Next Article in Journal
Non-Stoichiometric Polymer-Cyclodextrin Inclusion Compounds: Constraints Placed on Un-Included Chain Portions Tethered at Both Ends and Their Relation to Polymer Brushes
Next Article in Special Issue
Stimuli-Responsive Polymers and Colloids under Electric and Magnetic Fields
Previous Article in Journal
Polyamide 11/Poly(vinylidene fluoride)/Vinyl Acetate-Maleic Anhydride Copolymer as Novel Blends Flexible Materials for Capacitors
Previous Article in Special Issue
Temperature-Responsive Polymer Modified Surface for Cell Sheet Engineering
 
 
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Communication

Release of Insulin from Calcium Carbonate Microspheres with and without Layer-by-Layer Thin Coatings

Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki, Aoba-ku, Sendai 980-8578, Japan
*
Author to whom correspondence should be addressed.
Polymers 2014, 6(8), 2157-2165; https://doi.org/10.3390/polym6082157
Submission received: 25 June 2014 / Revised: 29 July 2014 / Accepted: 29 July 2014 / Published: 11 August 2014
(This article belongs to the Special Issue Stimuli-Responsive Polymers and Colloids)

Abstract

:
The release of insulin from insulin-containing CaCO3 microspheres was investigated. The microspheres were prepared by mixing aqueous solutions of CaCl2 and Na2CO3 in the presence of insulin. The surface of the insulin-containing CaCO3 microspheres was coated with a layer-by-layer thin film consisting of poly(allylamine hydrochloride) and poly(styrene sulfonate) to regulate the release kinetics of insulin. The release rate of insulin from the coated CaCO3 microspheres was significantly suppressed compared with that of uncoated CaCO3 microspheres, and depended on the thickness of the films. Rhombohedral calcite crystals of CaCO3 formed from the microspheres during the release of insulin, suggesting that the CaCO3 microspheres dissolved and recrystallized during the release of insulin.

Graphical Abstract

1. Introduction

Organic and inorganic nano- and micro-particles have been extensively studied for the development of catalysts, biosensors, reagents for imaging, and drug delivery systems [1,2,3,4,5,6,7,8]. CaCO3 microspheres are widely used owing to their facile preparation, biocompatibility, and low cost [9,10,11]. CaCO3 microspheres are promising materials for encapsulating biological molecules such as proteins, because the microspheres can be prepared in aqueous media under mild conditions. Protein-loaded CaCO3 microspheres have been prepared by mixing a Na2CO3 solution and protein-containing CaCl2 solution at room temperature, exploiting the limited solubility of CaCO3 in water [12,13,14,15,16]. In addition, protein-loaded CaCO3 microspheres can be used for preparing polymer microcapsules by coating the surface of CaCO3 microspheres with polyelectrolyte layer-by-layer (LbL) films, and then dissolving the core in solution [17,18,19,20]. The amount of proteins loaded in CaCO3 microspheres depends on the preparation conditions, including the concentration of proteins and salts in the solutions, the relative volume of the solutions, and the reaction time. These parameters must be optimized to obtain CaCO3 microspheres containing the desired amount of proteins. Therefore, we have optimized the operational variables for the preparation of CaCO3 microspheres using insulin as a model protein.
The release of drugs and proteins from CaCO3 microspheres and polymer microcapsules has been studied for developing controlled delivery systems. For example, the release of doxorubicin (DOX) from CaCO3 microspheres with and without polymer coatings has been investigated for temperature- and pH-sensitive release systems [21]. The release profile of DOX depended on the temperature and pH of the solution owing to the stimuli-sensitive nature of the polymer coatings, showing that CaCO3 microspheres are useful as vehicles for controlled drug delivery. In the present study, we have prepared insulin-loaded CaCO3 microspheres and coated the surface with LbL thin films consisting of poly(allylamine hydrochloride) (PAH) and poly(sodium styrenesulfonate) (PSS) to regulate the kinetics of insulin release. We report the effects of solution pH and LbL film coatings on the release profile of insulin.

2. Experimental

2.1. Materials

PAH (MW, ~70,000) and PSS (MW, ~70,000) were purchased from Nitto Bouseki Co. Ltd. (Tokyo, Japan) and Sowa Science Co. Ltd. (Tokyo, Japan), respectively. Insulin (human, recombinant) was obtained from Wako Pure Chemical Co. Ltd. (Osaka, Japan). All other reagents used were of the highest grade available. Fluorescein-labelled insulin (F-insulin) was prepared by the coupling reaction of fluorescein isothiocyanate and insulin according to a previously reported procedure [22].

2.2. Preparation of Uncoated and LbL Film-Coated CaCO3 Microspheres

CaCO3 microspheres containing insulin were prepared by mixing 0.2 M Na2CO3 aqueous solution (10 mL) and 0.2 M CaCl2 aqueous solution (10 mL) containing insulin (0.5–5 mg). The mixture was stirred for 30 min at ambient temperature. The precipitated CaCO3 microspheres were filtered off and dried. The amount of insulin loaded in the CaCO3 microspheres was determined by high-performance liquid chromatography of a dialyzed solution of microspheres (80 mg) in 1 M HCl (Shimadzu, LC-20AB (Kyoto, Japan) with COSMOSIL 5Diol-II packed column (Nacalai USA, Inc., San Diego, CA, USA), 1 mM carbonate buffer at pH 8.0 and 1 mM acetate buffer at pH 4.0 as eluents). The surface of CaCO3 microspheres was coated with the LbL films by immersing CaCO3 microspheres alternately in 0.5 mg·mL−1 PAH solution (10 mM HEPES buffer at pH 7.4) and in 0.5 mg·mL−1 PSS solution (10 mM HEPES buffer at pH 7.4) for 15 min each. After each deposition, CaCO3 microspheres were rinsed for 5 min in the working buffer. Sedimentation or aggregation of the microspheres did not occur during the film deposition and ζ-potential measurement.
2.3. ζ-Potential and Scanning Electron Microscopy
To monitor the film deposition, ζ-potentials of LbL film-coated CaCO3 microspheres were recorded with a ζ-potential analyzer (Zeecom/ZC-2000, Microtec, Funabashi, Japan). Scanning electron microscope (SEM; S-3200N, Hitachi Co., Tokyo, Japan) images of CaCO3 microspheres and crystals were obtained for platinum-sputtered samples at 15 kV.

2.4. Release of Insulin from Microspheres

In vitro release of insulin was studied using F-insulin and the amount of released insulin was determined by UV-visible spectroscopy. F-insulin-loaded CaCO3 microspheres (100 mg) were dispersed in 10 mM HEPES buffer (5 mL) at pH 7.4 under gentle stirring. The dispersion was centrifuged every 60 min and the absorption intensity at 494 nm of the supernatant was recorded to determine the amount of F-insulin released.

3. Results and Discussion

Insulin-loaded CaCO3 microspheres were prepared by using CaCl2 solutions containing varying amounts of insulin to evaluate the effect of insulin concentration on the loading of insulin in the microspheres. Table 1 shows the weights of CaCO3 microspheres produced by the reaction and their insulin contents. The reaction produced 184–188 mg of CaCO3 microspheres, which corresponded to a 92%–94% yield. Thus, CaCO3 microspheres were obtained nearly quantitatively with this protocol. The insulin loading in the microspheres increased with the insulin concentration in the CaCl2 solution. The insulin loading was approximately 18 mg/g in the CaCO3 microspheres for 5 mg of insulin in 10 mL CaCl2 solution, showing that 64% of the insulin was immobilized in the CaCO3 microspheres. The insulin loading in the CaCO3 microspheres was lower when CaCl2 solutions containing smaller amount of insulin were used. In addition, we have evaluated the effect of additives on the preparation of insulin-containing CaCO3 microspheres. When Na2CO3 solutions (10 mL) containing 1–40 mg additives such as dextran sulfate (DS), PSS, or PAH were employed, CaCO3 microspheres were successfully prepared. However, the loading of insulin in the microspheres could not be improved by the addition of these polymers. Therefore, in the following experiments, CaCO3 microspheres were prepared using 5 mg insulin in 10 mL CaCl2 solution without additives.
Figure 1 shows the release profiles of insulin from CaCO3 microspheres without LbL film coating in solutions of pH 6.0, 7.4, and 9.0. The release of insulin was suppressed in the first 300 min, irrespective of the pH of the solution. After the inductive period, the release rate of insulin depended on the solution pH. The release was faster at pH 6.0 than at pH 7.4 and 9.0. This may arise from the difference in solubility of CaCO3 microspheres at pH 6.0–9.0. In insulin-containing CaCO3 microspheres, the CaCO3 core dissolves in solutions of pH 6.5 or lower, whereas CaCO3 is practically insoluble at higher pH [23]. The insulin is probably released from the CaCO3 microspheres at the same time as the CaCO3 core partially dissolves. Figure 2 shows SEM images of insulin-containing CaCO3 microspheres before and after the microspheres were immersed in the buffer solution at pH 7.4. The as-prepared CaCO3 microspheres were spherical with a rough surface, which is typical for vaterite morphology [24]. However, after soaking the CaCO3 microspheres in the buffer solution, the microspheres changed to rhombohedral crystals characteristic of calcite [25]. It is clear that the phase transition in the crystal form of CaCO3 occurred during the insulin release in the buffer solution as a result of the simultaneous partial dissolution of CaCO3 microspheres and precipitation of calcite crystals. A similar phase transition in CaCO3 microspheres has recently been reported [24]. The crystalline phase of CaCO3 readily changes from metastable vaterite to stable calcite in solution [26,27]. These results suggest that the dissolution of the CaCO3 core is involved in determining the release rate of insulin from the microspheres.
Table 1. Preparation of insulin-containing CaCO3 microspheres (1).
Table 1. Preparation of insulin-containing CaCO3 microspheres (1).
Insulin in CaCl2 Solution (mg/10 mL)CaCO3 Precipitated (2) (mg)Insulin Loading in CaCO3 (2) (mg/g)
0.51851.4 ± 0.3
1.01842.6 ± 0.4
2.01884.4 ± 0.2
5.018617.5 ± 0.8
(1) CaCO3 microspheres were prepared by mixing 0.2 M Na2CO3 (10 mL) and 0.2 M CaCl2 (10 mL) containing insulin (0.5–5.0 mg); (2) Average values of three preparations are listed.
Figure 1. Amount of insulin released from uncoated CaCO3 microspheres in buffer solutions at pH 6.0 (■), 7.4 (●), and 9.0 (▲). Average values of three measurements are plotted.
Figure 1. Amount of insulin released from uncoated CaCO3 microspheres in buffer solutions at pH 6.0 (■), 7.4 (●), and 9.0 (▲). Average values of three measurements are plotted.
Polymers 06 02157 g001
The surface of insulin-loaded CaCO3 microspheres was coated with LbL films consisting of PAH and PSS to evaluate the effect of LbL film coatings on the insulin release. Figure 3 shows the ζ-potentials of LbL film-coated CaCO3 microspheres as a function of the number of bilayers. The unmodified microspheres showed a negative potential, and the potential was reversed upon deposition of first PAH layer because of the positive charge of PAH. The sign of the ζ-potential alternated depending on the sign of electric charges of polymeric materials deposited on the outermost surface of the microspheres, suggesting the successful formation of the LbL film coatings on the surface of the microspheres [28]. It is reasonable to assume that PAH and PSS are deposited on the surface through electrostatic bonds. Figure 4 shows SEM images of (PAH/PSS)5 film-coated CaCO3 microspheres, in which microspheres are well-dispersed without significant aggregation. The partial aggregation of the microspheres observed in the SEM images might probably be caused during drying process for preparing SEM samples.
Figure 2. SEM images of insulin-containing CaCO3 microspheres (a) before and (b) after the microspheres were immersed in buffer solution for insulin release.
Figure 2. SEM images of insulin-containing CaCO3 microspheres (a) before and (b) after the microspheres were immersed in buffer solution for insulin release.
Polymers 06 02157 g002
Figure 3. ζ-Potentials of (PAH/PSS)n film-coated CaCO3 microspheres at pH 7.4. The average values of ζ-potentials for ca. 50 particles are plotted with standard deviations. The outermost surface of the microspheres was covered with PSS for the integer bilayer numbers.
Figure 3. ζ-Potentials of (PAH/PSS)n film-coated CaCO3 microspheres at pH 7.4. The average values of ζ-potentials for ca. 50 particles are plotted with standard deviations. The outermost surface of the microspheres was covered with PSS for the integer bilayer numbers.
Polymers 06 02157 g003
Figure 5 shows the effects of LbL film coatings on the release of insulin from CaCO3 microspheres. The LbL film coatings significantly suppressed the release of insulin. The amount of insulin released from the (PAH/PSS)1 film-coated CaCO3 microspheres after 7 h was approximately 40% of that released from uncoated microspheres, showing the substantial effect of the film coating. The effects of thicker (PAH/PSS)3 and (PAH/PSS)5 films were more significant; the amount of released insulin after 7 h was less than 5% of that released from uncoated microspheres. These results suggest that the transport of insulin across the LbL films determined the overall release rate from the microspheres. The significant variations in the amounts of released insulin from uncoated CaCO3 microspheres at 300 and 360 min may result from the fact that a burst release of insulin occurred at this stage after induction period. The effects of the LbL film coating and its thickness on the stability and permeability of ions and drugs have been reported [29,30,31,32,33,34]. However, the suppressive effect of the film coatings on the release is more clearly demonstrated here for insulin, probably because of the large size of the protein drug. The phase transition of CaCO3 microspheres to calcite crystals during the insulin release was also observed for the LbL film-coated CaCO3 microspheres (data not shown). Thus, the release rate of insulin from CaCO3 microspheres can be regulated by coating the surface of microspheres with LbL films.
Figure 4. SEM images of (PAH/PSS)5 film-coated CaCO3 microspheres.
Figure 4. SEM images of (PAH/PSS)5 film-coated CaCO3 microspheres.
Polymers 06 02157 g004
Figure 5. Amount of insulin released from (PAH/PSS)n film-coated CaCO3 microspheres in buffer solutions at pH 7.4. The number of bilayers (n): 0 (○), 1 (◆), 3 (■), and 5 (●). Average values of three measurements are plotted.
Figure 5. Amount of insulin released from (PAH/PSS)n film-coated CaCO3 microspheres in buffer solutions at pH 7.4. The number of bilayers (n): 0 (○), 1 (◆), 3 (■), and 5 (●). Average values of three measurements are plotted.
Polymers 06 02157 g005

4. Conclusions

We have prepared insulin-containing CaCO3 microspheres with and without polymer film coatings. The release of insulin from the microspheres depended on the pH of the medium and the thickness of the polymer film coating on the surface. The release rate of insulin from uncoated CaCO3 microspheres was faster at pH 6.0 than in neutral and basic solutions, probably because of the higher solubility of CaCO3 in weakly acidic solutions. SEM images showed that a phase transition in CaCO3 microspheres from vaterite to calcite crystals occurred during the release of insulin in the solution. The polymer thin films on the surface of the CaCO3 microspheres substantially suppressed the release of insulin, depending on the thickness of the films. The results suggest LbL film coatings are effective for regulating the release rate of macromolecular drugs such as insulin from CaCO3 microspheres.

Acknowledgments

This work was supported in part by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (grant numbers 24390006 and 25460031).

Author Contributions

All authors were involved equally in the experimental works and the manuscript preparation.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Siangproh, W.; Dungchai, W.; Rattanarat, P.; Chailapakul, O. Nanoparticle-based electrochemical detection in conventional and miniaturized systems and their bioanalytical applications: A review. Anal. Chim. Acta 2011, 690, 10–25. [Google Scholar]
  2. Zhou, Y.; Chai, Y.; Yuan, R.; Mao, L.; Yuan, Y.; Han, J. Glucose oxidase and ferrocene labels immobilized at Au/TiO2 nanocomposites with high load amount and activity for sensitive immunoelectrochemical measurement of proGRP biomarker. Biosens. Bioelectron. 2011, 26, 3838–3844. [Google Scholar]
  3. Egawa, Y.; Seki, T.; Takahashi, S.; Anzai, J. Electrochemical and optical sugar sensors based on phenylboronic acid and its derivatives. Mater. Sci. Eng. C 2011, 31, 1257–1264. [Google Scholar] [CrossRef]
  4. Arya, S.K.; Saha, S.; Ramirez-Vick, J.E.; Gupta, V.; Bhansali, S.; Singhg, S.P. Recent advances in ZnO nanoparticles and thin films for biosensor applications: Review. Anal. Chim. Acta 2012, 737, 1–21. [Google Scholar]
  5. Takahashi, S.; Sato, K.; Anzai, J. Layer-by-layer construction of protein architectures through avidin-biotin and lectin-sugar interactions for biosensor applications. Anal. Bioanal. Chem. 2012, 402, 1749–1758. [Google Scholar]
  6. Sun, W.; Sun, Z.; Zhang, L.; Qi, X.; Li, G.; Wu, J.; Wang, M. Application of Fe3O4 mesoporous sphere modified carbon ionic liquid electrode as electrochemical hemoglobin biosensor. Colloids Surf. B 2013, 101, 177–182. [Google Scholar] [CrossRef]
  7. Takahashi, S.; Anzai, J. Recent progress in ferrocene-modified thin films and nanoparticles for biosensors. Materials 2013, 6, 5742–5762. [Google Scholar] [CrossRef]
  8. Ishihara, T.; Takahashi, M.; Higaki, M.; Mizushima, Y. Efficient encapsulation of a water-soluble corticosteroid in biodegradable nanoparticles. Int. J. Pharm. 2009, 365, 200–205. [Google Scholar]
  9. Cai, W.; Xu, Q.; Zhao, X.; Zhu, J.; Chen, H. Porous gold-nanopartcle-CaCO3 hybrid material: Preparation, characterization, and application for horseradish peroxide assembly and direct electrochemistry. Chem. Mater. 2006, 18, 279–284. [Google Scholar]
  10. Lu, Z.; Zhang, J.; Ma, Y.; Song, S.; Gu, W. Biomimetic mineralization of calcium carbonate/carboxymethylcellulose microspheres for lysozyme immobilization. Mater. Sci. Eng. C 2012, 32, 1982–1987. [Google Scholar] [CrossRef]
  11. Rauch, M.W.; Dressler, M.; Scheel, H.; van Opdenbosch, D.; Zollfrank, C. Mineralization of calcium carbonates in cellulose gel membranes. Eur. J. Inorg. Chem. 2012, 32, 5192–5198. [Google Scholar]
  12. Sukhorukov, G.B.; Volodkin, D.V.; Günther, A.M.; Petrov, A.I.; Shenoy, D.B.; Möhwald, H. Poroud calcium carbonate microparticles as templates for encapsulation of bioactive compounds. J. Mater. Chem. 2004, 14, 2073–2081. [Google Scholar]
  13. Johnston, A.P.R.; Cortez, C.; Angelatos, A.S.; Caruso, F. Layer-by-layer engineered capsules and their applications. Curr. Opin. Colloid Interface Sci. 2006, 11, 203–209. [Google Scholar]
  14. Karamitros, C.S.; Yashchenok, A.M.; Möhwald, H.; Skirtach, A.G.; Konrad, M. Preserving catalytic activity and enhancing biochemical stability of the therapeutic enzyme asparaginase by biocompatible multilayered polyelectrolyte microcapsules. Biomacromolecules 2013, 14, 4398–4406. [Google Scholar]
  15. Peng, C.; Zhao, Q.; Gao, C. Sustained delivery of doxorubicin by porous CaCO3 and chitosan/alginate multilayers-coated CaCO3 microparticles. Colloids Surf. A 2010, 353, 132–139. [Google Scholar] [CrossRef]
  16. Sato, K.; Kodama, D.; Endo, Y.; Anzai, J. Preparation of insulin-containing microcapsules by a layer-by-layer deposition of concanavalin A and glycogen. J. Nanosci. Nanotechnol. 2009, 9, 386–390. [Google Scholar]
  17. Sato, K.; Takahashi, S.; Anzai, J. Layer-by-layer thin films and microcapsules for biosensors and controlled release. Anal. Sci. 2012, 28, 929–938. [Google Scholar]
  18. Wang, X.; Shi, J.; Jiang, Z.; Li, Z.; Zhang, W.; Song, X.; Ai, Q.; Wu, H. Preparation of ultrathin, robust protein microcapsules through template-mediated interfacial reaction between amine and catechol groups. Biomacromolecules 2013, 14, 3861–3869. [Google Scholar]
  19. Yashchenok, A.; Parakhonskiy, B.; Donatan, S.; Kohler, D.; Skirtach, A.; Möhwald, H. Polyelectrolyte multilayer microcapsules template on spherical, elliptical and square calcium carbonate particles. J. Mater. Chem. B 2013, 1, 1223–1228. [Google Scholar]
  20. Endo, Y.; Sato, K.; Anzai, J. Preparation of avidin-containing polyelectrolyte microcapsules and their uptake and release properties. Polym. Bull. 2011, 66, 711–720. [Google Scholar]
  21. Du, C.; Shi, J.; Shi, J.; Zhang, L.; Cao, S. PUA/PSS multilayer coated CaCO3 microparticles as smart drug delivery vehicles. Mater. Sci. Eng. C 2013, 33, 3745–3752. [Google Scholar] [CrossRef]
  22. Zhang, X.; Guan, Y.; Zhang, Y. Dynamically bonded layer-by-layer films for self-regulated insulin release. J. Mater. Chem. 2012, 22, 16299–16305. [Google Scholar]
  23. Schmidt, S.; Uhlig, K.; Duschl, C.; Volodkin, D. Stability and cell uptake of calcium carbonate template insulin microparticles. Acta Biomater. 2014, 10, 1423–1430. [Google Scholar]
  24. Fujiwara, M.; Shiokawa, K.; Araki, M.; Ashitaka, N.; Morigaki, K.; Kubota, T.; Nakahara, Y. Encapsulation of proteins into CaCO3 by phase transition from vaterite to calcite. Cryst. Growth Des. 2010, 10, 4030–4037. [Google Scholar]
  25. Cölfen, H.; Qi, L. A systematic examination of the morphogenesis of calcium carbonate in the presence of a double-hydrophilic block copolymer. Chem. Euro. J. 2001, 7, 106–116. [Google Scholar]
  26. Volodkin, D.V.; Larionova, N.I.; Sukhorukov, G.B. Protein encapsulation via CaCO3 microparticles templating. Biomacromolecules 2004, 5, 1962–1972. [Google Scholar]
  27. Nan, Z.; Chen, X.; Yang, Q.; Wang, X.; Shi, Z.; Hou, W. Structure transition from aragonite to vaterite and calcite by the assistance of SDBS. J. Colloid Interface Sci. 2008, 325, 331–336. [Google Scholar]
  28. Hashide, R.; Yoshida, K.; Hasebe, Y.; Seno, M.; Takahashi, S.; Sato, K.; Anzai, J. Poly(lactic acid) microparticles coated with insulin-containing layer-by-layer films and their pH-dependent insulin release. J. Nanosci. Nanotechnol. 2014, 14, 3100–3105. [Google Scholar]
  29. Yoshida, K.; Hasebe, Y.; Takahashi, S.; Sato, K.; Anzai, J. Layer-by-layer deposited nano- and micro-assemblies for insulin delivery: A review. Mater. Sci. Eng. C 2014, 34, 384–392. [Google Scholar]
  30. Wang, C.; He, C.; Tong, Z.; Liu, X.; Ren, B.; Zeng, F. Combination of adsorption by porous CaCO3 microparticles and encapsulation by polyelectrolyte multilayer films for sustained drug delivery. Int. J. Pharm. 2006, 308, 160–167. [Google Scholar]
  31. Tong, W.; Dong, W.; Cao, C.; Möhwald, H. Charge-controlled permeability of polyelectrolyte microcapsules. J. Phys. Chem. B 2005, 109, 13159–13165. [Google Scholar]
  32. Li, J.; Jiang, Z.; Wu, H.; Long, L.; Jiang, Y.; Zhang, L. Improving the recycling and storage stability of enzyme by encapsulation in mesoporous CaCO3-alginate composite gel. Comp. Sci. Technol. 2009, 69, 539–544. [Google Scholar]
  33. Sato, K.; Shiba, T.; Anzai, J. Preparation of free-suspended polyelectrolyte multilayer films using an alginate scaffold and their ion permeability. Mater. Sci. Eng. C 2012, 5, 696–705. [Google Scholar]
  34. Tran, M.K.; Hassani, L.N.; Calvignac, B.; Beuvier, T.; Hindré, F.; Boury, F. Lyosozyme encapsulation within PLAGA and CaCO3 microparticles using supercritical CO2 medium. J. Supercrit. Fluids 2013, 79, 159–169. [Google Scholar]

Share and Cite

MDPI and ACS Style

Sato, K.; Seno, M.; Anzai, J.-I. Release of Insulin from Calcium Carbonate Microspheres with and without Layer-by-Layer Thin Coatings. Polymers 2014, 6, 2157-2165. https://doi.org/10.3390/polym6082157

AMA Style

Sato K, Seno M, Anzai J-I. Release of Insulin from Calcium Carbonate Microspheres with and without Layer-by-Layer Thin Coatings. Polymers. 2014; 6(8):2157-2165. https://doi.org/10.3390/polym6082157

Chicago/Turabian Style

Sato, Katsuhiko, Masaru Seno, and Jun-Ichi Anzai. 2014. "Release of Insulin from Calcium Carbonate Microspheres with and without Layer-by-Layer Thin Coatings" Polymers 6, no. 8: 2157-2165. https://doi.org/10.3390/polym6082157

Article Metrics

Back to TopTop