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CaCO3 hybrid micro-particles as carriers for water-soluble bioactive molecules
Colloids and Surfaces B: Biointerfaces 157 (2017) 481–489
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Fabrication of PLA/CaCO3 hybrid micro-particles as carriers for water-soluble bioactive molecules Valeriya L. Kudryavtseva b,c,1 , Li Zhao a,1 , Sergei I. Tverdokhlebov c , Gleb B. Sukhorukov a,∗ a b c
School of Engineering and Materials Science, Queen Mary University of London, Mile End Road, London, E1 4NS, United Kingdom National Research Tomsk Polytechnic University, RASA Center in Tomsk, Tomsk, 634050, Russia National Research Tomsk Polytechnic University, Department of Experimental Physics, Tomsk, 634050, Russia
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Article history: Received 7 March 2017 Received in revised form 27 May 2017 Accepted 11 June 2017 Available online 17 June 2017 Keywords: Biodegradable Micro-capsules Polylactic acid Calcium carbonate Encapsulation
a b s t r a c t We propose the use of polylactic acid/calcium carbonate (PLA/CaCO3 ) hybrid micro-particles for achieving improved encapsulation of water-soluble substances. Biodegradable porous CaCO3 microparticles can be loaded with wide range of bioactive substance. Thus, the formation of hydrophobic polymeric shell on surface of these loaded microparticles results on encapsulation and, hence, sealing internal cargo and preventing their release in aqueous media. In this study, to encapsulate proteins, we explore the solidin-oil-in-water emulsion method for fabricating core/shell PLA/CaCO3 systems. We used CaCO3 particles as a protective core for encapsulated bovine serum albumin, which served as a model protein system. We prepared a PLA coating using dichloromethane as an organic solvent and polyvinyl alcohol as a surfactant for emulsification; in addition, we varied experimental parameters such as surfactant concentration and polymer-to-CaCO3 ratio to determine their effect on particle-size distribution, encapsulation efficiency and capsule permeability. The results show that the particle size decreased and the size distribution narrowed as the surfactant concentration increased in the external aqueous phase. In addition, when the CaCO3 /PLA mass ratio dropped below 0.8, the hybrid micro-particles were more likely to resist treatment by ethylenediaminetetraacetic acid and thus retained their bioactive cargos within the polymer-coated micro-particles. © 2017 Elsevier B.V. All rights reserved.
1. Introduction In recent decades, research on the development of delivery systems for various bioactive compounds has drawn significant attention because of the increasing demand for more sophisticated constructs for storage, protection and on-site and timely deployment [1–4]. Consequently, a variety of polymeric carriers has been developed on the macro- and nano-scale, including polymeric micelles [5,6], multi-layer micro-capsules [7,8] and liposomes [9,10]. To improve the retention of loaded cargo, the permeability of vehicles must be reduced while the cargo is being delivered. However, the fabrication of polymeric micro-carriers with low permeability remains an unresolved issue, as does the fabrication of leak-proof delivery systems [11–13].
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (G.B. Sukhorukov). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2017.06.011 0927-7765/© 2017 Elsevier B.V. All rights reserved.
To address this problem, we investigate the use of a hydrophobic polymer layer as a promising solution to separate encapsulated cargos from external aqueous media. Various techniques are already available for this purpose, for example, surface-initiated polymerisation, which creates polymer shells in situ [14–17]. However, specific monomers can be used for certain types of polymerisation; this restricts the choice of materials from which the shell may be fabricated. Self-assembled micelles also contain a hydrophobic part; however, the entangled hydrophilic chains probably absorb water molecules, thereby providing a route for water molecules to diffuse into the core. Conversely, no limitation on the use of polymers is encountered when using the emulsion method, which separates the shell layer from the stabilizing layer so that the hydrophobic layer completely isolates the interior [18]. Emulsion methods are generally categorized into two types, namely single-emulsion methods and double-emulsion methods [19,20]. The former is widely used for encapsulating hydrophobic molecules, and the latter is more efficient for loading hydrophilic substances onto primary formed emulsions [21–25]. Thus, the encapsulation method must be chosen according to the nature of the cargo molecules. Regardless of the emulsion method used
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and the cargo molecules involved, the final emulsification step always involves an aqueous solution containing an emulsifier. In this step, a stabilizing layer coats the particles, which is essential to prevent aggregation of the particles. To encapsulate solid particles, the cargo may be dispersed in an oil phase to form a solid-in-oil emulsion; this is followed by a second emulsion process involving the use of water. Thus, the entire process is called the solid-in-oil-in-water (S/O/W) double-emulsion method. Unlike the water-in-oil-in-water (W/O/W) and oil-in-water (O/W) emulsion processes, wherein cargos are encapsulated directly during the formation of polymer shells, the active molecules are incorporated into the solid particles prior to the emulsion process in the S/O/W technique [26–31]. This approach may be used to encapsulate a broader range of substances, especially sensitive molecules that require protection for storage until further use. Porous calcium carbonate (CaCO3 ) micro-particles have been used as carriers for various bioactive molecules, including proteins and drugs. They can be fabricated in pre-defined sizes ranging from 400 nm to several microns [32–35]. Micro-sized CaCO3 particles have been intensively used as templates in the layer-by-layer process [32]. The use of CaCO3 particles allows a wide range of substances to be co-precipitated during particle formation from CaCl2 and NaCO3 [36–39]. Moreover, because calcium is the main component of bones, CaCO3 has been used clinically in the treatment of bone-related diseases [40,41]. The internal volume of pore in CaCO3 particles is up to 50% of the total particle volume; this allows them to hold various molecules of interest in their pores, while the hydrophobic coating over the microparticles protects the encapsulated molecules from the aqueous exterior. This study aims to use the S/O/W emulsion method to fabricate polymer-coated micro-particles with low permeability [42]. To investigate the possibility of drug delivery, we use CaCO3 particles as a protective core containing encapsulated bovine serum albumin (BSA) as a model protein system. We coated CaCO3 particles with polylactic acid (PLA), which acted as a hydrophobic layer, to seal pores and protect the cargo of bioactive molecules. PLA is a biodegradable polymer, and it is widely used in drugdelivery systems where the active substance (mostly substances with poor water solubility) is trapped in the bulk of PLA. When PLA degrades, CaCO3 may also dissolve, especially under a slightly acidic pH. BSA is thus released after the PLA layer degrades. In addition, this release may be accelerated by the decomposition of CaCO3 . We use scanning electron microscopy (SEM), confocal fluorescence laser scanning microscopy (CLSM) and thermogravimetric analysis (TGA) to study these structures and their stability for protein encapsulation against various dissolving agents.
2. Materials and methods 2.1. Materials PLA Ingeo 4044D was supplied by NatureWorks LLC. Dichloromethane (DCM), polyvinyl alcohol [PVA, molecular weight (MW)] = 89000–98000 g/mol), BSA (MW = ∼66000 g/mol), fluorescein isothiocyanate isomer I (FITC, MW = 389.38 g/mol), ethylenediaminetetraacetic acid (EDTA) and other chemicals were purchased from Sigma-Aldrich. All the materials were used as obtained without further purification; CaCO3 micro-particles were synthesised using sodium carbonate (Na2 CO3 ) and calcium chloride (CaCl2 ) purchased from Sigma-Aldrich and used without further purification. The water used in the experiments and solution preparation was purified using a Milli-Q system with a resistance of 18.2 M cm.
2.2. Labelling of BSA using FITC One hundred and sixty milligram of BSA was dissolved in 40 ml of PBS at a pH of 8, and 5 mg of FITC was dissolved in 5 ml of ethanol. These two solutions were then mixed and incubated in a refrigerator for 12 h, followed by dialyzing against deionised (DI) water for 72 h. The solution obtained was stored in the dark until further use. 2.3. Co-precipitation of BSA–FITC in CaCO3 micro-particles Aqueous solutions of CaCl2 and Na2 CO3 (0.615 ml, 1 M) were mixed with 1 ml of the BSA–FITC solution and 1.5 ml of DI water, as described in [43]. According to the procedure the resulted CaCO3 particles contain up to 107 –108 protein molecules per particle. The mixed solution was then vigorously stirred for 30 s before being washed three times with DI water and ethanol. Then, the micro-particle dispersion was centrifuged and the supernatant was discarded. Finally, the micro-particles obtained were dried overnight in an oven (90 ◦ C). 2.4. Preparation of PLA-coated micro-particles using the S/O/W emulsion process A given amount of pre-dried CaCO3 particles was mixed with a DCM solution containing 8% PLA, and this dispersion was sonicated in an ultrasonic bath for 2 min. Then, a PVA aqueous solution was added, which led to the formation of a two-phase liquid mixture with the PVA solution being the upper phase because the density of water in it was lower than that in the polymer solution. The emulsions were sonicated for 30 s at 20 Hz in a mechanical sonicator. The resulting emulsions were then left at ambient temperature for 20 min to let the solvent evaporate completely. Finally, the polymer-coated-CaCO3 -particle dispersion was washed intensively with DI water and stored in the refrigerator until further use. 2.5. Characterisation of micro-spheres The surface morphology and internal morphology of the microspheres were examined via SEM (FEI QUANTA 400 ESEM) using a backscattered electron (BSE) detector after the samples were pre-coated with a thin layer of carbon. Confocal laser scanning microscopy (Leica) was used to investigate the distribution of BSA–FITC CaCO3 particles within the micro-spheres as well as the permeability of micro-particles. The micro-capsule diameter and diameter distribution were determined based on three images captured from different fields of view using the ImageJ 1.38 software. Elemental analysis of the micro-spheres was performed using energy-dispersive spectroscopy (EDS; Oxford INCA Energy 400 equipped in the aforementioned SEM device). TGA of PLA microspheres was performed using a TGA thermal analyser SDT Q600. Samples with a weight of ∼5–10 mg were heated in an aluminium crucible from 30 to 800 ◦ C at a rate of 10 ◦ C/min under airflow. The measurements were repeated three times, and the data were averaged. 3. Results and discussion Fig. 1 shows a schematic of the fabrication process for microparticles coated with PLA using the single-emulsion method. In all experiments, the concentration of the PLA–DCM solution was 8% w/w and the organic-to-aqueous-phase ratio was 1/2 w/w. PLA is present in the organic phase along with CaCO3 micro-particles (Fig. 2). It covers the CaCO3 micro-particles when the solvent evaporates from the organic phase under vigorous mechanical sonication. The mean diameter of the CaCO3 particles is 1.11 ± 0.011 m, and
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Fig. 1. Schematic of the emulsion method used for fabricating PLA-coated-CaCO3 -particle dispersion.
the mean diameter of the droplets formed is ∼2 m. Thus, the PLA/CaCO3 system does not form a Pickering emulsion, for which the size of the stabilizing particles should be negligible with respect to the size of the droplets and polymer particles [44]. Since PLA is hydrophobic, which would very likely lead to aggregation of the micro-particles obtained in the aqueous solution, PVA, which is a hydrophilic surfactant, is added to the aqueous phase. PVA adsorbs onto particles as an additional layer atop PLA and yields well-dispersed micro-particles with a polymer coating. 3.1. Effect of PVA content on the micro-particles To determine the optimal amount of PVA to be used, the polymer-coated micro-particles were prepared using four different concentrations of PVA (0%, 0.1%, 1% and 2.5% mass percent of PVA relative to PLA). For each sample, the mass of the CaCO3 particles was set at 20% (1:5 ratio) relative to PLA. As can be seen in Fig. 3, spherical micro-particles are obtained in all the samples. The SEM
Fig. 2. SEM image of the CaCO3 particles fabricated via co-synthesis with BSA.
images show that the CaCO3 particles are well encapsulated in the polymer particles. In addition, no significant difference appears in particle morphology between each sample, and the mean diameters of the resulting particles indicate that the particles are of similar size, regardless of the PVA concentration. However, the size distribution of the micro-particles narrows as the PVA percentage increases, demonstrating that PVA exerts a positive effect. More importantly, with increasing PVA content, a lesser amount of PLA precipitates in the particle dispersion after the emulsion process, indicating that a large amount of PLA polymer is stabilised on the micro-particles. Furthermore, PLA-coated micro-particles without a PVA stabilizing layer tend to aggregate very quickly, whereas a PVA content of 2.5% results in fairly well-dispersed samples even after weeks of preparation. Thus, we used a PVA content of 2.5% throughout the experiments discussed hereafter. 3.2. Effect of CaCO3 content on the morphology and encapsulation of micro-particles To study how the CaCO3 content affects the polymer-coated micro-particles, six samples were prepared with the following mass ratios of CaCO3 relative to PLA: 0, 0.1 (1:10), 0.2 (1:5), 0.4 (2:5), 0.8 (4:5) and 0.12 (6:5). All samples were prepared using the standard emulsion process described in Fig. 1. BSA–FITC was incorporated into CaCO3 cores during their formation for the permeability study. The morphologies, elemental compositions and permeability of the resulting micro-particles were then studied using SEM, EDS, CLSM and TGA. Because the main task was the encapsulation of CaCO3 particles using PLA coating, the positions of CaCO3 particles after the emulsion process should be known. Thus, we used a BSE detector to obtain Z-contrast images to brighter appearance of the higherZ phases; therefore, because the atomic number of Ca is higher than those of C, O and H, CaCO3 particles should appear brighter in the image (Fig. S1). Based on the results shown in Fig. 4, we find that the diameter of spherical particles in all samples ranges from hundreds of nano-metres to several micro-metres. The SEM image and EDS spectrum of the sample prepared without CaCO3 suggest that no Ca-containing particles were present. As expected, some bright contrast appears in the image of the particles for all the remaining samples; this is considered to originate from the
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Fig. 3. SEM images and size distributions of PLA-coated micro-particles with (a) 0%, (b) 0.1%, (c) 1% and (d) 2.5% PVA content as a surface stabiliser. All percentages indicate the mass ratio of PVA relative to PLA.
Fig. 4. SEM images (obtained from the BSE detector) of PLA-coated micro-particles with various mass ratios of CaCO3 particles relative to PLA: (a) 0, (b) 0.1, (c) 0.2, (d) 0.4, (e) 0.8 and (f) 1.2.
CaCO3 particles, whose presence is confirmed by EDS (see Fig. 4b–f). In addition, more bright contrast appears with increasing number of CaCO3 particles. Fig. 3f shows that most of the micro-particles obtained contain CaCO3 particles with a CaCO3 /PLA mass ratio of
1.2, which may also be deduced from the corresponding EDS spectrum. Moreover, because more CaCO3 particles form in the sample, fewer small particles of PLA only form after the emulsion process; this is attributed to the majority of the PLA being deposited onto the
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Fig. 5. Overlayed CLSM images of PLA-coated micro-particles with various mass ratios of CaCO3 particles relative to PLA: (a) 0.1, (b) 0.2, (c) 0.4, (d) 0.8 and (e) 1.2.
Fig. 6. SEM images (obtained from the BSE detector) of PLA-coated micro-particles with various mass ratios of CaCO3 particles relative to PLA after EDTA treatment: (a) 0.1, (b) 0.2, (c) 0.4, (d) 0.8 and (e) 1.2.
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Fig. 7. Overlayed CLSM images of PLA-coated micro-particles with various mass ratios of CaCO3 particles relative to PLA after EDTA treatment: (a) 0.1, (b) 0.2, (c) 0.4, (d) 0.8, (e) 1.2.
CaCO3 cores. Thus, less PLA is left to form pure-polymer particles. Fig. S2 shows SEM images of PLA-coated CaCO3 micro-particles; these images were acquired using a secondary electron (SE) detector and the BSE detector. CLSM was also applied to check whether the incorporated BSA–FITC remains trapped in the micro-particles after the emulsion process. The results (see Fig. S3) clearly show that in all five samples, BSA–FITC is sealed within the micro-particles, as indicated by the solid fluorescent circles. The overlayed CLSM images clearly demonstrate the presence of fluorescent particles in each sample (Fig. 5). CLSM images of the samples with a higher CaCO3 content show more fluorescent particles. 3.3. Investigation of the permeability of the PLA-coated micro-particles The vaterite polymorph of CaCO3 is known to recrystallise into calcite in a few hours. However, CaCO3 particles remain spherical in water if the polymer deposition step is performed as soon as the particles are fabricated. Once the particles are coated with the polymer, the particle shape is protected and ion diffusion is restricted by the surrounding polymer shell, as seen in the SEM images (Fig. S1). Next, PLA micro-capsules were treated with EDTA solution to see if it dissolves the encapsulated CaCO3 particles. Each sample was dispersed in a 0.2-M EDTA solution for 2 h and washed with DI water before further characterisation. SEM, EDS, CLSM and TGA were used to characterise the microcapsules after treating them with EDTA. In general, compared with the micro-capsules before EDTA treatment, only a small number of CaCO3 particles were dissolved in the all investigated samples. We attribute this result to the morphology and non-homogeneous formation of the PLA particles, what is difficult to control and that leads to more defects in the PLA shells formed over microparticles.
However, despite this effect, a significant number of CaCO3 particles are still retained within the PLA shells after EDTA treatment (see Fig. 6), especially in samples with a CaCO3 /PLA mass ratio ≤ 0.8. However, almost half of the CaCO3 particles disappear after EDTA treatment in the sample with a CaCO3 /PLA mass ratio of 1.2, wherein the number of CaCO3 particles that remain is even fewer than that in samples with a mass ratio of 0.4 or 0.8. This probably results from the higher CaCO3 content, which would reduce the amount of PLA polymer adsorbed onto each CaCO3 particle, thereby resulting in a thinner PLA layer that is more permeable to EDTA molecules. For the first four samples, the CLSM images show no obvious changes in the samples before and after EDTA treatment (Fig. 7 and S4). In contrast, very few fluorescent circles appear in the sample with a CaCO3 /PLA mass ratio of 1.2 after EDTA treatment. The PLA/CaCO3 particles in the sample with a mass ratio of 1.2 have a much higher permeability compared with the particles in the other samples. We assume that once the CaCO3 particles dissolve, a thin PLA layer with possible defects cannot retain BSA–FITC within the polymer shell. Also, such an effect may be explained by the appearance of crater-structured surface formations when the CaCO3 /PLA mass ratio exceeds 0.8 and the PLA layer becomes thinner (Fig. 8). Despite this, even at a higher mass ratio of 1.2, the breakage or rupture of the PLA layer is not observed in most cases. The thermal stability of PLA micro-capsules was studied using TGA, in which the samples were compared before and after EDTA treatment. Fig. 9 shows sample weight loss as a function of temperature. Upon heating, CaCO3 undergoes a reaction whereby carbon dioxide (CO2 ) is released from the material, leaving only calcium oxide (CaO) after the heating process. Therefore, 44% mass loss with respect to the total mass is expected for CaCO3 micro-particles. For pure CaCO3 , mass loss is minimum until the temperature exceeds 550 ◦ C; in addition, at 800 ◦ C, where the decomposition of CaCO3 is complete, the mass loss is 38.71 ± 1.21 wt.%, which is close to
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Fig. 8. SEM images (obtained from the BSE detector) of PLA-coated micro-particles with various mass ratios of CaCO3 particles relative to PLA: (a) 0, (b) 0.2, (c) 0.8 and (d) 1.2.
Fig. 9. TGA curves of (a) pure CaCO3 and PLA, (b) PLA-coated micro-particles with a mass ratio of 0.8 for CaCO3 particles relative to PLA.
the theoretical value (Fig. 9a). Fig. 7a shows that pure PLA decomposes completely above 400 ◦ C and the percent mass remaining is 1.57 ± 0.12 wt.%. The mass loss in PLA/CaCO3 micro-particles occurs in two stages: at ∼200 ◦ C due to the loss of PLA and at 750 ◦ C due to the release of CO2 . The total amount of CaCO3 in the sample is
the sum of the amount of the residual (CaO) and the mass loss after 750 ◦ C (CO2 ). Fig. 9b shows that PLA-coated micro-particles with a CaCO3 /PLA mass ratio of 0.8 resist EDTA treatment because the amount of CaCO3 does not decrease significantly (considering the measurement error).
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4. Conclusion We deposited a low-permeability PLA coating onto CaCO3 micro-particles containing co-precipitated BSA–FITC using the S/O/W emulsion process, thereby sealing the pores and drastically reducing the shell permeability. Adding an additional outermost PVA layer proved to be helpful in stabilizing the obtained coated micro-particles; in addition, this process narrowed the size distribution of the resulting PLA/CaCO3 particles, with 2.5% being the optimal PVA concentration for particle formulation. The variable ratio of the CaCO3 micro-particle content to the amount of PLA in different samples was studied to better understand particle formation. In all the samples studied, BSA–FITC appeared in the PLA particles with a CaCO3 /PLA mass ratio ranging from 0.1 to 1.2. At a CaCO3 /PLA mass ratio less than 0.8, polymer-coated microparticles are more likely to resist EDTA treatment and retain their bioactive cargos, whereas, owing to the thin layer of PLA on each CaCO3 particle and the appearance of pores in the PLA shell, the PLA-coated particles can be affected by EDTA at a CaCO3 /PLA mass ratio of 1.2. Therefore, the CaCO3 /PLA mass ratio of 0.8 was found to be optimal in terms of the high payload of proteins contained in the micro-particles and the stability of the resulting microparticles against dissolution. Both components (PLA and CaCO3 ) are biodegradable; CaCO3 is a component bone, which means that this micro-capsule system can be used to store and deliver bone-growth factors or other drug molecules to treat bone-related diseases while giving additional mechanical support for delivering cargos. Other delivery strategies, e.g. targeted delivery, can also be implemented by incorporating magnetic nanoparticles within the PLA shell for subsequent magnetic navigation of drug-loaded micro-capsules to the site of action. We thus expect that PLA-coated CaCO3 microparticles containing bioactive molecules can be used for various biomedical purposes, such as in drug storage and delivery systems. Therefore, the hybrid micro-capsules developed herein may potentially be used for the encapsulation of a wide range of water-soluble substances, such as proteins, insulin, cytostatics and polysaccharides.
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Acknowledgements
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This work was supported in part by the Russian Governmental Program “Nauka” N: 11.7293.2017/8.9. Li Zhao is grateful for a CSC Scholarship for funding.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2017.06. 011.
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Fabrication of PLA/CaCO3 hybrid micro-particles as carriers for water-soluble bioactive molecules Valeriya L. Kudryavtseva b,c,1 , Li Zhao a,1 , Sergei I. Tverdokhlebov c , Gleb B. Sukhorukov a,∗ a b c
School of Engineering and Materials Science, Queen Mary University of London, Mile End Road, London, E1 4NS, United Kingdom National Research Tomsk Polytechnic University, RASA Center in Tomsk, Tomsk, 634050, Russia National Research Tomsk Polytechnic University, Department of Experimental Physics, Tomsk, 634050, Russia
a r t i c l e
i n f o
Article history: Received 7 March 2017 Received in revised form 27 May 2017 Accepted 11 June 2017 Available online 17 June 2017 Keywords: Biodegradable Micro-capsules Polylactic acid Calcium carbonate Encapsulation
a b s t r a c t We propose the use of polylactic acid/calcium carbonate (PLA/CaCO3 ) hybrid micro-particles for achieving improved encapsulation of water-soluble substances. Biodegradable porous CaCO3 microparticles can be loaded with wide range of bioactive substance. Thus, the formation of hydrophobic polymeric shell on surface of these loaded microparticles results on encapsulation and, hence, sealing internal cargo and preventing their release in aqueous media. In this study, to encapsulate proteins, we explore the solidin-oil-in-water emulsion method for fabricating core/shell PLA/CaCO3 systems. We used CaCO3 particles as a protective core for encapsulated bovine serum albumin, which served as a model protein system. We prepared a PLA coating using dichloromethane as an organic solvent and polyvinyl alcohol as a surfactant for emulsification; in addition, we varied experimental parameters such as surfactant concentration and polymer-to-CaCO3 ratio to determine their effect on particle-size distribution, encapsulation efficiency and capsule permeability. The results show that the particle size decreased and the size distribution narrowed as the surfactant concentration increased in the external aqueous phase. In addition, when the CaCO3 /PLA mass ratio dropped below 0.8, the hybrid micro-particles were more likely to resist treatment by ethylenediaminetetraacetic acid and thus retained their bioactive cargos within the polymer-coated micro-particles. © 2017 Elsevier B.V. All rights reserved.
1. Introduction In recent decades, research on the development of delivery systems for various bioactive compounds has drawn significant attention because of the increasing demand for more sophisticated constructs for storage, protection and on-site and timely deployment [1–4]. Consequently, a variety of polymeric carriers has been developed on the macro- and nano-scale, including polymeric micelles [5,6], multi-layer micro-capsules [7,8] and liposomes [9,10]. To improve the retention of loaded cargo, the permeability of vehicles must be reduced while the cargo is being delivered. However, the fabrication of polymeric micro-carriers with low permeability remains an unresolved issue, as does the fabrication of leak-proof delivery systems [11–13].
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (G.B. Sukhorukov). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2017.06.011 0927-7765/© 2017 Elsevier B.V. All rights reserved.
To address this problem, we investigate the use of a hydrophobic polymer layer as a promising solution to separate encapsulated cargos from external aqueous media. Various techniques are already available for this purpose, for example, surface-initiated polymerisation, which creates polymer shells in situ [14–17]. However, specific monomers can be used for certain types of polymerisation; this restricts the choice of materials from which the shell may be fabricated. Self-assembled micelles also contain a hydrophobic part; however, the entangled hydrophilic chains probably absorb water molecules, thereby providing a route for water molecules to diffuse into the core. Conversely, no limitation on the use of polymers is encountered when using the emulsion method, which separates the shell layer from the stabilizing layer so that the hydrophobic layer completely isolates the interior [18]. Emulsion methods are generally categorized into two types, namely single-emulsion methods and double-emulsion methods [19,20]. The former is widely used for encapsulating hydrophobic molecules, and the latter is more efficient for loading hydrophilic substances onto primary formed emulsions [21–25]. Thus, the encapsulation method must be chosen according to the nature of the cargo molecules. Regardless of the emulsion method used
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and the cargo molecules involved, the final emulsification step always involves an aqueous solution containing an emulsifier. In this step, a stabilizing layer coats the particles, which is essential to prevent aggregation of the particles. To encapsulate solid particles, the cargo may be dispersed in an oil phase to form a solid-in-oil emulsion; this is followed by a second emulsion process involving the use of water. Thus, the entire process is called the solid-in-oil-in-water (S/O/W) double-emulsion method. Unlike the water-in-oil-in-water (W/O/W) and oil-in-water (O/W) emulsion processes, wherein cargos are encapsulated directly during the formation of polymer shells, the active molecules are incorporated into the solid particles prior to the emulsion process in the S/O/W technique [26–31]. This approach may be used to encapsulate a broader range of substances, especially sensitive molecules that require protection for storage until further use. Porous calcium carbonate (CaCO3 ) micro-particles have been used as carriers for various bioactive molecules, including proteins and drugs. They can be fabricated in pre-defined sizes ranging from 400 nm to several microns [32–35]. Micro-sized CaCO3 particles have been intensively used as templates in the layer-by-layer process [32]. The use of CaCO3 particles allows a wide range of substances to be co-precipitated during particle formation from CaCl2 and NaCO3 [36–39]. Moreover, because calcium is the main component of bones, CaCO3 has been used clinically in the treatment of bone-related diseases [40,41]. The internal volume of pore in CaCO3 particles is up to 50% of the total particle volume; this allows them to hold various molecules of interest in their pores, while the hydrophobic coating over the microparticles protects the encapsulated molecules from the aqueous exterior. This study aims to use the S/O/W emulsion method to fabricate polymer-coated micro-particles with low permeability [42]. To investigate the possibility of drug delivery, we use CaCO3 particles as a protective core containing encapsulated bovine serum albumin (BSA) as a model protein system. We coated CaCO3 particles with polylactic acid (PLA), which acted as a hydrophobic layer, to seal pores and protect the cargo of bioactive molecules. PLA is a biodegradable polymer, and it is widely used in drugdelivery systems where the active substance (mostly substances with poor water solubility) is trapped in the bulk of PLA. When PLA degrades, CaCO3 may also dissolve, especially under a slightly acidic pH. BSA is thus released after the PLA layer degrades. In addition, this release may be accelerated by the decomposition of CaCO3 . We use scanning electron microscopy (SEM), confocal fluorescence laser scanning microscopy (CLSM) and thermogravimetric analysis (TGA) to study these structures and their stability for protein encapsulation against various dissolving agents.
2. Materials and methods 2.1. Materials PLA Ingeo 4044D was supplied by NatureWorks LLC. Dichloromethane (DCM), polyvinyl alcohol [PVA, molecular weight (MW)] = 89000–98000 g/mol), BSA (MW = ∼66000 g/mol), fluorescein isothiocyanate isomer I (FITC, MW = 389.38 g/mol), ethylenediaminetetraacetic acid (EDTA) and other chemicals were purchased from Sigma-Aldrich. All the materials were used as obtained without further purification; CaCO3 micro-particles were synthesised using sodium carbonate (Na2 CO3 ) and calcium chloride (CaCl2 ) purchased from Sigma-Aldrich and used without further purification. The water used in the experiments and solution preparation was purified using a Milli-Q system with a resistance of 18.2 M cm.
2.2. Labelling of BSA using FITC One hundred and sixty milligram of BSA was dissolved in 40 ml of PBS at a pH of 8, and 5 mg of FITC was dissolved in 5 ml of ethanol. These two solutions were then mixed and incubated in a refrigerator for 12 h, followed by dialyzing against deionised (DI) water for 72 h. The solution obtained was stored in the dark until further use. 2.3. Co-precipitation of BSA–FITC in CaCO3 micro-particles Aqueous solutions of CaCl2 and Na2 CO3 (0.615 ml, 1 M) were mixed with 1 ml of the BSA–FITC solution and 1.5 ml of DI water, as described in [43]. According to the procedure the resulted CaCO3 particles contain up to 107 –108 protein molecules per particle. The mixed solution was then vigorously stirred for 30 s before being washed three times with DI water and ethanol. Then, the micro-particle dispersion was centrifuged and the supernatant was discarded. Finally, the micro-particles obtained were dried overnight in an oven (90 ◦ C). 2.4. Preparation of PLA-coated micro-particles using the S/O/W emulsion process A given amount of pre-dried CaCO3 particles was mixed with a DCM solution containing 8% PLA, and this dispersion was sonicated in an ultrasonic bath for 2 min. Then, a PVA aqueous solution was added, which led to the formation of a two-phase liquid mixture with the PVA solution being the upper phase because the density of water in it was lower than that in the polymer solution. The emulsions were sonicated for 30 s at 20 Hz in a mechanical sonicator. The resulting emulsions were then left at ambient temperature for 20 min to let the solvent evaporate completely. Finally, the polymer-coated-CaCO3 -particle dispersion was washed intensively with DI water and stored in the refrigerator until further use. 2.5. Characterisation of micro-spheres The surface morphology and internal morphology of the microspheres were examined via SEM (FEI QUANTA 400 ESEM) using a backscattered electron (BSE) detector after the samples were pre-coated with a thin layer of carbon. Confocal laser scanning microscopy (Leica) was used to investigate the distribution of BSA–FITC CaCO3 particles within the micro-spheres as well as the permeability of micro-particles. The micro-capsule diameter and diameter distribution were determined based on three images captured from different fields of view using the ImageJ 1.38 software. Elemental analysis of the micro-spheres was performed using energy-dispersive spectroscopy (EDS; Oxford INCA Energy 400 equipped in the aforementioned SEM device). TGA of PLA microspheres was performed using a TGA thermal analyser SDT Q600. Samples with a weight of ∼5–10 mg were heated in an aluminium crucible from 30 to 800 ◦ C at a rate of 10 ◦ C/min under airflow. The measurements were repeated three times, and the data were averaged. 3. Results and discussion Fig. 1 shows a schematic of the fabrication process for microparticles coated with PLA using the single-emulsion method. In all experiments, the concentration of the PLA–DCM solution was 8% w/w and the organic-to-aqueous-phase ratio was 1/2 w/w. PLA is present in the organic phase along with CaCO3 micro-particles (Fig. 2). It covers the CaCO3 micro-particles when the solvent evaporates from the organic phase under vigorous mechanical sonication. The mean diameter of the CaCO3 particles is 1.11 ± 0.011 m, and
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Fig. 1. Schematic of the emulsion method used for fabricating PLA-coated-CaCO3 -particle dispersion.
the mean diameter of the droplets formed is ∼2 m. Thus, the PLA/CaCO3 system does not form a Pickering emulsion, for which the size of the stabilizing particles should be negligible with respect to the size of the droplets and polymer particles [44]. Since PLA is hydrophobic, which would very likely lead to aggregation of the micro-particles obtained in the aqueous solution, PVA, which is a hydrophilic surfactant, is added to the aqueous phase. PVA adsorbs onto particles as an additional layer atop PLA and yields well-dispersed micro-particles with a polymer coating. 3.1. Effect of PVA content on the micro-particles To determine the optimal amount of PVA to be used, the polymer-coated micro-particles were prepared using four different concentrations of PVA (0%, 0.1%, 1% and 2.5% mass percent of PVA relative to PLA). For each sample, the mass of the CaCO3 particles was set at 20% (1:5 ratio) relative to PLA. As can be seen in Fig. 3, spherical micro-particles are obtained in all the samples. The SEM
Fig. 2. SEM image of the CaCO3 particles fabricated via co-synthesis with BSA.
images show that the CaCO3 particles are well encapsulated in the polymer particles. In addition, no significant difference appears in particle morphology between each sample, and the mean diameters of the resulting particles indicate that the particles are of similar size, regardless of the PVA concentration. However, the size distribution of the micro-particles narrows as the PVA percentage increases, demonstrating that PVA exerts a positive effect. More importantly, with increasing PVA content, a lesser amount of PLA precipitates in the particle dispersion after the emulsion process, indicating that a large amount of PLA polymer is stabilised on the micro-particles. Furthermore, PLA-coated micro-particles without a PVA stabilizing layer tend to aggregate very quickly, whereas a PVA content of 2.5% results in fairly well-dispersed samples even after weeks of preparation. Thus, we used a PVA content of 2.5% throughout the experiments discussed hereafter. 3.2. Effect of CaCO3 content on the morphology and encapsulation of micro-particles To study how the CaCO3 content affects the polymer-coated micro-particles, six samples were prepared with the following mass ratios of CaCO3 relative to PLA: 0, 0.1 (1:10), 0.2 (1:5), 0.4 (2:5), 0.8 (4:5) and 0.12 (6:5). All samples were prepared using the standard emulsion process described in Fig. 1. BSA–FITC was incorporated into CaCO3 cores during their formation for the permeability study. The morphologies, elemental compositions and permeability of the resulting micro-particles were then studied using SEM, EDS, CLSM and TGA. Because the main task was the encapsulation of CaCO3 particles using PLA coating, the positions of CaCO3 particles after the emulsion process should be known. Thus, we used a BSE detector to obtain Z-contrast images to brighter appearance of the higherZ phases; therefore, because the atomic number of Ca is higher than those of C, O and H, CaCO3 particles should appear brighter in the image (Fig. S1). Based on the results shown in Fig. 4, we find that the diameter of spherical particles in all samples ranges from hundreds of nano-metres to several micro-metres. The SEM image and EDS spectrum of the sample prepared without CaCO3 suggest that no Ca-containing particles were present. As expected, some bright contrast appears in the image of the particles for all the remaining samples; this is considered to originate from the
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Fig. 3. SEM images and size distributions of PLA-coated micro-particles with (a) 0%, (b) 0.1%, (c) 1% and (d) 2.5% PVA content as a surface stabiliser. All percentages indicate the mass ratio of PVA relative to PLA.
Fig. 4. SEM images (obtained from the BSE detector) of PLA-coated micro-particles with various mass ratios of CaCO3 particles relative to PLA: (a) 0, (b) 0.1, (c) 0.2, (d) 0.4, (e) 0.8 and (f) 1.2.
CaCO3 particles, whose presence is confirmed by EDS (see Fig. 4b–f). In addition, more bright contrast appears with increasing number of CaCO3 particles. Fig. 3f shows that most of the micro-particles obtained contain CaCO3 particles with a CaCO3 /PLA mass ratio of
1.2, which may also be deduced from the corresponding EDS spectrum. Moreover, because more CaCO3 particles form in the sample, fewer small particles of PLA only form after the emulsion process; this is attributed to the majority of the PLA being deposited onto the
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Fig. 5. Overlayed CLSM images of PLA-coated micro-particles with various mass ratios of CaCO3 particles relative to PLA: (a) 0.1, (b) 0.2, (c) 0.4, (d) 0.8 and (e) 1.2.
Fig. 6. SEM images (obtained from the BSE detector) of PLA-coated micro-particles with various mass ratios of CaCO3 particles relative to PLA after EDTA treatment: (a) 0.1, (b) 0.2, (c) 0.4, (d) 0.8 and (e) 1.2.
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Fig. 7. Overlayed CLSM images of PLA-coated micro-particles with various mass ratios of CaCO3 particles relative to PLA after EDTA treatment: (a) 0.1, (b) 0.2, (c) 0.4, (d) 0.8, (e) 1.2.
CaCO3 cores. Thus, less PLA is left to form pure-polymer particles. Fig. S2 shows SEM images of PLA-coated CaCO3 micro-particles; these images were acquired using a secondary electron (SE) detector and the BSE detector. CLSM was also applied to check whether the incorporated BSA–FITC remains trapped in the micro-particles after the emulsion process. The results (see Fig. S3) clearly show that in all five samples, BSA–FITC is sealed within the micro-particles, as indicated by the solid fluorescent circles. The overlayed CLSM images clearly demonstrate the presence of fluorescent particles in each sample (Fig. 5). CLSM images of the samples with a higher CaCO3 content show more fluorescent particles. 3.3. Investigation of the permeability of the PLA-coated micro-particles The vaterite polymorph of CaCO3 is known to recrystallise into calcite in a few hours. However, CaCO3 particles remain spherical in water if the polymer deposition step is performed as soon as the particles are fabricated. Once the particles are coated with the polymer, the particle shape is protected and ion diffusion is restricted by the surrounding polymer shell, as seen in the SEM images (Fig. S1). Next, PLA micro-capsules were treated with EDTA solution to see if it dissolves the encapsulated CaCO3 particles. Each sample was dispersed in a 0.2-M EDTA solution for 2 h and washed with DI water before further characterisation. SEM, EDS, CLSM and TGA were used to characterise the microcapsules after treating them with EDTA. In general, compared with the micro-capsules before EDTA treatment, only a small number of CaCO3 particles were dissolved in the all investigated samples. We attribute this result to the morphology and non-homogeneous formation of the PLA particles, what is difficult to control and that leads to more defects in the PLA shells formed over microparticles.
However, despite this effect, a significant number of CaCO3 particles are still retained within the PLA shells after EDTA treatment (see Fig. 6), especially in samples with a CaCO3 /PLA mass ratio ≤ 0.8. However, almost half of the CaCO3 particles disappear after EDTA treatment in the sample with a CaCO3 /PLA mass ratio of 1.2, wherein the number of CaCO3 particles that remain is even fewer than that in samples with a mass ratio of 0.4 or 0.8. This probably results from the higher CaCO3 content, which would reduce the amount of PLA polymer adsorbed onto each CaCO3 particle, thereby resulting in a thinner PLA layer that is more permeable to EDTA molecules. For the first four samples, the CLSM images show no obvious changes in the samples before and after EDTA treatment (Fig. 7 and S4). In contrast, very few fluorescent circles appear in the sample with a CaCO3 /PLA mass ratio of 1.2 after EDTA treatment. The PLA/CaCO3 particles in the sample with a mass ratio of 1.2 have a much higher permeability compared with the particles in the other samples. We assume that once the CaCO3 particles dissolve, a thin PLA layer with possible defects cannot retain BSA–FITC within the polymer shell. Also, such an effect may be explained by the appearance of crater-structured surface formations when the CaCO3 /PLA mass ratio exceeds 0.8 and the PLA layer becomes thinner (Fig. 8). Despite this, even at a higher mass ratio of 1.2, the breakage or rupture of the PLA layer is not observed in most cases. The thermal stability of PLA micro-capsules was studied using TGA, in which the samples were compared before and after EDTA treatment. Fig. 9 shows sample weight loss as a function of temperature. Upon heating, CaCO3 undergoes a reaction whereby carbon dioxide (CO2 ) is released from the material, leaving only calcium oxide (CaO) after the heating process. Therefore, 44% mass loss with respect to the total mass is expected for CaCO3 micro-particles. For pure CaCO3 , mass loss is minimum until the temperature exceeds 550 ◦ C; in addition, at 800 ◦ C, where the decomposition of CaCO3 is complete, the mass loss is 38.71 ± 1.21 wt.%, which is close to
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Fig. 8. SEM images (obtained from the BSE detector) of PLA-coated micro-particles with various mass ratios of CaCO3 particles relative to PLA: (a) 0, (b) 0.2, (c) 0.8 and (d) 1.2.
Fig. 9. TGA curves of (a) pure CaCO3 and PLA, (b) PLA-coated micro-particles with a mass ratio of 0.8 for CaCO3 particles relative to PLA.
the theoretical value (Fig. 9a). Fig. 7a shows that pure PLA decomposes completely above 400 ◦ C and the percent mass remaining is 1.57 ± 0.12 wt.%. The mass loss in PLA/CaCO3 micro-particles occurs in two stages: at ∼200 ◦ C due to the loss of PLA and at 750 ◦ C due to the release of CO2 . The total amount of CaCO3 in the sample is
the sum of the amount of the residual (CaO) and the mass loss after 750 ◦ C (CO2 ). Fig. 9b shows that PLA-coated micro-particles with a CaCO3 /PLA mass ratio of 0.8 resist EDTA treatment because the amount of CaCO3 does not decrease significantly (considering the measurement error).
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4. Conclusion We deposited a low-permeability PLA coating onto CaCO3 micro-particles containing co-precipitated BSA–FITC using the S/O/W emulsion process, thereby sealing the pores and drastically reducing the shell permeability. Adding an additional outermost PVA layer proved to be helpful in stabilizing the obtained coated micro-particles; in addition, this process narrowed the size distribution of the resulting PLA/CaCO3 particles, with 2.5% being the optimal PVA concentration for particle formulation. The variable ratio of the CaCO3 micro-particle content to the amount of PLA in different samples was studied to better understand particle formation. In all the samples studied, BSA–FITC appeared in the PLA particles with a CaCO3 /PLA mass ratio ranging from 0.1 to 1.2. At a CaCO3 /PLA mass ratio less than 0.8, polymer-coated microparticles are more likely to resist EDTA treatment and retain their bioactive cargos, whereas, owing to the thin layer of PLA on each CaCO3 particle and the appearance of pores in the PLA shell, the PLA-coated particles can be affected by EDTA at a CaCO3 /PLA mass ratio of 1.2. Therefore, the CaCO3 /PLA mass ratio of 0.8 was found to be optimal in terms of the high payload of proteins contained in the micro-particles and the stability of the resulting microparticles against dissolution. Both components (PLA and CaCO3 ) are biodegradable; CaCO3 is a component bone, which means that this micro-capsule system can be used to store and deliver bone-growth factors or other drug molecules to treat bone-related diseases while giving additional mechanical support for delivering cargos. Other delivery strategies, e.g. targeted delivery, can also be implemented by incorporating magnetic nanoparticles within the PLA shell for subsequent magnetic navigation of drug-loaded micro-capsules to the site of action. We thus expect that PLA-coated CaCO3 microparticles containing bioactive molecules can be used for various biomedical purposes, such as in drug storage and delivery systems. Therefore, the hybrid micro-capsules developed herein may potentially be used for the encapsulation of a wide range of water-soluble substances, such as proteins, insulin, cytostatics and polysaccharides.
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Acknowledgements
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This work was supported in part by the Russian Governmental Program “Nauka” N: 11.7293.2017/8.9. Li Zhao is grateful for a CSC Scholarship for funding.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2017.06. 011.
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