when calcium carbonate was mineralized in the chitosan nanoparticle suspension. The extent of vaterite stabilization depends on the cross-linking fraction of the chitosan nanoparticles
SC RI PT
and the crystallization conditions.
KEYWORDS: chitosan; calcium carbonate; biomineralization; hydrogel; nanoparticle;
A
CC
EP
TE
D
M
A
N
U
miniemulsion
2
1. Introduction Calcium-based biominerals, such as calcium carbonate (CaCO3), are the main inorganic structural component of marine organisms including mollusks, crustaceans, and coccolith producing algae [1-3]. Frequently, these calcium carbonate biominerals are grown from an insoluble substrate, such as the polysaccharide chitin [4, 5]. Due to the limited processability of
SC RI PT
pure chitin, a deacetylated form, chitosan, is required for research and commercial uses. Chitosan chemistry has been studied extensively, and cross-linked chitosan networks have been formed by addition of tripolyphosphate, genipin, glutaraldehyde, carbodiimide, and diglycidyl ether [6-10]. In bioinspired mineralization of calcium-based materials, films of both chitin and chitosan have been used as substrates in the crystallization of calcium carbonate and hydroxyapatite [11-13]. The polymorph selection and the morphology of CaCO3 may be affected by additives present
U
during nucleation and growth, leading to aragonite and vaterite stabilization over the
N
thermodynamically favorable calcite [14, 15].
Both chitosan nanoparticles and calcium carbonate have shown significant potential in
A
biomedical applications, including enamel remineralization and cancer therapies [16-20].
M
However, nanoparticles of chitosan have been difficult to form because of limited processability of chitosan in water and organic solvents [21]. Nanoparticles formed via the miniemulsion
D
synthetic route have been used in medicine, structural materials, and crystallization [22-26]. The nanoparticles [27].
TE
miniemulsion technique produces droplet micro-reactors that can more easily form chitosan Herein, stable suspensions of swellable chitosan nanoparticles were produced by the inverse
EP
miniemulsion method. The cross-link density was modified by changing the amount of crosslinking agent (1,4-butanediol diglycidyl ether) from 5 to 20 wt% of the total solid nanoparticle
CC
content. The swelling behavior and colloidal stability of the chitosan nanoparticles were estimated from dynamic light scattering (DLS) and ζ-potential measurements at pH values of 4
A
and 7. The swelling behavior of chitosan nanoparticles suggested a potential use of the nanoparticles as both a drug delivery matrix and a scaffold for crystallization [27-31]. Chitosan nanoparticles were subsequently added to the medium prior to crystallization of calcium carbonate. The cross-linking density and the carbonate source affected the stabilization of the polymorph vaterite during crystallization.
3
2. Materials and methods 2.1. Materials Low molecular weight chitosan (Sigma Aldrich, 50,000–190,000 Da, 75–85% deacetylated), acetic acid (Sigma Aldrich, puriss.), 1,4-butanediol diglycidyl ether (BDE, Sigma Aldrich,
SC RI PT
≥95%), polyglycerol polyricinoleate (PGPR, DANISCO), cyclohexane (VWR), calcium chloride dihydrate (Sigma Aldrich, ≥99%), sodium carbonate (Fisher Scientific, ≥99.5%), sodium hydrogen carbonate (Fisher Scientific, ≥99.5%), and potassium hydroxide (Riedel deHaën) were used as-received. De-ionized water was used for the miniemulsion procedures. Ultrapure MilliQ
U
water (>18.5 MΩ∙cm) was used for precipitation studies.
N
2.2. Synthesis of chitosan hydrogel nanoparticles
A
Chitosan cross-linking relied on the reaction of the primary amines from chitosan and a di-
M
epoxide cross-linker, 1,4-butanediol diglycidyl ether (Scheme 1). Two separate emulsions were prepared to prevent the cross-linking reaction from occurring until nanosized droplets were formed. The dispersed phase of the chitosan emulsion consisted of chitosan (5 wt%) dissolved in
D
a 2 wt% acetic acid aqueous solution. For the BDE emulsion, the desired amount of cross-linking
TE
agent (5, 10, 15, 20 wt% with respect to chitosan weight) was dissolved in de-ionized water. 650 μL of chitosan or BDE solution was placed into a cylindrical vial containing 6.2 g of
EP
cyclohexane and 55 mg of the non-ionic surfactant PGPR (0.88 wt% in cyclohexane). Emulsions were formed by ultrasonication (Branson W 450 digital sonifier) using a 1/2" sonication tip for 2
CC
min with a pulse sequence (10 s on and 2 s off). The two emulsions (chitosan and BDE) were mixed and immediately ultrasonicated again using the same procedure. The final emulsions were allowed to mix under magnetic stirring at 800 rpm for 24 h. During those 24 h, cross-linking
A
between the amine and glycidal ether groups occurred. Purification of the cross-linked chitosan nanoparticles required that nanoparticle/cyclohexane
suspensions were added to 10 mL of 1-propanol and 20 mL of de-ionized water. The resulting dilute azeotropic suspension was distilled at reduced pressure until the chitosan particles were suspended only within water. The chitosan/water suspensions were transferred to dialysis tubing of a molecular weight cut off of 13,000 Da. Dialysis took place in de-ionized water containing 4
1 wt% acetic acid for 1 week. After 1 week, de-ionized water was changed twice daily for three days with no acetic acid. A final exchange of water was performed with ultrapure water. The suspensions from the dialysis tubing were collected into glass vials. Two phases were observed: (1) a translucent suspension of particles in water, and (2) an opaque, white waxy substance. The waxy substance was assumed to be free PGPR as it was not soluble in water. Suspensions were
SC RI PT
filtered through 5 µm syringe filters to remove the waxy substance and remove any aggregates from the suspension. Fourier transform infrared spectra (FTIR) spectra of nanoparticles are shown in Fig. S1.
2.3. Crystallization of calcium carbonate crystallization
U
Cross-linked chitosan particles corresponding to a content of 10 ppm were added to a 2.5 mM
N
CaCl2 solution at pH 7. 1.0 mL of 1.0 M CaCl2 was added to 16 mL of MilliQ water. The pH was adjusted to a value of 7 by addition of KOH and additional ultrapure water was added till the
A
total solution volume was adjusted to 18 mL. After mixing for at least 3 h, 1.0 mL of 1.0 M
M
Na2CO3 or NaHCO3 was added with 1.0 mL of ultrapure water, so that the total solution concentration was 20 mL.
D
Crystallization took place under stirring (300 rpm) for 24 h at room temperature.
TE
Precipitation experiments were performed in reaction vessels with tightly sealed screw on caps in order to minimize exchange of CO2 between the reactions and the atmosphere. Crystals of calcium carbonate were collected by centrifugation at 4000 rpm for 20 min using a Rotixa 50 RS
CC
EP
swinging-bucket rotor centrifuge. Precipitants were dried under vacuum for 14 h.
2.4. Characterization methods Particle sizes were measured with dynamic light scattering (DLS) by using a PSS Nicomp
A
Particle Sizer 380 in 90° scattering mode at 25 °C. Dispersions for DLS measurements had a solid content of 0.1−0.5 wt% nanoparticles in water. The zeta potential was determined by electrophoretic mobility measurements in a Malvern ZetaSizer Nano-Z instrument. The pH of suspensions was controlled by adding acetic acid to ultrapure water based on calculated values for pH 4. The ionic strength of the solution was controlled to contain 0.01 mM KCl. 5
The nanoparticles and the morphology of calcium carbonate crystals were analyzed by scanning electron microscopy (SEM) on a Zeiss 1530 Gemini LEO microscope on samples deposited on Si wafers with no coating. Transmission electron microscopy (TEM) was carried out in a JEOL 1400 microscope with an accelerating voltage of 120 kV was used to analyze dried particles dropcast onto carbon coated copper grids.
SC RI PT
X-ray diffraction (XRD) patterns were registered for powder samples on a Mylar film by using a Guinier imaging plate camera (G670) from HUBER, with an incident X-ray beam with a Cu Kα wavelength of 1.5406 Å. Data was smoothed using a least-mean-square fitting algorithm in Origin to reduce the overall noise generated due to air and Mylar film.
U
3. Results and discussion
N
3.1. Synthesis of chitosan nanoparticles
A
We attempted the preparation of pH-swellable chitosan nanoparticles by cross-linking chitosan
M
amino groups to either sulfo-N-hydroxysuccinimide functionalized polyethylene glycol via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide coupling or to 1,4-butanediol diglycidyl ether. Although both routes led to positive results, the nanoparticle yield was greater when using
D
1,4-butanediol diglycidal ether. Accordingly, further chitosan nanoparticles with various
TE
amounts of cross-linker were prepared by the epoxy–amine reaction between the chitosan amino groups and the 1,4-butanediol diglycidyl ether epoxide groups (Scheme 1).
EP
The two reaction agents were separately pre-emulsifed and then combined under sonication to generate “nanoreactors” for the cross-linking of the chitosan. After reaction, a majority of the
CC
excess surfactant and nanoparticle aggregates were removed by solvent exchange to ultrapure
A
water, followed by filtration through a 5 µm filter, and finally dialysis.
6
SC RI PT
N
U
Scheme 1: Chitosan reaction with 1,4-butanediol diglycidyl ether.
A
3.2. pH-dependent swelling behavior of chitosan nanoparticles
M
The pH-dependent swelling properties of the chitosan nanoparticles were confirmed by DLS measurements at pH values of 4 and 7. The average hydrodynamic diameter (dH) of the
D
nanoparticles at a pH of 7 was ~350 nm (Table 1); it does not significantly depend on the amount
TE
of cross-linker. TEM images indicated that the polydispersity in the particle size was rather high, with many nanoparticles between 50 and 100 nm, a smaller fraction around 350 nm, and a minority of particles greater than 1 µm (Fig. 1 and Fig. S2). This observation is consistent with
EP
the high polydispersity obtained for chitosan nanoparticles by other groups [10, 32]. At a value of pH 4, which is below the pKa of chitosan (around 6.4), the size of the chitosan nanoparticles
CC
increased by ~50 nm when compared to values at neutral pH, as shown in Table 1. The zeta potential of chitosan nanoparticles at a pH of 4 were greater than +40 mV regardless
A
of the cross-linker concentration, indicating good stability. At pH values above the pKa value, deprotonation of the amino groups results in a charge decrease, which would typically correlate with a loss of colloidal stability [27]. At a pH value of 7, the suspensions had zeta potential values within the rapid flocculation regime (0–5 mV). While flocculation likely began at a pH value of 7, it was not rapid. Only after a period of 1 to 3 weeks, nanoparticles displayed larger aggregates that would slowly precipitate out of suspension. After flocculation, SEM and DLS 7
analysis indicated that the number of particles larger than ~1 µm had increased. At pH values greater than 7, chitosan nanoparticles aggregated quickly and precipitated out of suspension. Chitosan nanoparticles aggregated and sedimented more quickly as their cross-linking density
SC RI PT
increased [33].
Table 1: Particle sizes and zeta potential of chitosan nanoparticles at different cross-linker concentration
pH = 4
pH = 7
pH = 4
pH = 7
5%
371 (±40%)
353 (±34%)
44 (±3%)
3.5 (±26%)
CP2
10 %
395 (±59%)
322 (±68%)
46 (±1%)
4.3 (±19%)
CP3
15 %
404 (±31%)
357 (±46%)
43 (±9%)
6.9 (±6%)
CP4
20 %
403 (±47%)
344 (±53%)
46 (±5%)
4.2 (±26%)
A
M
Determined by DLS
CP20
b)
A
CC
EP
a)
TE
CP19
N
CP1
D
a
ζ potential (mV)
dHa (nm)
Cross-linker Concentration
U
Sample
CP19
CP20
Fig. 1. TEM images of as-prepared chitosan nanoparticles a) small aggregates of sub-100 nm particles, b) a greater distribution of nanoparticle sizes.
8
3.3 Crystallization of calcium carbonate in the presence of chitosan nanoparticles CaCO3 is typically formed under basic conditions. At pH values below 7, carbonic acid and CO2 are favored over hydrogencarbonate and carbonate ions in solution [34]. Due to the aggregation
SC RI PT
of chitosan nanoparticles at pH values greater than 7, it was not possible to crystallize calcium carbonate under a typical pH value of 8.5–10. Thus, chitosan nanoparticles and calcium ions were placed into a suspension at an initial pH of 7, and the addition of a carbonate salt led to higher pH value upon dissolution [14].
Sodium hydrogencarbonate and sodium carbonate salts were chosen as carbonate sources for their impact on the pH value of the solution after dissociation. At the concentration used in this
U
work, sodium hydrogencarbonate raised a pH value of the suspension to a value of 8.5, while the
N
sodium carbonate increased the pH towards 10. The dissociation of the proton from HCO3− to form carbonate ions (CO32−) also slows down the kinetics of CaCO3 formation, which we had
M
oligopeptides) and calcium chloride [14].
A
observed previously when using these two carbonate salts in the presence of amino acids (or
Chitosan nanoparticles equivalent to a concentration of 10 ppm were added to solutions of
D
[Ca2+] = 2.5 mM. To initiate crystallization, either NaHCO3 or Na2CO3 was added to the
TE
Ca2+/chitosan suspensions. Crystal shapes typical for calcite and vaterite were observed by SEM after the crystallization experiments (Fig. 2 and Fig. S3). The ability of chitosan nanoparticles to stabilize the kinetically favorable but thermodynamically unfavorable vaterite polymorph was
EP
found to be dependent on both the cross-linking density and the carbonate source used, as seen by the XRD patterns of the CaCO3 crystals formed with the differing conditions, shown in Fig. 3.
CC
Upon addition of Na2CO3, vaterite stabilization was not observed from the XRD patterns until nanoparticles prepared with 15 and 20 wt% cross-linking agent were used. In contrast, when
A
using NaHCO3, the vaterite phase was stabilized only at 5 and 10 wt% of the cross-linking agent. Kinetics and pH value play a role in crystallization. As observed by zeta potential the chitosan nanoparticles quickly lost stability in suspension as the pH increased to basic pH values greater than 7. As mentioned before, Na2CO3 increased the pH value of the particle suspensions to 10, and the chitosan nanoparticles quickly began to flocculate. However the driving force for CaCO3 crystallization was also high and CaCO3 crystals grew concurrently with the quickly aggregating 9
chitosan nanoparticles. A more cross-linked substrate to grow from was necessary for the stabilization of the vaterite crystals, possibly due to its increased rigidity [35, 36]. A strong interaction between the chitosan nanoparticles and the growing CaCO3 crystals was observed by the large amount of surface pitting on the calcite crystals, despite the low amount of chitosan nanoparticles added (Fig. 2b).
SC RI PT
On the other hand vaterite was stabilized at lower nanoparticle stiffness (i.e., cross-linking fraction) when NaHCO3 was used as a carbonate source. At the lower pH value of 8.5, chitosan nanoparticles flocculated slower than at pH 10 (although complete flocculation occurred over the course of a few hours). However, HCO3− ions, not fully dissociated to carbonate ions, led to slower crystallization. The interaction of less cross-linked nanoparticles, which remained colloidally stable for longer times than the more rigid substrates, had an impact on the
U
stabilization of vaterite. At higher cross-linking concentrations, vaterite was not stabilized.
N
While the harder substrates acted as better nucleation surfaces for crystals with Na2CO3, when using NaHCO3, it was necessary to have faster interaction times between the chitosan
A
nanoparticles and the Ca2+ and mineralizing CO32− ions as the particles quickly sedimented out
M
of solution. The softer substrates of the lower cross-linked chitosan nanoparticles interact with
TE
along the softer particles.
D
the ions and growing clusters of CaCO3 for longer times, thus resulting in higher supersaturation
b)
A
CC
EP
a)
Fig. 2. SEM images of CaCO3 show a) vaterite and calcite co-mineralization along with pitting of calcite crystals due to nanoparticles interaction with the growing CaCO3 crystals, b) zoomed in area of region highlighted in red from image a.
10
(b) NaHCO3
V (112)
CP4 (20 wt% cross-linker)
C (018) C (116)
C (202)
CP3 (15 wt% cross-linker)
SC RI PT
C (113)
V (300)
CP4 (20 wt% cross-linker)
C (110)
V (116)
C (104)
C (106)
V (110)
V (113)
C (012)
(a) Na2CO3
Intensity (a.u.)
Intensity (a.u.)
CP3 (15 wt% cross-linker)
CP2 (10 wt% cross-linker)
CP2 (10 wt% cross-linker)
CP1 (5 wt% cross-linker)
25
30
35
40
45
U
CP1 (5 wt% cross-linker)
50
25
30
35 40 2 (
45
50
A
N
2 ()
Fig. 3. XRD patterns showing the stabilization of vaterite (V: vaterite, C: calcite) in chitosan
M
nanoparticles suspensions containing calcium ions upon addition of a) Na2CO3 and b) NaHCO3.
4. Conclusions
TE
D
The particle content was in all cases 10 ppm.
We have shown a simple method to produce a large amount of pH swellable, cross-linked
EP
chitosan nanoparticles, which have been used as substrates during the crystallization of CaCO3. Promising stabilization of vaterite and obvious interactions with growing calcite crystals at
CC
contents of chitosan nanoparticles as low as 10 ppm suggest that these nanoparticles should be further evaluated for their ability to affect bioinspired crystallization. In this sense, chitin is
A
already observed in nature to act as a potent substrate for growth of CaCO3. These swellable nanoparticles could find a potential for use in a plethora of smart materials, with particular focus in drug delivery systems that contain polymorph specific calcium carbonate.
Acknowledgements
11
The authors thank Michael Steiert for the assistance with X-ray diffraction. The authors also thank the Max Planck Society for funding of the Max Planck Partner Group on Colloidal Methods for Multifunctional Materials (CM3) at the University of Valencia, headed by RME. RME acknowledges the financial support from the Spanish Ministry of Economy, Industry and
SC RI PT
Competitiveness through a Ramón y Cajal grant (grant no. RYC-2013-13451),
References
[1] S. Sviben, A. Gal, M.A. Hood, L. Bertinetti, Y. Politi, M. Bennet, P. Krishnamoorthy, A. Schertel, R. Wirth, A. Sorrentino, E. Pereiro, D. Faivre, A. Scheffel, A vacuole-like compartment concentrates a disordered calcium phase in a key coccolithophorid alga, Nature Commun. 7
U
(2016) 11228.
N
[2] S. Mann, Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry,
A
Oxford University Press, 2001.
[3] M.A. Hood, H. Leemreize, A. Scheffel, D. Faivre, Lattice distortions in coccolith calcite
M
crystals originate from occlusion of biomacromolecules, J. Struct. Biol. 196 (2016) 147-154. [4] L. Addadi, D. Joester, F. Nudelman, S. Weiner, Mollusk Shell Formation: A Source of New
D
Concepts for Understanding Biomineralization Processes, Chem. Eur. J. 12 (2006) 980-987.
TE
[5] N.H. Munro, K.M. McGrath, Biomimetic approach to forming chitin/aragonite composites, Chem. Commun. 48 (2012) 4716-4718.
EP
[6] R.A. Muzzarelli, Genipin-crosslinked chitosan hydrogels as biomedical and pharmaceutical aids, Carbohyd. Polym. 77 (2009) 1-9. [7] G.A. Roberts, K.E. Taylor, Chitosan gels, 3. The formation of gels by reaction of chitosan
CC
with glutaraldehyde, Makromol. Chem. 190 (1989) 951-960. [8] M. Rafat, F. Li, P. Fagerholm, N.S. Lagali, M.A. Watsky, R. Munger, T. Matsuura, M.
A
Griffith, PEG-stabilized carbodiimide crosslinked collagen–chitosan hydrogels for corneal tissue engineering, Biomaterials 29 (2008) 3960-3972. [9] F.-L. Mi, S.-S. Shyu, C.-T. Chen, J.-Y. Schoung, Porous chitosan microsphere for controlling the antigen release of Newcastle disease vaccine: preparation of antigen-adsorbed microsphere and in vitro release, Biomaterials 20 (1999) 1603-1612.
12
[10] V. Kamat, D. Bodas, K. Paknikar, Chitosan nanoparticles synthesis caught in action using microdroplet reactions Sci. Rep., 6 (2016). [11] X. An, C. Cao, Coeffect of silk fibroin and self-assembly monolayers on the biomineralization of calcium carbonate, J. Phys. Chem. C 112 (2008) 15844-15849. [12] G. Falini, S. Weiner, L. Addadi, Chitin-silk fibroin interactions: relevance to calcium
SC RI PT
carbonate formation in invertebrates, Calcif. Tissue Int. 72 (2003) 548-554.
[13] E.C. Keene, J.S. Evans, L.A. Estroff, Silk Fibroin Hydrogels Coupled with the n16N− βChitin Complex: An in Vitro Organic Matrix for Controlling Calcium Carbonate Mineralization, Cryst. Growth Des. 10 (2010) 5169-5175.
[14] M.A. Hood, K. Landfester, R. Muñoz-Espí, The Role of Residue Acidity on the Stabilization of Vaterite by Amino Acids and Oligopeptides, Cryst. Growth Des. 14 (2014)
U
1077-1085.
N
[15] H. Lu, M.A. Hood, S. Mauri, J.E. Baio, M. Bonn, R. Muñoz-Espí, T. Weidner, Biomimetic vaterite formation at surfaces structurally templated by oligo(glutamic acid) peptides, Chem.
A
Commun. 51 (2015) 15902-15905.
M
[16] W.E. Rudzinski, A. Palacios, A. Ahmed, M.A. Lane, T.M. Aminabhavi, Targeted delivery of small interfering RNA to colon cancer cells using chitosan and PEGylated chitosan
D
nanoparticles, Carbohyd. Polym. 147 (2016) 323-332.
TE
[17] A. Abruzzo, G. Zuccheri, F. Belluti, S. Provenzano, L. Verardi, F. Bigucci, T. Cerchiara, B. Luppi, N. Calonghi, Chitosan nanoparticles for lipophilic anticancer drug delivery: Development, characterization and in vitro studies on HT29 cancer cells, Colloids Surf. B, 145
EP
(2016) 362-372.
[18] Q. Ruan, Y. Zhang, X. Yang, S. Nutt, J. Moradian-Oldak, An amelogenin–chitosan matrix
CC
promotes assembly of an enamel-like layer with a dense interface, Acta Biomater., 9 (2013) 7289-7297.
A
[19] Y. Huang, Y. Duan, Y. Qian, R. Huang, Z. Yang, Y. Li, Z. Zhou, Remineralization efficacy of a toothpaste containing 8% arginine and calcium carbonate on enamel surface, Amer. J. Dent. 26 (2013) 291-297. [20] X.w. He, T. Liu, Y.x. Chen, D.j. Cheng, X.r. Li, Y. Xiao, Y.l. Feng, Calcium carbonate nanoparticle delivering vascular endothelial growth factor-C siRNA effectively inhibits lymphangiogenesis and growth of gastric cancer in vivo, Cancer Gene Ther, 15 (2008) 193-202. 13
[21] M. Rinaudo, Chitin and chitosan: properties and applications, Progr. Polym. Sci., 31 (2006) 603-632. [22] M. Hood, M. Mari, R. Muñoz-Espí, Synthetic Strategies in the Preparation of Polymer/Inorganic Hybrid Nanoparticles, Materials 7 (2014) 4057. [23] K. Landfester, D. Crespy, Miniemulsion Polymerization, in:
SC RI PT
Technology, Wiley-VCH, 2006.
Materials Science and
[24] R. Muñoz-Espí, C.K. Weiss, K. Landfester, Inorganic nanoparticles prepared in miniemulsion, Curr. Opin. Colloid Interf. Sci., 17 (2012) 212-224.
[25] M.A. Hood, N. Encinas, D. Vollmer, R. Graf, K. Landfester, R. Muñoz‐Espí, Controlling hydrophobicity of silica nanocapsules prepared from organosilanes, Colloids Surf. A. 532 (2017) 172–177
U
[26] M.A. Hood, U. Paiphansiri, D. Schaeffel, K. Koynov, M. Kappl, K. Landfester, R. Muñoz-
N
Espí, Hybrid Poly(urethane–urea)/Silica Nanocapsules with pH-Sensitive Gateways, Chem. Mater. 27 (2015) 4311-4318.
A
[27] N. Solomko, O. Budishevska, S. Voronov, K. Landfester, A. Musyanovych, pH-Sensitive
M
Chitosan-based Hydrogel Nanoparticles through Miniemulsion Polymerization Mediated by Peroxide Containing Macromonomer, Macromol. Biosci. 14 (2014) 1076-1083.
D
[28] S.-C. Chen, Y.-C. Wu, F.-L. Mi, Y.-H. Lin, L.-C. Yu, H.-W. Sung, A novel pH-sensitive
TE
hydrogel composed of N, O-carboxymethyl chitosan and alginate cross-linked by genipin for protein drug delivery, J. Control. Release 96 (2004) 285-300. [29] A.M. De Campos, A. Sánchez, M.a.J. Alonso, Chitosan nanoparticles: a new vehicle for the
EP
improvement of the delivery of drugs to the ocular surface. Application to cyclosporin A, Int. J. Pharmac. 224 (2001) 159-168.
CC
[30] K.A. Janes, M.P. Fresneau, A. Marazuela, A. Fabra, M.a.J. Alonso, Chitosan nanoparticles as delivery systems for doxorubicin, J. Control. Release 73 (2001) 255-267.
A
[31] L.C. Preiss, L. Werber, V. Fischer, S. Hanif, K. Landfester, Y. Mastai, R. Muñoz-Espí, Amino-Acid-Based Chiral Nanoparticles for Enantioselective Crystallization, Adv. Mater. 27 (2015) 2728-2732. [32] A. Rampino, M. Borgogna, P. Blasi, B. Bellich, A. Cesàro, Chitosan nanoparticles: preparation, size evolution and stability, Int. J. Pharmac. 455 (2013) 219-228.
14
[33] H. Jonassen, A.-L. Kjøniksen, M. Hiorth, Stability of Chitosan Nanoparticles Cross-Linked with Tripolyphosphate, Biomacromolecules 13 (2012) 3747-3756. [34] P. Atkins, Shriver and Atkins' Inorganic Chemistry, OUP Oxford, 2010. [35] X.Y. Liu, K. Tsukamoto, M. Sorai, New Kinetics of CaCO3 Nucleation and Microgravity Effect, Langmuir 16 (2000) 5499-5502.
SC RI PT
[36] Y.Y. Kim, L. Ribeiro, F. Maillot, O. Ward, S.J. Eichhorn, F.C. Meldrum, Bio‐Inspired Synthesis and Mechanical Properties of Calcite–Polymer Particle Composites, Adv. Mater. 22
A
CC
EP
TE
D
M
A
N
U
(2010) 2082-2086.
15
S0927-7757(17)31145-7 https://doi.org/10.1016/j.colsurfa.2017.12.048 COLSUA 22172
To appear in:
Colloids and Surfaces A: Physicochem. Eng. Aspects
Received date: Revised date: Accepted date:
13-10-2017 16-12-2017 18-12-2017
Please cite this article as: Hood MA, Landfester K, Mu˜noz-Esp´ı R, Chitosan nanoparticles affect polymorph selection in crystallization of calcium carbonate, Colloids and Surfaces A: Physicochemical and Engineering Aspects (2010), https://doi.org/10.1016/j.colsurfa.2017.12.048 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chitosan nanoparticles affect polymorph selection in crystallization of calcium carbonate Matthew A. Hood1, Katharina Landfester1, and Rafael Muñoz-Espí1,2*
2
Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany
SC RI PT
1
Institute of Materials Science (ICMUV), Universitat de València, c/ Catedràtic José Beltrán 2, 46980 Paterna, València, Spain * E-mail: [email protected]
+ CO32− Ca2+
Ca2+ Ca2+
Ca2+
CaCO3
Ca2+
A
CC
EP
TE
D
M
A
N
U
Graphical abstract
Abstract. Stable suspensions of chitosan nanoparticles were produced by the inverse miniemulsion technique and used as substrates for the crystallization of various polymorphs of calcium carbonate (CaCO3). These bioinspired chitosan nanoparticles were cross-linked with 1,4-butanediol diglycidyl ether, had an average particle diameter of 100–350 nm, and displayed 1
pH sensitive swelling behavior. X-ray diffraction demonstrated that vaterite and calcite were obtained when calcium carbonate was mineralized in the chitosan nanoparticle suspension. The extent of vaterite stabilization depends on the cross-linking fraction of the chitosan nanoparticles
SC RI PT
and the crystallization conditions.
KEYWORDS: chitosan; calcium carbonate; biomineralization; hydrogel; nanoparticle;
A
CC
EP
TE
D
M
A
N
U
miniemulsion
2
1. Introduction Calcium-based biominerals, such as calcium carbonate (CaCO3), are the main inorganic structural component of marine organisms including mollusks, crustaceans, and coccolith producing algae [1-3]. Frequently, these calcium carbonate biominerals are grown from an insoluble substrate, such as the polysaccharide chitin [4, 5]. Due to the limited processability of
SC RI PT
pure chitin, a deacetylated form, chitosan, is required for research and commercial uses. Chitosan chemistry has been studied extensively, and cross-linked chitosan networks have been formed by addition of tripolyphosphate, genipin, glutaraldehyde, carbodiimide, and diglycidyl ether [6-10]. In bioinspired mineralization of calcium-based materials, films of both chitin and chitosan have been used as substrates in the crystallization of calcium carbonate and hydroxyapatite [11-13]. The polymorph selection and the morphology of CaCO3 may be affected by additives present
U
during nucleation and growth, leading to aragonite and vaterite stabilization over the
N
thermodynamically favorable calcite [14, 15].
Both chitosan nanoparticles and calcium carbonate have shown significant potential in
A
biomedical applications, including enamel remineralization and cancer therapies [16-20].
M
However, nanoparticles of chitosan have been difficult to form because of limited processability of chitosan in water and organic solvents [21]. Nanoparticles formed via the miniemulsion
D
synthetic route have been used in medicine, structural materials, and crystallization [22-26]. The nanoparticles [27].
TE
miniemulsion technique produces droplet micro-reactors that can more easily form chitosan Herein, stable suspensions of swellable chitosan nanoparticles were produced by the inverse
EP
miniemulsion method. The cross-link density was modified by changing the amount of crosslinking agent (1,4-butanediol diglycidyl ether) from 5 to 20 wt% of the total solid nanoparticle
CC
content. The swelling behavior and colloidal stability of the chitosan nanoparticles were estimated from dynamic light scattering (DLS) and ζ-potential measurements at pH values of 4
A
and 7. The swelling behavior of chitosan nanoparticles suggested a potential use of the nanoparticles as both a drug delivery matrix and a scaffold for crystallization [27-31]. Chitosan nanoparticles were subsequently added to the medium prior to crystallization of calcium carbonate. The cross-linking density and the carbonate source affected the stabilization of the polymorph vaterite during crystallization.
3
2. Materials and methods 2.1. Materials Low molecular weight chitosan (Sigma Aldrich, 50,000–190,000 Da, 75–85% deacetylated), acetic acid (Sigma Aldrich, puriss.), 1,4-butanediol diglycidyl ether (BDE, Sigma Aldrich,
SC RI PT
≥95%), polyglycerol polyricinoleate (PGPR, DANISCO), cyclohexane (VWR), calcium chloride dihydrate (Sigma Aldrich, ≥99%), sodium carbonate (Fisher Scientific, ≥99.5%), sodium hydrogen carbonate (Fisher Scientific, ≥99.5%), and potassium hydroxide (Riedel deHaën) were used as-received. De-ionized water was used for the miniemulsion procedures. Ultrapure MilliQ
U
water (>18.5 MΩ∙cm) was used for precipitation studies.
N
2.2. Synthesis of chitosan hydrogel nanoparticles
A
Chitosan cross-linking relied on the reaction of the primary amines from chitosan and a di-
M
epoxide cross-linker, 1,4-butanediol diglycidyl ether (Scheme 1). Two separate emulsions were prepared to prevent the cross-linking reaction from occurring until nanosized droplets were formed. The dispersed phase of the chitosan emulsion consisted of chitosan (5 wt%) dissolved in
D
a 2 wt% acetic acid aqueous solution. For the BDE emulsion, the desired amount of cross-linking
TE
agent (5, 10, 15, 20 wt% with respect to chitosan weight) was dissolved in de-ionized water. 650 μL of chitosan or BDE solution was placed into a cylindrical vial containing 6.2 g of
EP
cyclohexane and 55 mg of the non-ionic surfactant PGPR (0.88 wt% in cyclohexane). Emulsions were formed by ultrasonication (Branson W 450 digital sonifier) using a 1/2" sonication tip for 2
CC
min with a pulse sequence (10 s on and 2 s off). The two emulsions (chitosan and BDE) were mixed and immediately ultrasonicated again using the same procedure. The final emulsions were allowed to mix under magnetic stirring at 800 rpm for 24 h. During those 24 h, cross-linking
A
between the amine and glycidal ether groups occurred. Purification of the cross-linked chitosan nanoparticles required that nanoparticle/cyclohexane
suspensions were added to 10 mL of 1-propanol and 20 mL of de-ionized water. The resulting dilute azeotropic suspension was distilled at reduced pressure until the chitosan particles were suspended only within water. The chitosan/water suspensions were transferred to dialysis tubing of a molecular weight cut off of 13,000 Da. Dialysis took place in de-ionized water containing 4
1 wt% acetic acid for 1 week. After 1 week, de-ionized water was changed twice daily for three days with no acetic acid. A final exchange of water was performed with ultrapure water. The suspensions from the dialysis tubing were collected into glass vials. Two phases were observed: (1) a translucent suspension of particles in water, and (2) an opaque, white waxy substance. The waxy substance was assumed to be free PGPR as it was not soluble in water. Suspensions were
SC RI PT
filtered through 5 µm syringe filters to remove the waxy substance and remove any aggregates from the suspension. Fourier transform infrared spectra (FTIR) spectra of nanoparticles are shown in Fig. S1.
2.3. Crystallization of calcium carbonate crystallization
U
Cross-linked chitosan particles corresponding to a content of 10 ppm were added to a 2.5 mM
N
CaCl2 solution at pH 7. 1.0 mL of 1.0 M CaCl2 was added to 16 mL of MilliQ water. The pH was adjusted to a value of 7 by addition of KOH and additional ultrapure water was added till the
A
total solution volume was adjusted to 18 mL. After mixing for at least 3 h, 1.0 mL of 1.0 M
M
Na2CO3 or NaHCO3 was added with 1.0 mL of ultrapure water, so that the total solution concentration was 20 mL.
D
Crystallization took place under stirring (300 rpm) for 24 h at room temperature.
TE
Precipitation experiments were performed in reaction vessels with tightly sealed screw on caps in order to minimize exchange of CO2 between the reactions and the atmosphere. Crystals of calcium carbonate were collected by centrifugation at 4000 rpm for 20 min using a Rotixa 50 RS
CC
EP
swinging-bucket rotor centrifuge. Precipitants were dried under vacuum for 14 h.
2.4. Characterization methods Particle sizes were measured with dynamic light scattering (DLS) by using a PSS Nicomp
A
Particle Sizer 380 in 90° scattering mode at 25 °C. Dispersions for DLS measurements had a solid content of 0.1−0.5 wt% nanoparticles in water. The zeta potential was determined by electrophoretic mobility measurements in a Malvern ZetaSizer Nano-Z instrument. The pH of suspensions was controlled by adding acetic acid to ultrapure water based on calculated values for pH 4. The ionic strength of the solution was controlled to contain 0.01 mM KCl. 5
The nanoparticles and the morphology of calcium carbonate crystals were analyzed by scanning electron microscopy (SEM) on a Zeiss 1530 Gemini LEO microscope on samples deposited on Si wafers with no coating. Transmission electron microscopy (TEM) was carried out in a JEOL 1400 microscope with an accelerating voltage of 120 kV was used to analyze dried particles dropcast onto carbon coated copper grids.
SC RI PT
X-ray diffraction (XRD) patterns were registered for powder samples on a Mylar film by using a Guinier imaging plate camera (G670) from HUBER, with an incident X-ray beam with a Cu Kα wavelength of 1.5406 Å. Data was smoothed using a least-mean-square fitting algorithm in Origin to reduce the overall noise generated due to air and Mylar film.
U
3. Results and discussion
N
3.1. Synthesis of chitosan nanoparticles
A
We attempted the preparation of pH-swellable chitosan nanoparticles by cross-linking chitosan
M
amino groups to either sulfo-N-hydroxysuccinimide functionalized polyethylene glycol via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide coupling or to 1,4-butanediol diglycidyl ether. Although both routes led to positive results, the nanoparticle yield was greater when using
D
1,4-butanediol diglycidal ether. Accordingly, further chitosan nanoparticles with various
TE
amounts of cross-linker were prepared by the epoxy–amine reaction between the chitosan amino groups and the 1,4-butanediol diglycidyl ether epoxide groups (Scheme 1).
EP
The two reaction agents were separately pre-emulsifed and then combined under sonication to generate “nanoreactors” for the cross-linking of the chitosan. After reaction, a majority of the
CC
excess surfactant and nanoparticle aggregates were removed by solvent exchange to ultrapure
A
water, followed by filtration through a 5 µm filter, and finally dialysis.
6
SC RI PT
N
U
Scheme 1: Chitosan reaction with 1,4-butanediol diglycidyl ether.
A
3.2. pH-dependent swelling behavior of chitosan nanoparticles
M
The pH-dependent swelling properties of the chitosan nanoparticles were confirmed by DLS measurements at pH values of 4 and 7. The average hydrodynamic diameter (dH) of the
D
nanoparticles at a pH of 7 was ~350 nm (Table 1); it does not significantly depend on the amount
TE
of cross-linker. TEM images indicated that the polydispersity in the particle size was rather high, with many nanoparticles between 50 and 100 nm, a smaller fraction around 350 nm, and a minority of particles greater than 1 µm (Fig. 1 and Fig. S2). This observation is consistent with
EP
the high polydispersity obtained for chitosan nanoparticles by other groups [10, 32]. At a value of pH 4, which is below the pKa of chitosan (around 6.4), the size of the chitosan nanoparticles
CC
increased by ~50 nm when compared to values at neutral pH, as shown in Table 1. The zeta potential of chitosan nanoparticles at a pH of 4 were greater than +40 mV regardless
A
of the cross-linker concentration, indicating good stability. At pH values above the pKa value, deprotonation of the amino groups results in a charge decrease, which would typically correlate with a loss of colloidal stability [27]. At a pH value of 7, the suspensions had zeta potential values within the rapid flocculation regime (0–5 mV). While flocculation likely began at a pH value of 7, it was not rapid. Only after a period of 1 to 3 weeks, nanoparticles displayed larger aggregates that would slowly precipitate out of suspension. After flocculation, SEM and DLS 7
analysis indicated that the number of particles larger than ~1 µm had increased. At pH values greater than 7, chitosan nanoparticles aggregated quickly and precipitated out of suspension. Chitosan nanoparticles aggregated and sedimented more quickly as their cross-linking density
SC RI PT
increased [33].
Table 1: Particle sizes and zeta potential of chitosan nanoparticles at different cross-linker concentration
pH = 4
pH = 7
pH = 4
pH = 7
5%
371 (±40%)
353 (±34%)
44 (±3%)
3.5 (±26%)
CP2
10 %
395 (±59%)
322 (±68%)
46 (±1%)
4.3 (±19%)
CP3
15 %
404 (±31%)
357 (±46%)
43 (±9%)
6.9 (±6%)
CP4
20 %
403 (±47%)
344 (±53%)
46 (±5%)
4.2 (±26%)
A
M
Determined by DLS
CP20
b)
A
CC
EP
a)
TE
CP19
N
CP1
D
a
ζ potential (mV)
dHa (nm)
Cross-linker Concentration
U
Sample
CP19
CP20
Fig. 1. TEM images of as-prepared chitosan nanoparticles a) small aggregates of sub-100 nm particles, b) a greater distribution of nanoparticle sizes.
8
3.3 Crystallization of calcium carbonate in the presence of chitosan nanoparticles CaCO3 is typically formed under basic conditions. At pH values below 7, carbonic acid and CO2 are favored over hydrogencarbonate and carbonate ions in solution [34]. Due to the aggregation
SC RI PT
of chitosan nanoparticles at pH values greater than 7, it was not possible to crystallize calcium carbonate under a typical pH value of 8.5–10. Thus, chitosan nanoparticles and calcium ions were placed into a suspension at an initial pH of 7, and the addition of a carbonate salt led to higher pH value upon dissolution [14].
Sodium hydrogencarbonate and sodium carbonate salts were chosen as carbonate sources for their impact on the pH value of the solution after dissociation. At the concentration used in this
U
work, sodium hydrogencarbonate raised a pH value of the suspension to a value of 8.5, while the
N
sodium carbonate increased the pH towards 10. The dissociation of the proton from HCO3− to form carbonate ions (CO32−) also slows down the kinetics of CaCO3 formation, which we had
M
oligopeptides) and calcium chloride [14].
A
observed previously when using these two carbonate salts in the presence of amino acids (or
Chitosan nanoparticles equivalent to a concentration of 10 ppm were added to solutions of
D
[Ca2+] = 2.5 mM. To initiate crystallization, either NaHCO3 or Na2CO3 was added to the
TE
Ca2+/chitosan suspensions. Crystal shapes typical for calcite and vaterite were observed by SEM after the crystallization experiments (Fig. 2 and Fig. S3). The ability of chitosan nanoparticles to stabilize the kinetically favorable but thermodynamically unfavorable vaterite polymorph was
EP
found to be dependent on both the cross-linking density and the carbonate source used, as seen by the XRD patterns of the CaCO3 crystals formed with the differing conditions, shown in Fig. 3.
CC
Upon addition of Na2CO3, vaterite stabilization was not observed from the XRD patterns until nanoparticles prepared with 15 and 20 wt% cross-linking agent were used. In contrast, when
A
using NaHCO3, the vaterite phase was stabilized only at 5 and 10 wt% of the cross-linking agent. Kinetics and pH value play a role in crystallization. As observed by zeta potential the chitosan nanoparticles quickly lost stability in suspension as the pH increased to basic pH values greater than 7. As mentioned before, Na2CO3 increased the pH value of the particle suspensions to 10, and the chitosan nanoparticles quickly began to flocculate. However the driving force for CaCO3 crystallization was also high and CaCO3 crystals grew concurrently with the quickly aggregating 9
chitosan nanoparticles. A more cross-linked substrate to grow from was necessary for the stabilization of the vaterite crystals, possibly due to its increased rigidity [35, 36]. A strong interaction between the chitosan nanoparticles and the growing CaCO3 crystals was observed by the large amount of surface pitting on the calcite crystals, despite the low amount of chitosan nanoparticles added (Fig. 2b).
SC RI PT
On the other hand vaterite was stabilized at lower nanoparticle stiffness (i.e., cross-linking fraction) when NaHCO3 was used as a carbonate source. At the lower pH value of 8.5, chitosan nanoparticles flocculated slower than at pH 10 (although complete flocculation occurred over the course of a few hours). However, HCO3− ions, not fully dissociated to carbonate ions, led to slower crystallization. The interaction of less cross-linked nanoparticles, which remained colloidally stable for longer times than the more rigid substrates, had an impact on the
U
stabilization of vaterite. At higher cross-linking concentrations, vaterite was not stabilized.
N
While the harder substrates acted as better nucleation surfaces for crystals with Na2CO3, when using NaHCO3, it was necessary to have faster interaction times between the chitosan
A
nanoparticles and the Ca2+ and mineralizing CO32− ions as the particles quickly sedimented out
M
of solution. The softer substrates of the lower cross-linked chitosan nanoparticles interact with
TE
along the softer particles.
D
the ions and growing clusters of CaCO3 for longer times, thus resulting in higher supersaturation
b)
A
CC
EP
a)
Fig. 2. SEM images of CaCO3 show a) vaterite and calcite co-mineralization along with pitting of calcite crystals due to nanoparticles interaction with the growing CaCO3 crystals, b) zoomed in area of region highlighted in red from image a.
10
(b) NaHCO3
V (112)
CP4 (20 wt% cross-linker)
C (018) C (116)
C (202)
CP3 (15 wt% cross-linker)
SC RI PT
C (113)
V (300)
CP4 (20 wt% cross-linker)
C (110)
V (116)
C (104)
C (106)
V (110)
V (113)
C (012)
(a) Na2CO3
Intensity (a.u.)
Intensity (a.u.)
CP3 (15 wt% cross-linker)
CP2 (10 wt% cross-linker)
CP2 (10 wt% cross-linker)
CP1 (5 wt% cross-linker)
25
30
35
40
45
U
CP1 (5 wt% cross-linker)
50
25
30
35 40 2 (
45
50
A
N
2 ()
Fig. 3. XRD patterns showing the stabilization of vaterite (V: vaterite, C: calcite) in chitosan
M
nanoparticles suspensions containing calcium ions upon addition of a) Na2CO3 and b) NaHCO3.
4. Conclusions
TE
D
The particle content was in all cases 10 ppm.
We have shown a simple method to produce a large amount of pH swellable, cross-linked
EP
chitosan nanoparticles, which have been used as substrates during the crystallization of CaCO3. Promising stabilization of vaterite and obvious interactions with growing calcite crystals at
CC
contents of chitosan nanoparticles as low as 10 ppm suggest that these nanoparticles should be further evaluated for their ability to affect bioinspired crystallization. In this sense, chitin is
A
already observed in nature to act as a potent substrate for growth of CaCO3. These swellable nanoparticles could find a potential for use in a plethora of smart materials, with particular focus in drug delivery systems that contain polymorph specific calcium carbonate.
Acknowledgements
11
The authors thank Michael Steiert for the assistance with X-ray diffraction. The authors also thank the Max Planck Society for funding of the Max Planck Partner Group on Colloidal Methods for Multifunctional Materials (CM3) at the University of Valencia, headed by RME. RME acknowledges the financial support from the Spanish Ministry of Economy, Industry and
SC RI PT
Competitiveness through a Ramón y Cajal grant (grant no. RYC-2013-13451),
References
[1] S. Sviben, A. Gal, M.A. Hood, L. Bertinetti, Y. Politi, M. Bennet, P. Krishnamoorthy, A. Schertel, R. Wirth, A. Sorrentino, E. Pereiro, D. Faivre, A. Scheffel, A vacuole-like compartment concentrates a disordered calcium phase in a key coccolithophorid alga, Nature Commun. 7
U
(2016) 11228.
N
[2] S. Mann, Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry,
A
Oxford University Press, 2001.
[3] M.A. Hood, H. Leemreize, A. Scheffel, D. Faivre, Lattice distortions in coccolith calcite
M
crystals originate from occlusion of biomacromolecules, J. Struct. Biol. 196 (2016) 147-154. [4] L. Addadi, D. Joester, F. Nudelman, S. Weiner, Mollusk Shell Formation: A Source of New
D
Concepts for Understanding Biomineralization Processes, Chem. Eur. J. 12 (2006) 980-987.
TE
[5] N.H. Munro, K.M. McGrath, Biomimetic approach to forming chitin/aragonite composites, Chem. Commun. 48 (2012) 4716-4718.
EP
[6] R.A. Muzzarelli, Genipin-crosslinked chitosan hydrogels as biomedical and pharmaceutical aids, Carbohyd. Polym. 77 (2009) 1-9. [7] G.A. Roberts, K.E. Taylor, Chitosan gels, 3. The formation of gels by reaction of chitosan
CC
with glutaraldehyde, Makromol. Chem. 190 (1989) 951-960. [8] M. Rafat, F. Li, P. Fagerholm, N.S. Lagali, M.A. Watsky, R. Munger, T. Matsuura, M.
A
Griffith, PEG-stabilized carbodiimide crosslinked collagen–chitosan hydrogels for corneal tissue engineering, Biomaterials 29 (2008) 3960-3972. [9] F.-L. Mi, S.-S. Shyu, C.-T. Chen, J.-Y. Schoung, Porous chitosan microsphere for controlling the antigen release of Newcastle disease vaccine: preparation of antigen-adsorbed microsphere and in vitro release, Biomaterials 20 (1999) 1603-1612.
12
[10] V. Kamat, D. Bodas, K. Paknikar, Chitosan nanoparticles synthesis caught in action using microdroplet reactions Sci. Rep., 6 (2016). [11] X. An, C. Cao, Coeffect of silk fibroin and self-assembly monolayers on the biomineralization of calcium carbonate, J. Phys. Chem. C 112 (2008) 15844-15849. [12] G. Falini, S. Weiner, L. Addadi, Chitin-silk fibroin interactions: relevance to calcium
SC RI PT
carbonate formation in invertebrates, Calcif. Tissue Int. 72 (2003) 548-554.
[13] E.C. Keene, J.S. Evans, L.A. Estroff, Silk Fibroin Hydrogels Coupled with the n16N− βChitin Complex: An in Vitro Organic Matrix for Controlling Calcium Carbonate Mineralization, Cryst. Growth Des. 10 (2010) 5169-5175.
[14] M.A. Hood, K. Landfester, R. Muñoz-Espí, The Role of Residue Acidity on the Stabilization of Vaterite by Amino Acids and Oligopeptides, Cryst. Growth Des. 14 (2014)
U
1077-1085.
N
[15] H. Lu, M.A. Hood, S. Mauri, J.E. Baio, M. Bonn, R. Muñoz-Espí, T. Weidner, Biomimetic vaterite formation at surfaces structurally templated by oligo(glutamic acid) peptides, Chem.
A
Commun. 51 (2015) 15902-15905.
M
[16] W.E. Rudzinski, A. Palacios, A. Ahmed, M.A. Lane, T.M. Aminabhavi, Targeted delivery of small interfering RNA to colon cancer cells using chitosan and PEGylated chitosan
D
nanoparticles, Carbohyd. Polym. 147 (2016) 323-332.
TE
[17] A. Abruzzo, G. Zuccheri, F. Belluti, S. Provenzano, L. Verardi, F. Bigucci, T. Cerchiara, B. Luppi, N. Calonghi, Chitosan nanoparticles for lipophilic anticancer drug delivery: Development, characterization and in vitro studies on HT29 cancer cells, Colloids Surf. B, 145
EP
(2016) 362-372.
[18] Q. Ruan, Y. Zhang, X. Yang, S. Nutt, J. Moradian-Oldak, An amelogenin–chitosan matrix
CC
promotes assembly of an enamel-like layer with a dense interface, Acta Biomater., 9 (2013) 7289-7297.
A
[19] Y. Huang, Y. Duan, Y. Qian, R. Huang, Z. Yang, Y. Li, Z. Zhou, Remineralization efficacy of a toothpaste containing 8% arginine and calcium carbonate on enamel surface, Amer. J. Dent. 26 (2013) 291-297. [20] X.w. He, T. Liu, Y.x. Chen, D.j. Cheng, X.r. Li, Y. Xiao, Y.l. Feng, Calcium carbonate nanoparticle delivering vascular endothelial growth factor-C siRNA effectively inhibits lymphangiogenesis and growth of gastric cancer in vivo, Cancer Gene Ther, 15 (2008) 193-202. 13
[21] M. Rinaudo, Chitin and chitosan: properties and applications, Progr. Polym. Sci., 31 (2006) 603-632. [22] M. Hood, M. Mari, R. Muñoz-Espí, Synthetic Strategies in the Preparation of Polymer/Inorganic Hybrid Nanoparticles, Materials 7 (2014) 4057. [23] K. Landfester, D. Crespy, Miniemulsion Polymerization, in:
SC RI PT
Technology, Wiley-VCH, 2006.
Materials Science and
[24] R. Muñoz-Espí, C.K. Weiss, K. Landfester, Inorganic nanoparticles prepared in miniemulsion, Curr. Opin. Colloid Interf. Sci., 17 (2012) 212-224.
[25] M.A. Hood, N. Encinas, D. Vollmer, R. Graf, K. Landfester, R. Muñoz‐Espí, Controlling hydrophobicity of silica nanocapsules prepared from organosilanes, Colloids Surf. A. 532 (2017) 172–177
U
[26] M.A. Hood, U. Paiphansiri, D. Schaeffel, K. Koynov, M. Kappl, K. Landfester, R. Muñoz-
N
Espí, Hybrid Poly(urethane–urea)/Silica Nanocapsules with pH-Sensitive Gateways, Chem. Mater. 27 (2015) 4311-4318.
A
[27] N. Solomko, O. Budishevska, S. Voronov, K. Landfester, A. Musyanovych, pH-Sensitive
M
Chitosan-based Hydrogel Nanoparticles through Miniemulsion Polymerization Mediated by Peroxide Containing Macromonomer, Macromol. Biosci. 14 (2014) 1076-1083.
D
[28] S.-C. Chen, Y.-C. Wu, F.-L. Mi, Y.-H. Lin, L.-C. Yu, H.-W. Sung, A novel pH-sensitive
TE
hydrogel composed of N, O-carboxymethyl chitosan and alginate cross-linked by genipin for protein drug delivery, J. Control. Release 96 (2004) 285-300. [29] A.M. De Campos, A. Sánchez, M.a.J. Alonso, Chitosan nanoparticles: a new vehicle for the
EP
improvement of the delivery of drugs to the ocular surface. Application to cyclosporin A, Int. J. Pharmac. 224 (2001) 159-168.
CC
[30] K.A. Janes, M.P. Fresneau, A. Marazuela, A. Fabra, M.a.J. Alonso, Chitosan nanoparticles as delivery systems for doxorubicin, J. Control. Release 73 (2001) 255-267.
A
[31] L.C. Preiss, L. Werber, V. Fischer, S. Hanif, K. Landfester, Y. Mastai, R. Muñoz-Espí, Amino-Acid-Based Chiral Nanoparticles for Enantioselective Crystallization, Adv. Mater. 27 (2015) 2728-2732. [32] A. Rampino, M. Borgogna, P. Blasi, B. Bellich, A. Cesàro, Chitosan nanoparticles: preparation, size evolution and stability, Int. J. Pharmac. 455 (2013) 219-228.
14
[33] H. Jonassen, A.-L. Kjøniksen, M. Hiorth, Stability of Chitosan Nanoparticles Cross-Linked with Tripolyphosphate, Biomacromolecules 13 (2012) 3747-3756. [34] P. Atkins, Shriver and Atkins' Inorganic Chemistry, OUP Oxford, 2010. [35] X.Y. Liu, K. Tsukamoto, M. Sorai, New Kinetics of CaCO3 Nucleation and Microgravity Effect, Langmuir 16 (2000) 5499-5502.
SC RI PT
[36] Y.Y. Kim, L. Ribeiro, F. Maillot, O. Ward, S.J. Eichhorn, F.C. Meldrum, Bio‐Inspired Synthesis and Mechanical Properties of Calcite–Polymer Particle Composites, Adv. Mater. 22
A
CC
EP
TE
D
M
A
N
U
(2010) 2082-2086.
15