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Poly-L-ornithine/fucoidan-coated calcium carbonate microparticles by layer-by-layer self-assembly technique for cancer theranostics

Poly-L-ornithine/fucoidan-coated calcium carbonate microparticles by layer-by-layer self-assembly... Recently, the layer-by-layer (LbL) self-assembly technology has attracted the enormous interest of researchers in synthesizing various pharmaceutical dosage forms. Herewith, we designed a biocompatible drug delivery system containing the calcium carbonate microparticles (CaCO MPs) that coated with the alternatively charged polyelectrolytes, i.e., poly-L- ornithine (PLO)/fucoidan by LbL self-assembly process (LbL MPs). Upon coating with the polyelectrolytes, the mean particle size of MPs obtained from SEM observations increased from 1.91 to 2.03 μm, and the surface of LbL MPs was smoothened compared to naked CaCO MPs. In addition, the reversible zeta potential changes have confirmed the accomplishment of layer upon a layer assembly. To evaluate the efficiency of cancer therapeutics, we loaded doxorubicin (Dox) in the LbL MPs, which resulted in high (69.7%) drug encapsulation efficiency. The controlled release of Dox resulted in the significant antiproliferative efficiency in breast cancer cell line (MCF-7 cells), demonstrating the potential of applying this innovative drug delivery system in the biomedical field. 1 Introduction treatment are still surgery, radiotherapy and chemotherapy. As one of the main therapy methods, chemotherapy using Cancer is one of the leading causes of death globally, various anti-tumor drugs is commonly practiced after sur- accounting for millions of deaths every year. According to gery and radiation therapy. However, it faces limitations the statistics in 2017 from The National Central Cancer such as damaging the surrounding healthy normal cells, Registry of China, breast cancer and thyroid cancer are tissues, and organs such as kidney (mitomycin C) or the prevalent in women, where the occurrence and death rates heart [1, 2]. More often, the conventional delivery of anti- of breast cancer in small towns, medium towns and big tumor drugs exhibit undesirable cytotoxicity with low effi- cities are 29.9%, 8.44%; 39.6%, 9.59%, and 59.7%, 12.78% ciency leading to increasing the frequency of its respectively. So conquering cancer is the direction of many administration, which has created a stringent requirement scientists in the world. At present, the main ways of cancer for the new delivery strategy that can achieve the desired sustained release of drugs. Amongst various carriers for antitumor drug delivery, inorganic nanoparticles (for example, Fe O nanoparticles 3 4 * Yuangang Liu [3], mesoporous silica nanoparticles (MSN) [4], carbon ygliu@hqu.edu.cn nanotubes [5], quantum dots [6] et al.) have attracted the * Shibin Wang attention of researchers due to their advantages such as ideal sbwang@hqu.edu.cn biocompatibility as well as biodegradability and tunable morphological properties such as surface porosity [7–12]. College of Chemical Engineering, Huaqiao University, Xiamen 361021, China These carriers have a potential impact in the pharmaceutical field for the delivery of various drugs because of their College of Materials Science and Engineering, Huaqiao University, Xiamen 361021, China unique advantages. CaCO MPs exhibit favorable pH sen- sitive dissolution behavior, i.e., the degradation in the acidic Institute of Pharmaceutical Engineering, Huaqiao University, Xiamen 361021, China environment facilitating the intracellular drug release [13]. In a case, Volodkin et al. encapsulated and delivered pro- Fujian Provincial Key Laboratory of Biochemical Technology, Xiamen 361021, China teins via porous CaCO MPs [14]. In another way, 1234567890();,: 1234567890();,: 68 Page 2 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:68 Sukhorukov et al. prepared porous CaCO MPs as templates Dalian, China. PLO (M = 30–70 kDa), sodium carbonate, 3 w for the encapsulation of bioactive compounds [15]. and calcium chloride were obtained from Sigma. Co. Ltd In the application of drug carriers, the surface of the (St. Louis, MO, USA). Fucoidan (Mw = 200–400 kDa) was carriers will be modified to achieve the different functions. purchased from, Jiejing Group, Shandong, China. All Amongst many surface modification methods, LbL techni- solutions were prepared in water supplied from an ionic que is perhaps one of the most widely used simplified exchange system coupled to a Milli-Q-plus® purification processes for the surface modification of biomaterials and is system, Millipore Corp., electrical resistivity 18.2 MΩ cm. beneficial in producing numerous assembled supramole- Dulbecco’s modified eagle medium (DMEM), Fetal cular structures [16]. Recent advancements of LbL techni- bovine serum (FBS), penicillin, and streptomycin were que to building various multilayers have demonstrated that purchased from GIBCO/BRL Life Technologies (Grand this method is highly versatile [17, 18]. The successful Island, NY, USA). MCF-7 cells (Human breast cancer cells) formation of polyelectrolyte multilayers can be achieved by and C2C12 cells (Mouse myoblast cells) were obtained successive deposition of polycations (poly allylamine from the Type Culture Collection of the Chinese Academy hydrochloride [19], poly dimethyl diallyl ammonium of Sciences (CAS), Shanghai, China. All the cell lines were chloride [20], polyethyleneimine [21], and chitosan [22]) cultured in DMEM in a humidified incubator supplied with and polyanions (poly styrene sulfonate [23], poly acrylic 5% CO and maintained at 37 °C. Cell Counting Kit-8 acid [22], and poly methacrylic acid [24]) alternatively. (CCK-8), fluorescein isothiocyanate (FITC)-annexin V/ PLO as polycation consisting of -ornithine alone has a Propidium iodide (PI) Cell Apoptosis Kit were purchased primary amine on its side chain. The amino acid monomer from KeyGene Biotech, Nanjing, China. DAPI Staining of PLO is shorter in structure and likely allows PLO to bind Solution was acquired from Solarbio Co, Beijing, China. more efficiently to the membrane, which in turn results in increased membrane thickness and strength [25]. PLO also 2.2 Preparation of LbL MPs can alter the localization of tight junction proteins and enhance the permeation of hydrophilic macromolecules Initially, CaCO MPs were prepared using CMC by fol- [26–29]. So PLO is an interesting candidate for biomedical lowing the reported co-precipitation method [17]. CMC was applications [30]. Fucoidan as polyanion utilized in this completely dissolved in ultrapure water, then sodium car- work has an ability to induce apoptosis in cancer. There was bonate solution was added and stirred (1200 rpm) for a report that the anticancer effects of fucoidan varied 30 min, and then calcium chloride solution was added depending on its structure, while it can also target multiple dropwise to the mixture to precipitate uniform CaCO MPs. receptors or signaling molecules in various cell types, Eventually, PLO (0.1 mg/mL in water, pH-4) and fucoidan including tumor cells and immune cells demonstrating that (0.1 mg/mL in water, pH-9) were coated alternatively on the this polymer was suitable for cancer prevention or treatment surface of the above-prepared CaCO MPs to yield LbL [31]. In this work, we synthesized the biocompatible CaCO films by incubating the MPs in the respective solutions for MPs and further coated with alternative deposition of PLO 15 min each. After each deposition, CaCO MPs were and fucoidan on the surface of CaCO MPs core by the LbL rinsed twice in water to remove the adsorbed polymer technique resulting in LbL MPs. Furthermore, we investi- chains. The LbL assembly was repeated for four cycles and gated the in vitro release as well as antitumor effects of then dried in vacuum-assisted freeze drier to obtain the LbL doxorubicin (Dox) loaded in the LbL MPs. The results MPs. showed that the delivery system presented great bio- compatibility and anticancer effect, demonstrating that the 2.3 Characterization of LbL MPs LbL MPs had the potential application in pharmaceutical field. To investigate the morphology and size of the CaCO MPs and LbL MPs, scanning electron microscopic (SEM: S- 4800, Hitachi) and transmission electron microscopic 2 Materials and methods (TEM: H-7650, Hitachi) images were captured. The sam- ples were sputter-coated with a layer of gold for SEM 2.1 Materials observation. The samples for TEM observations were loa- ded on a 300 mesh copper grids at 100 kV. Alternatively All the chemicals, reagents, and organic solvents were of charged polyelectrolyte film deposition on the CaCO MPs the analytical grade of the highest purity and were obtained were confirmed by measuring the zeta-potential values after and used as received without any further purification. successive coating by a zeta-potential analyzer (ZetaPALS, Doxorubicin hydrochloride, carboxymethyl chitosan Malvern Instruments Co. Ltd.). The samples of LbL MPs (CMC) (10–80 mPa·s), were obtained from Meilunbio, after each adsorption were measured by suspending in Journal of Materials Science: Materials in Medicine (2018) 29:68 Page 3 of 10 68 ultrapure water, and the readings were taken for three 2.6 Biocompatibility studies independent experiments. The particle size of LbL MPs was measured from the SEM image. Functional group con- 2.6.1 Cell viability firmations of the different LbL coating on the surface of the CaCO MPs were elucidated by Fourier transform Cell viability of various LbL MPs was determined by Cell infrared (FT-IR) spectra, which were recorded on iS 50, Counting Kit-8 (CCK-8) assay using the mouse myoblast Nicolet FT-IR spectrophotometer using the standard KBr (C2C12) cell line [32]. 100 μL of medium containing cells method. Sampling was done by mixing the dried MPs with at a density of 8 × 10 cells/well were seeded into a 96-well the KBr, ground into a fine powder, pressed into transparent plate and incubated for 24 h at 37 °C in a humidified pellets and then subjected to scanning at a range of atmosphere maintained at 5% CO for proper cell attach- −1 4000–400 cm . ment. Subsequently, the culture media was replaced with 100 μL of FBS free medium containing CaCO and LbL MPs at various concentrations. After the cells were co- 2.4 Drug loading and encapsulation efficiency incubated with MPs for 48 h, the medium was removed, cells were washed using fresh medium, and CCK-8 reagent Dox was loaded using the following procedure. We accu- (10 μL) mixed with the fresh medium was added to each rately weighed 100 mg of LbL MPs and then incubated in well. After 3 h of incubation, the absorbance of the solution 10 mL of Dox solution (1 mg/mL), stirred at room tem- was measured using a microplate reader at 450 nm. Cell perature overnight. Finally, the resultant product was cen- viability was calculated by using the following formula: trifuged and washed twice with water and then dried using OD  OD treated free ð3Þ vacuum freeze drier. The Dox concentration of the super- Cell viability ¼ 100%; OD  OD control free natant was determined by UV-Vis spectroscopy at 480 nm with the pre-recorded calibration curve and the loading where OD is the absorbance of the group of treated amount and encapsulation efficiency of Dox calculated by microparticles, OD is the absorbance of the group of free the following expressions: not cells in well, OD is the control group. control Dox  Dox fed supernatant Loading amountðÞ % ¼  100; 2.6.2 Hemolysis test LbL  Dox ð1Þ The method for hemolysis assay has been reported pre- where Dox is the total weight of Dox fed, Dox is viously [33, 34]. A mixed sample of 5 mL normal saline and fed supernatant the weight of non-encapsulated free Dox, and LbL-Dox is 4 mL of fresh rabbit blood was centrifuged, and the red the weight of microparticles after loading Dox. blood cells (RBCs) were washed with PBS and diluted to thin the blood. To evaluate the hemolysis of various con- Dox  Dox fed supernatant centrations of MPs, the leaching of red hemoglobin Encapsulation efficiencyðÞ % ¼ 100; Dox in the supernatant was determined by measuring the fed ð2Þ absorption of positive and negative control experiments by incubation of RBCs with dd-H2O and phosphate buffered where Dox is the total weight of Dox fed, Dox is saline (PBS), respectively. The MPs for treatment were fed supernatant the weight of non-encapsulated free Dox. initially incubated with normal saline for 30 min, and then 0.2 mL diluted rabbit blood was added to the respective 2.5 In vitro drug release concentration and incubated for 60 min at 37 °C. Further, the red blood cells were isolated by centrifuging at The Dox-loaded LbL MPs (2 mg) were suspended in 2500 rpm for 5 min, and the absorbance of supernatant was phosphate-buffered solution (PBS) (pH 7.4, 30 mL) and measured at 545 nm. The hemolysis rate was calculated by placed in a dialysis bag. Further, the medium was the following formula: placed in a horizontal shaker maintained at 37 °C and OD OD treated negative control Hemolysis rateðÞ % ¼ 100; stirred at 150 rpm. Aliquots of the sample (3 mL) were OD OD positive control negative control periodically removed, and the sample was replaced with ð4Þ the same volume of fresh medium for a further period. The amount of released Dox was analyzed periodically using UV-Vis spectroscopy as mentioned above. All the Where OD is the absorbance of the group of micro- treated tests, including measurements, were carried out in particles, OD is the absorbance of the group of negative control triplicate. PBS, OD is the absorbance of the group of dd-H O. positive control 2 68 Page 4 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:68 Percentage hemolysis values were calculated from three 2.8 Cellular uptake study separate experiments. MCF-7 cells in 2 mL of culture medium were seeded in the 2.7 Apoptosis measurement well (2 × 10 cells / mL) of a 6-well plate and incubated at 37 °C for 24 h. Then the wells were treated with a particular Apoptosis of LbL MPs was measured using FITC-annexin agent (pure Dox and drug loaded LbL MPs with Dox V and PI containing Apoptosis Kit. MCF-7 cells were concentrations of 5 μg/mL). After co-incubation for 4 h at seeded into a 6-well plate placing glass slides in the well at 37 °C, the medium was removed, and the cells were washed a density of 2 × 10 cells/mL and incubated for 24 h. After three times with PBS. The cell nuclei were stained with proper cell attachment on a glass slide, the media in the DAPI solution for 15 min. Subsequently, the cells were wells were replaced with the 5 μg/mL of samples (i.e., of washed with PBS three times. A confocal laser scanning Dox and LbL-Dox). Cells without treatment were taken as a microscope was used to observe the samples under mag- control experiment. After 24 h of incubation, the cells were nification of 630. washed with PBS. Then, 100 μL of binding buffer, 5 μLof the annexin V conjugate, and 5 μL of the PI working solution were added to the cells were observed using a 3 Results confocal laser scanning microscope (CLSM, Leica TCS SP8). 3.1 Physical characterization of LbL MPs For the cell viability assay, MCF-7 cells seeded into 96- well plates were treated with samples of Dox and LbL DOX Surface morphology from SEM as well as TEM images of at 37 °C for 24 h, respectively. Other details are as described the CaCO and LbL MPs are depicted in Fig. 1a-c. CaCO 3 3 in Section 2.6.1. The statistical significance between two MPs were uniform spherical microstructured particles with sets of data was calculated by Student’s t-test. A p value < an average size of around 2 μm (Fig. 1a) and the size 0.05 was considered statistically significant. slightly increased after coating with the polyelectrolytes. Fig. 1 Morphological analysis of various MPs. SEM images of (a) CaCO MPs, (b) LbL MPs, (c) TEM image of LbL MPs. Particle size distribution of (d) CaCO MPs, (e) LbL MPs obtained from SEM image 3 Journal of Materials Science: Materials in Medicine (2018) 29:68 Page 5 of 10 68 Inset image of CaCO MPs illustrates the rough surface (Fig. 1a-inset), which is beneficial to form compact multi- layers around CaCO core MPs via LbL method. After assembling 4 bilayers of PLO/fucoidan alternatively, the surface of the LbL MPs was smoothed (Fig. 1b-inset) and obviously different from the uncoated MPs demonstrating the successful incorporation of PLO/fucoidan multilayers around the CaCO MPs. Further, we have also confirmed the polyelectrolyte coating over the core MPs by TEM measurements. Figure 1c illustrates that the appearance of organic polyelectrolyte coating is misty in nature around the dark CaCO core microstructure at around 0.5 μm thickness. Particle size distribution analysis demonstrated that the average size of LbLMPs (Fig. 1e) was 2.03 μm, which is slightly larger than that of CaCO ones (Fig. 1d), 1.91 μm. The subtle increase in the size of MPs is attributed to the formation of PLO/fucoidan multilayers around the CaCO MPs. The surface of CaCO MPs was coated with PLO and fucoidan alternatively layer-upon-a-layer successively. To evaluate the successive depositions of polyelectrolytes on CaCO core, the surface charge of LbL MP was recorded after each coating. Figure 2a illustrates the zeta-potential values of LbL polyelectrolyte films coated over CaCO MPs and confirms the number of bilayers. The core CaCO MPs showed a negative potential (−24 ± 4 mV) due to carbonate ions, and the potential was reversed upon deposition of first PLO layer due to its positive charge (16 ± 3 mV). Further, the negatively charged fucoidan deposition resulted in the negative potential (−22 ± 3 mV). The process of deposition repeated more 3 times, and the zeta potential values upon successive coating followed similar pattern demonstrating the successful deposition of polyelectrolyte bilayers on CaCO core. FT-IR spectra in Fig. 2b revealed the functional group modifications of obtained core MPs and LbL MPs, i.e., bi- layered polyelectrolyte (PLO/fucoidan) matrix deposition Fig. 2 a Zeta potential values of LbL MPs (CaCO -(PLO/fucoidan) ) 3 4 on the CaCO core. In all the spectra, a strong band centered −1 after alternate deposition of each PLO or fucoidan layer. b FT-IR around 3400 cm is due to the O–H stretching of absorbed spectra of (a) CaCO MPs, (b) PLO, (c) fucoidan and (d) LbL MPs −1 water molecules. The absorption peaks at 745 and 875 cm are attributed to the in-plane and out of plane bending 2− vibration peaks of CO , respectively. A broad peak at −1 1450 cm exemplifies the antisymmetric stretching vibra- were still observed on the LbL MPs after deposition of 2− tion peak of CO , indicating the crystalline form of bilayers on core MPs subsequently demonstrating the suc- −1 CaCO (Fig. 2B-a). An absorption peak at around 3420 cm cessful assembly of polyelectrolytes (Fig. 2B-d) by elec- represents the amine (–NH ) group in CMC. Figure 2B-b trostatic absorption. illustrates the characteristic absorption peaks of PLO. The −1 absorbance peaks at 1640, 1520 and 1250 cm were due to 3.2 Dox loading efficiency in LbL MPs and its release C = O and C-N stretching vibration and were in agreement with the previous report from Sapna et al. [35]. In addition, In the present work, we immobilized the anti-tumor drug −1 the characteristic peaks of fucoidan at 1260 and 1630 cm Dox, and the loading efficiency was 69.7 ± 1.3%, giving the were assigned to stretching vibration peak of C-N and C = Dox loading amount at around 6.5% of the LbL MPs. The O respectively (Fig. 2B-c). All the above-discussed peaks negatively charged fucoidan assembled on the surface of the 68 Page 6 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:68 LbL MPs provides additional attractive forces for Dox 3.4 In vitro antitumor assay immobilization. The LbL MPs have an ability to release the immobilized The antiproliferative effects of Dox-loaded LbL MPs (LbL- Dox in a controlled fashion due to extensive electrostatic Dox) were compared with the pure Dox in MCF-7 cells. interactions of polyelectrolytes and drug. In addition to This formulation resulted in the dose-dependent cytotoxi- multiple layers, the rough surface of core MPs might also city, i.e., the viable cell count decreased gradually with the accommodate a drug, which eventually results in its long increase in drug concentration, indicating that the Dox term diffusion effect. Interestingly, the in vitro release released effectively from LbL MPs (Fig. 4). Comparatively, behavior showed that the Dox released slowly and around the cell inhibitory effect (IC-50 value) of LbL MPs is higher 35% of the original drug for 150 h of incubation, but (approximately 2-fold) than pure Dox, representing the initially, for the first half an hour released rapidly due to solubility of Dox is enhanced significantly when delivered adsorbed drug molecules on bilayers. This phenomenon through this carrier. indicated that MPs are given with a long-term release Dox delivered from LbL MPs has shown noticeable anti- property. cancer effect, by triggering the induction of cell apoptosis. The mechanism lying behind the cell inhibition effect of 3.3 Biocompatibility study Dox and LbL-Dox were studied by using FITC-annexin V/ Dead Cell Apoptosis Kit. In this study, we stained MCF-7 The synthetic process is completely green and eco-friendly cells with FITC-annexin V and PI after the treatment of pure utilizing excellent biocompatible materials such as PLO, fucoidan, and CMC. However, there is a need in evaluating biocompatibility because the deposition of multiple layers and excessive charge interactions may result in cytotoxicity [36]. Fig. 3a elucidated the cytotoxicity study of CaCO MPs and LbL MPs at various concentrations in C2C12 cells. The results uncovered that the CaCO MPs and LbL MPs had no obvious inhibition effect on cells and with > 90% of viable cells, indicating that the carriers were in line with the biocompatibility standards. Furthermore, we evaluated the biocompatibility of MPs through a standard hemolysis test, which is a significant evaluation index for measuring the cytotoxicity of materials that can be feasible for in vivo correlation. Figure 3b showed the hemolytic properties of materials, which also closely interpret the solubility of carrier materials. The hemolysis rate of LbL MPs was less than 5%, even at a higher concentration (200 μg/mL), demonstrating that these Fig. 4 Cell viability (CCK-8 assay) of Dox and LbL-Dox at increasing concentrations in MCF-7 cells. *p < 0.05 MPs possess excellent hemocompatibility. Fig. 3 a Cell viability of C2C12 cells cultured in suspensions of CaCO and LbL MPs at increasing concentrations. b Hemolysis assay after the treatment with various concentrations of LbL MPs Journal of Materials Science: Materials in Medicine (2018) 29:68 Page 7 of 10 68 Dox and LbL-Dox for 24 h. After staining the cell popu- 4 Discussions lation with FITC-annexin V and PI, apoptotic cells show green fluorescence; dead cells show red and green fluores- The material design of our carrier is depicted in Fig. 7. cence under a CLSM. Based on this evaluation, the slicing Initially, we have synthesized well ordered, biocompatible, cells, dead cells, and cells that underwent apoptosis could spherical, and uniform-sized CaCO core MPs following be differentiated. In the control group (Fig. 5a), we co-precipitation method. Further, the alternatively charged observed a little red and green fluorescence. However, in polyelectrolyte layers namely, PLO and fucoidan were pure Dox treated cells (Fig. 5b), most of them were deposited successively on the CaCO MPs as shells around shrunken and round, and strong green fluorescence could be the core, and the sample was denoted as LbL MPs. Even- seen, indicating that cell death is due to apoptosis. How- tually, Dox was loaded into the LbL MPs, denoted as LbL- ever, most of the cells were dead in LbL-Dox treatment Dox.The synthesized MPs and LbL MPs were characterized group (Fig. 5c). using various physical characterization techniques, such as transmission and scanning electron microscopies (TEM and 3.5 Cellular uptake Study of Dox and LbL-Dox SEM, respectively) for surface morphology determination, Fourier transform infrared spectroscopy (FT-IR) for func- To study the cellular internalization of drug-loaded LbL tional group identification, Zeta potential measurements for MPs, the cells after being treated with different MPs with clarifying the surface charge after successive polyelectrolyte equivalent Dox concentrations for 4 h were visualized by deposition. An in vitro release study is performed to mea- CLSM. As shown in Fig. 6, the density of red fluorescence sure the Dox release in buffered sale mimicking the phy- from Dox in MCF-7 cells treated with pure Dox was siological conditions. Then, we report the biocompatibility apparently lower than LbL-Dox-treated cells. The images using call viability measurements and Hemolysis assay indicate that the fluorescence intensity of Dox is in proximity using fresh RBCs. Eventually, the anticancer evaluation to the nuclei in both the treatment elucidating the increased studies were performed using a CCK-8 assay for the intracellular Dox concentration. This result confirms the LbL determination of cell viability, the cell apoptosis determi- MPs have significantly enhanced drug delivery efficacy. nation using FITC annexin V and PI stains. Fig. 5 CLSM images of MCF-7 cells treated with a fresh medium (control), b pure Dox and c LbL-Dox (5 μg/mL Dox concentration) 68 Page 8 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:68 Fig. 6 Confocal images of MCF-7 cells after being treated with a pure Dox, b LbL-Dox for 4 h. Cell nuclei were stained with DAPI (blue-stained objects) After entering the tumor cells, the drug would undergo quick release triggered by acidic pH and a high con- centration of enzymes in the intracellular circumstance. In this study, the CaCO MPs were prepared by a very simple and efficient method based on the coprecipitation of CaCO and CMC. Further, these CaCO MPs were coated 3 3 with PLO and fucoidan by LbL technique. The experi- mental results demonstrated that LbL MPs worked as an efficient drug delivery micro-platform for breast cancer therapy. TEM image (Fig. 7c) indicated that the polymers were successfully assembled on the surface of the CaCO MPs and resulted in core-shell structure. The zeta-potential values confirmed the alternative deposition of different polyelectrolyte layers on the outermost surface of the MPs, demonstrating the successful formation of the LbL film coatings on the surface of the MPs and these layers were arranged through electrostatic attractions [38]. The results of FT-IR (Fig. 2a) indicated that there were no significant Fig. 7 Schematic representation showing the outline of the LbL design changes or result in hybrids through chemical reactions and the effect of MPs in the tumor environment between polymers. Dox immobilized in the LbL MPs resulted in slow release elucidating the interactions between For effective tumor therapy, a few factors such as the the drug and polyelectrolyte layers (Fig. 8). Eventually, the stability and tumor-specific drug release of carriers play a antitumor effects were performed to investigate the effi- crucial role. The formulations utilizing carriers for drug ciency of Dox, which corresponds to its release and effec- delivery could avoid degradation of drugs in an enzyme- tiveness against the tumor. rich plasma environment, in addition, the polyelectrolyte CaCO Mps are widely used to study phagocytosis layers coated over carriers provide an extra-barrier against because they possess narrow particle size distribution, leakage, excessive dilution of Dox. Most importantly, they highly stable in biological fluids, ease of labeling fluor- are internalized into cells by “stealth” endocytosis effect to escent moieties via modifying various functional groups on overcome multidrug resistance, i.e., the microparticles can the MPs [39, 40]. Dox, an anthracycline anticancer drug, prevent drug molecules from being recognized by cancer acts against various cancers through the interaction with cells because of the charged surface of polyelectrolyte [37]. DNA base pairs, resulting in its fragmentation, thus Journal of Materials Science: Materials in Medicine (2018) 29:68 Page 9 of 10 68 system using CaCO MPs. The drug delivery platform based on natural polymers coating over inorganic MPs with excellent biocompatibility and biodegradability is more promising and can emerge as an efficient drug delivery system. Acknowledgements This work was supported by Financial support from National marine economic innovation and development project (16PYY007SF17), the Science Research Foundation of National Health and Family Planning Commission of PRC & United Fujian Provincial Health and Education Project for Tacking the Key Research (WKJ2016-2-22), the Program for New Century Excellent Talents in Fujian Province University (2014FJ-NCET-ZR01), the Promotion Program for Young and Middle-aged Teachers in Science and Tech- nology Research of Huaqiao University (ZQN-PY108) and Subsidized Project for Postgraduates’ Innovative Fund in Scientific Research of Fig. 8 In vitro Dox release from LbL MPs at various intervals in Huaqiao University. simulated physiological fluids (PBS, pH 7.4) Compliance with ethical standards inducing apoptosis. The CLSM observation is in good Conflict of interest The authors declare that they have no conflict of agreement with the cell cytotoxicity results determined by interest. 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Poly-L-ornithine/fucoidan-coated calcium carbonate microparticles by layer-by-layer self-assembly technique for cancer theranostics

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Springer Journals
Copyright
Copyright © 2018 by Springer Science+Business Media, LLC, part of Springer Nature
Subject
Materials Science; Biomaterials; Biomedical Engineering; Regenerative Medicine/Tissue Engineering; Polymer Sciences; Ceramics, Glass, Composites, Natural Materials; Surfaces and Interfaces, Thin Films
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0957-4530
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1573-4838
DOI
10.1007/s10856-018-6075-z
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29748879
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Abstract

Recently, the layer-by-layer (LbL) self-assembly technology has attracted the enormous interest of researchers in synthesizing various pharmaceutical dosage forms. Herewith, we designed a biocompatible drug delivery system containing the calcium carbonate microparticles (CaCO MPs) that coated with the alternatively charged polyelectrolytes, i.e., poly-L- ornithine (PLO)/fucoidan by LbL self-assembly process (LbL MPs). Upon coating with the polyelectrolytes, the mean particle size of MPs obtained from SEM observations increased from 1.91 to 2.03 μm, and the surface of LbL MPs was smoothened compared to naked CaCO MPs. In addition, the reversible zeta potential changes have confirmed the accomplishment of layer upon a layer assembly. To evaluate the efficiency of cancer therapeutics, we loaded doxorubicin (Dox) in the LbL MPs, which resulted in high (69.7%) drug encapsulation efficiency. The controlled release of Dox resulted in the significant antiproliferative efficiency in breast cancer cell line (MCF-7 cells), demonstrating the potential of applying this innovative drug delivery system in the biomedical field. 1 Introduction treatment are still surgery, radiotherapy and chemotherapy. As one of the main therapy methods, chemotherapy using Cancer is one of the leading causes of death globally, various anti-tumor drugs is commonly practiced after sur- accounting for millions of deaths every year. According to gery and radiation therapy. However, it faces limitations the statistics in 2017 from The National Central Cancer such as damaging the surrounding healthy normal cells, Registry of China, breast cancer and thyroid cancer are tissues, and organs such as kidney (mitomycin C) or the prevalent in women, where the occurrence and death rates heart [1, 2]. More often, the conventional delivery of anti- of breast cancer in small towns, medium towns and big tumor drugs exhibit undesirable cytotoxicity with low effi- cities are 29.9%, 8.44%; 39.6%, 9.59%, and 59.7%, 12.78% ciency leading to increasing the frequency of its respectively. So conquering cancer is the direction of many administration, which has created a stringent requirement scientists in the world. At present, the main ways of cancer for the new delivery strategy that can achieve the desired sustained release of drugs. Amongst various carriers for antitumor drug delivery, inorganic nanoparticles (for example, Fe O nanoparticles 3 4 * Yuangang Liu [3], mesoporous silica nanoparticles (MSN) [4], carbon ygliu@hqu.edu.cn nanotubes [5], quantum dots [6] et al.) have attracted the * Shibin Wang attention of researchers due to their advantages such as ideal sbwang@hqu.edu.cn biocompatibility as well as biodegradability and tunable morphological properties such as surface porosity [7–12]. College of Chemical Engineering, Huaqiao University, Xiamen 361021, China These carriers have a potential impact in the pharmaceutical field for the delivery of various drugs because of their College of Materials Science and Engineering, Huaqiao University, Xiamen 361021, China unique advantages. CaCO MPs exhibit favorable pH sen- sitive dissolution behavior, i.e., the degradation in the acidic Institute of Pharmaceutical Engineering, Huaqiao University, Xiamen 361021, China environment facilitating the intracellular drug release [13]. In a case, Volodkin et al. encapsulated and delivered pro- Fujian Provincial Key Laboratory of Biochemical Technology, Xiamen 361021, China teins via porous CaCO MPs [14]. In another way, 1234567890();,: 1234567890();,: 68 Page 2 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:68 Sukhorukov et al. prepared porous CaCO MPs as templates Dalian, China. PLO (M = 30–70 kDa), sodium carbonate, 3 w for the encapsulation of bioactive compounds [15]. and calcium chloride were obtained from Sigma. Co. Ltd In the application of drug carriers, the surface of the (St. Louis, MO, USA). Fucoidan (Mw = 200–400 kDa) was carriers will be modified to achieve the different functions. purchased from, Jiejing Group, Shandong, China. All Amongst many surface modification methods, LbL techni- solutions were prepared in water supplied from an ionic que is perhaps one of the most widely used simplified exchange system coupled to a Milli-Q-plus® purification processes for the surface modification of biomaterials and is system, Millipore Corp., electrical resistivity 18.2 MΩ cm. beneficial in producing numerous assembled supramole- Dulbecco’s modified eagle medium (DMEM), Fetal cular structures [16]. Recent advancements of LbL techni- bovine serum (FBS), penicillin, and streptomycin were que to building various multilayers have demonstrated that purchased from GIBCO/BRL Life Technologies (Grand this method is highly versatile [17, 18]. The successful Island, NY, USA). MCF-7 cells (Human breast cancer cells) formation of polyelectrolyte multilayers can be achieved by and C2C12 cells (Mouse myoblast cells) were obtained successive deposition of polycations (poly allylamine from the Type Culture Collection of the Chinese Academy hydrochloride [19], poly dimethyl diallyl ammonium of Sciences (CAS), Shanghai, China. All the cell lines were chloride [20], polyethyleneimine [21], and chitosan [22]) cultured in DMEM in a humidified incubator supplied with and polyanions (poly styrene sulfonate [23], poly acrylic 5% CO and maintained at 37 °C. Cell Counting Kit-8 acid [22], and poly methacrylic acid [24]) alternatively. (CCK-8), fluorescein isothiocyanate (FITC)-annexin V/ PLO as polycation consisting of -ornithine alone has a Propidium iodide (PI) Cell Apoptosis Kit were purchased primary amine on its side chain. The amino acid monomer from KeyGene Biotech, Nanjing, China. DAPI Staining of PLO is shorter in structure and likely allows PLO to bind Solution was acquired from Solarbio Co, Beijing, China. more efficiently to the membrane, which in turn results in increased membrane thickness and strength [25]. PLO also 2.2 Preparation of LbL MPs can alter the localization of tight junction proteins and enhance the permeation of hydrophilic macromolecules Initially, CaCO MPs were prepared using CMC by fol- [26–29]. So PLO is an interesting candidate for biomedical lowing the reported co-precipitation method [17]. CMC was applications [30]. Fucoidan as polyanion utilized in this completely dissolved in ultrapure water, then sodium car- work has an ability to induce apoptosis in cancer. There was bonate solution was added and stirred (1200 rpm) for a report that the anticancer effects of fucoidan varied 30 min, and then calcium chloride solution was added depending on its structure, while it can also target multiple dropwise to the mixture to precipitate uniform CaCO MPs. receptors or signaling molecules in various cell types, Eventually, PLO (0.1 mg/mL in water, pH-4) and fucoidan including tumor cells and immune cells demonstrating that (0.1 mg/mL in water, pH-9) were coated alternatively on the this polymer was suitable for cancer prevention or treatment surface of the above-prepared CaCO MPs to yield LbL [31]. In this work, we synthesized the biocompatible CaCO films by incubating the MPs in the respective solutions for MPs and further coated with alternative deposition of PLO 15 min each. After each deposition, CaCO MPs were and fucoidan on the surface of CaCO MPs core by the LbL rinsed twice in water to remove the adsorbed polymer technique resulting in LbL MPs. Furthermore, we investi- chains. The LbL assembly was repeated for four cycles and gated the in vitro release as well as antitumor effects of then dried in vacuum-assisted freeze drier to obtain the LbL doxorubicin (Dox) loaded in the LbL MPs. The results MPs. showed that the delivery system presented great bio- compatibility and anticancer effect, demonstrating that the 2.3 Characterization of LbL MPs LbL MPs had the potential application in pharmaceutical field. To investigate the morphology and size of the CaCO MPs and LbL MPs, scanning electron microscopic (SEM: S- 4800, Hitachi) and transmission electron microscopic 2 Materials and methods (TEM: H-7650, Hitachi) images were captured. The sam- ples were sputter-coated with a layer of gold for SEM 2.1 Materials observation. The samples for TEM observations were loa- ded on a 300 mesh copper grids at 100 kV. Alternatively All the chemicals, reagents, and organic solvents were of charged polyelectrolyte film deposition on the CaCO MPs the analytical grade of the highest purity and were obtained were confirmed by measuring the zeta-potential values after and used as received without any further purification. successive coating by a zeta-potential analyzer (ZetaPALS, Doxorubicin hydrochloride, carboxymethyl chitosan Malvern Instruments Co. Ltd.). The samples of LbL MPs (CMC) (10–80 mPa·s), were obtained from Meilunbio, after each adsorption were measured by suspending in Journal of Materials Science: Materials in Medicine (2018) 29:68 Page 3 of 10 68 ultrapure water, and the readings were taken for three 2.6 Biocompatibility studies independent experiments. The particle size of LbL MPs was measured from the SEM image. Functional group con- 2.6.1 Cell viability firmations of the different LbL coating on the surface of the CaCO MPs were elucidated by Fourier transform Cell viability of various LbL MPs was determined by Cell infrared (FT-IR) spectra, which were recorded on iS 50, Counting Kit-8 (CCK-8) assay using the mouse myoblast Nicolet FT-IR spectrophotometer using the standard KBr (C2C12) cell line [32]. 100 μL of medium containing cells method. Sampling was done by mixing the dried MPs with at a density of 8 × 10 cells/well were seeded into a 96-well the KBr, ground into a fine powder, pressed into transparent plate and incubated for 24 h at 37 °C in a humidified pellets and then subjected to scanning at a range of atmosphere maintained at 5% CO for proper cell attach- −1 4000–400 cm . ment. Subsequently, the culture media was replaced with 100 μL of FBS free medium containing CaCO and LbL MPs at various concentrations. After the cells were co- 2.4 Drug loading and encapsulation efficiency incubated with MPs for 48 h, the medium was removed, cells were washed using fresh medium, and CCK-8 reagent Dox was loaded using the following procedure. We accu- (10 μL) mixed with the fresh medium was added to each rately weighed 100 mg of LbL MPs and then incubated in well. After 3 h of incubation, the absorbance of the solution 10 mL of Dox solution (1 mg/mL), stirred at room tem- was measured using a microplate reader at 450 nm. Cell perature overnight. Finally, the resultant product was cen- viability was calculated by using the following formula: trifuged and washed twice with water and then dried using OD  OD treated free ð3Þ vacuum freeze drier. The Dox concentration of the super- Cell viability ¼ 100%; OD  OD control free natant was determined by UV-Vis spectroscopy at 480 nm with the pre-recorded calibration curve and the loading where OD is the absorbance of the group of treated amount and encapsulation efficiency of Dox calculated by microparticles, OD is the absorbance of the group of free the following expressions: not cells in well, OD is the control group. control Dox  Dox fed supernatant Loading amountðÞ % ¼  100; 2.6.2 Hemolysis test LbL  Dox ð1Þ The method for hemolysis assay has been reported pre- where Dox is the total weight of Dox fed, Dox is viously [33, 34]. A mixed sample of 5 mL normal saline and fed supernatant the weight of non-encapsulated free Dox, and LbL-Dox is 4 mL of fresh rabbit blood was centrifuged, and the red the weight of microparticles after loading Dox. blood cells (RBCs) were washed with PBS and diluted to thin the blood. To evaluate the hemolysis of various con- Dox  Dox fed supernatant centrations of MPs, the leaching of red hemoglobin Encapsulation efficiencyðÞ % ¼ 100; Dox in the supernatant was determined by measuring the fed ð2Þ absorption of positive and negative control experiments by incubation of RBCs with dd-H2O and phosphate buffered where Dox is the total weight of Dox fed, Dox is saline (PBS), respectively. The MPs for treatment were fed supernatant the weight of non-encapsulated free Dox. initially incubated with normal saline for 30 min, and then 0.2 mL diluted rabbit blood was added to the respective 2.5 In vitro drug release concentration and incubated for 60 min at 37 °C. Further, the red blood cells were isolated by centrifuging at The Dox-loaded LbL MPs (2 mg) were suspended in 2500 rpm for 5 min, and the absorbance of supernatant was phosphate-buffered solution (PBS) (pH 7.4, 30 mL) and measured at 545 nm. The hemolysis rate was calculated by placed in a dialysis bag. Further, the medium was the following formula: placed in a horizontal shaker maintained at 37 °C and OD OD treated negative control Hemolysis rateðÞ % ¼ 100; stirred at 150 rpm. Aliquots of the sample (3 mL) were OD OD positive control negative control periodically removed, and the sample was replaced with ð4Þ the same volume of fresh medium for a further period. The amount of released Dox was analyzed periodically using UV-Vis spectroscopy as mentioned above. All the Where OD is the absorbance of the group of micro- treated tests, including measurements, were carried out in particles, OD is the absorbance of the group of negative control triplicate. PBS, OD is the absorbance of the group of dd-H O. positive control 2 68 Page 4 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:68 Percentage hemolysis values were calculated from three 2.8 Cellular uptake study separate experiments. MCF-7 cells in 2 mL of culture medium were seeded in the 2.7 Apoptosis measurement well (2 × 10 cells / mL) of a 6-well plate and incubated at 37 °C for 24 h. Then the wells were treated with a particular Apoptosis of LbL MPs was measured using FITC-annexin agent (pure Dox and drug loaded LbL MPs with Dox V and PI containing Apoptosis Kit. MCF-7 cells were concentrations of 5 μg/mL). After co-incubation for 4 h at seeded into a 6-well plate placing glass slides in the well at 37 °C, the medium was removed, and the cells were washed a density of 2 × 10 cells/mL and incubated for 24 h. After three times with PBS. The cell nuclei were stained with proper cell attachment on a glass slide, the media in the DAPI solution for 15 min. Subsequently, the cells were wells were replaced with the 5 μg/mL of samples (i.e., of washed with PBS three times. A confocal laser scanning Dox and LbL-Dox). Cells without treatment were taken as a microscope was used to observe the samples under mag- control experiment. After 24 h of incubation, the cells were nification of 630. washed with PBS. Then, 100 μL of binding buffer, 5 μLof the annexin V conjugate, and 5 μL of the PI working solution were added to the cells were observed using a 3 Results confocal laser scanning microscope (CLSM, Leica TCS SP8). 3.1 Physical characterization of LbL MPs For the cell viability assay, MCF-7 cells seeded into 96- well plates were treated with samples of Dox and LbL DOX Surface morphology from SEM as well as TEM images of at 37 °C for 24 h, respectively. Other details are as described the CaCO and LbL MPs are depicted in Fig. 1a-c. CaCO 3 3 in Section 2.6.1. The statistical significance between two MPs were uniform spherical microstructured particles with sets of data was calculated by Student’s t-test. A p value < an average size of around 2 μm (Fig. 1a) and the size 0.05 was considered statistically significant. slightly increased after coating with the polyelectrolytes. Fig. 1 Morphological analysis of various MPs. SEM images of (a) CaCO MPs, (b) LbL MPs, (c) TEM image of LbL MPs. Particle size distribution of (d) CaCO MPs, (e) LbL MPs obtained from SEM image 3 Journal of Materials Science: Materials in Medicine (2018) 29:68 Page 5 of 10 68 Inset image of CaCO MPs illustrates the rough surface (Fig. 1a-inset), which is beneficial to form compact multi- layers around CaCO core MPs via LbL method. After assembling 4 bilayers of PLO/fucoidan alternatively, the surface of the LbL MPs was smoothed (Fig. 1b-inset) and obviously different from the uncoated MPs demonstrating the successful incorporation of PLO/fucoidan multilayers around the CaCO MPs. Further, we have also confirmed the polyelectrolyte coating over the core MPs by TEM measurements. Figure 1c illustrates that the appearance of organic polyelectrolyte coating is misty in nature around the dark CaCO core microstructure at around 0.5 μm thickness. Particle size distribution analysis demonstrated that the average size of LbLMPs (Fig. 1e) was 2.03 μm, which is slightly larger than that of CaCO ones (Fig. 1d), 1.91 μm. The subtle increase in the size of MPs is attributed to the formation of PLO/fucoidan multilayers around the CaCO MPs. The surface of CaCO MPs was coated with PLO and fucoidan alternatively layer-upon-a-layer successively. To evaluate the successive depositions of polyelectrolytes on CaCO core, the surface charge of LbL MP was recorded after each coating. Figure 2a illustrates the zeta-potential values of LbL polyelectrolyte films coated over CaCO MPs and confirms the number of bilayers. The core CaCO MPs showed a negative potential (−24 ± 4 mV) due to carbonate ions, and the potential was reversed upon deposition of first PLO layer due to its positive charge (16 ± 3 mV). Further, the negatively charged fucoidan deposition resulted in the negative potential (−22 ± 3 mV). The process of deposition repeated more 3 times, and the zeta potential values upon successive coating followed similar pattern demonstrating the successful deposition of polyelectrolyte bilayers on CaCO core. FT-IR spectra in Fig. 2b revealed the functional group modifications of obtained core MPs and LbL MPs, i.e., bi- layered polyelectrolyte (PLO/fucoidan) matrix deposition Fig. 2 a Zeta potential values of LbL MPs (CaCO -(PLO/fucoidan) ) 3 4 on the CaCO core. In all the spectra, a strong band centered −1 after alternate deposition of each PLO or fucoidan layer. b FT-IR around 3400 cm is due to the O–H stretching of absorbed spectra of (a) CaCO MPs, (b) PLO, (c) fucoidan and (d) LbL MPs −1 water molecules. The absorption peaks at 745 and 875 cm are attributed to the in-plane and out of plane bending 2− vibration peaks of CO , respectively. A broad peak at −1 1450 cm exemplifies the antisymmetric stretching vibra- were still observed on the LbL MPs after deposition of 2− tion peak of CO , indicating the crystalline form of bilayers on core MPs subsequently demonstrating the suc- −1 CaCO (Fig. 2B-a). An absorption peak at around 3420 cm cessful assembly of polyelectrolytes (Fig. 2B-d) by elec- represents the amine (–NH ) group in CMC. Figure 2B-b trostatic absorption. illustrates the characteristic absorption peaks of PLO. The −1 absorbance peaks at 1640, 1520 and 1250 cm were due to 3.2 Dox loading efficiency in LbL MPs and its release C = O and C-N stretching vibration and were in agreement with the previous report from Sapna et al. [35]. In addition, In the present work, we immobilized the anti-tumor drug −1 the characteristic peaks of fucoidan at 1260 and 1630 cm Dox, and the loading efficiency was 69.7 ± 1.3%, giving the were assigned to stretching vibration peak of C-N and C = Dox loading amount at around 6.5% of the LbL MPs. The O respectively (Fig. 2B-c). All the above-discussed peaks negatively charged fucoidan assembled on the surface of the 68 Page 6 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:68 LbL MPs provides additional attractive forces for Dox 3.4 In vitro antitumor assay immobilization. The LbL MPs have an ability to release the immobilized The antiproliferative effects of Dox-loaded LbL MPs (LbL- Dox in a controlled fashion due to extensive electrostatic Dox) were compared with the pure Dox in MCF-7 cells. interactions of polyelectrolytes and drug. In addition to This formulation resulted in the dose-dependent cytotoxi- multiple layers, the rough surface of core MPs might also city, i.e., the viable cell count decreased gradually with the accommodate a drug, which eventually results in its long increase in drug concentration, indicating that the Dox term diffusion effect. Interestingly, the in vitro release released effectively from LbL MPs (Fig. 4). Comparatively, behavior showed that the Dox released slowly and around the cell inhibitory effect (IC-50 value) of LbL MPs is higher 35% of the original drug for 150 h of incubation, but (approximately 2-fold) than pure Dox, representing the initially, for the first half an hour released rapidly due to solubility of Dox is enhanced significantly when delivered adsorbed drug molecules on bilayers. This phenomenon through this carrier. indicated that MPs are given with a long-term release Dox delivered from LbL MPs has shown noticeable anti- property. cancer effect, by triggering the induction of cell apoptosis. The mechanism lying behind the cell inhibition effect of 3.3 Biocompatibility study Dox and LbL-Dox were studied by using FITC-annexin V/ Dead Cell Apoptosis Kit. In this study, we stained MCF-7 The synthetic process is completely green and eco-friendly cells with FITC-annexin V and PI after the treatment of pure utilizing excellent biocompatible materials such as PLO, fucoidan, and CMC. However, there is a need in evaluating biocompatibility because the deposition of multiple layers and excessive charge interactions may result in cytotoxicity [36]. Fig. 3a elucidated the cytotoxicity study of CaCO MPs and LbL MPs at various concentrations in C2C12 cells. The results uncovered that the CaCO MPs and LbL MPs had no obvious inhibition effect on cells and with > 90% of viable cells, indicating that the carriers were in line with the biocompatibility standards. Furthermore, we evaluated the biocompatibility of MPs through a standard hemolysis test, which is a significant evaluation index for measuring the cytotoxicity of materials that can be feasible for in vivo correlation. Figure 3b showed the hemolytic properties of materials, which also closely interpret the solubility of carrier materials. The hemolysis rate of LbL MPs was less than 5%, even at a higher concentration (200 μg/mL), demonstrating that these Fig. 4 Cell viability (CCK-8 assay) of Dox and LbL-Dox at increasing concentrations in MCF-7 cells. *p < 0.05 MPs possess excellent hemocompatibility. Fig. 3 a Cell viability of C2C12 cells cultured in suspensions of CaCO and LbL MPs at increasing concentrations. b Hemolysis assay after the treatment with various concentrations of LbL MPs Journal of Materials Science: Materials in Medicine (2018) 29:68 Page 7 of 10 68 Dox and LbL-Dox for 24 h. After staining the cell popu- 4 Discussions lation with FITC-annexin V and PI, apoptotic cells show green fluorescence; dead cells show red and green fluores- The material design of our carrier is depicted in Fig. 7. cence under a CLSM. Based on this evaluation, the slicing Initially, we have synthesized well ordered, biocompatible, cells, dead cells, and cells that underwent apoptosis could spherical, and uniform-sized CaCO core MPs following be differentiated. In the control group (Fig. 5a), we co-precipitation method. Further, the alternatively charged observed a little red and green fluorescence. However, in polyelectrolyte layers namely, PLO and fucoidan were pure Dox treated cells (Fig. 5b), most of them were deposited successively on the CaCO MPs as shells around shrunken and round, and strong green fluorescence could be the core, and the sample was denoted as LbL MPs. Even- seen, indicating that cell death is due to apoptosis. How- tually, Dox was loaded into the LbL MPs, denoted as LbL- ever, most of the cells were dead in LbL-Dox treatment Dox.The synthesized MPs and LbL MPs were characterized group (Fig. 5c). using various physical characterization techniques, such as transmission and scanning electron microscopies (TEM and 3.5 Cellular uptake Study of Dox and LbL-Dox SEM, respectively) for surface morphology determination, Fourier transform infrared spectroscopy (FT-IR) for func- To study the cellular internalization of drug-loaded LbL tional group identification, Zeta potential measurements for MPs, the cells after being treated with different MPs with clarifying the surface charge after successive polyelectrolyte equivalent Dox concentrations for 4 h were visualized by deposition. An in vitro release study is performed to mea- CLSM. As shown in Fig. 6, the density of red fluorescence sure the Dox release in buffered sale mimicking the phy- from Dox in MCF-7 cells treated with pure Dox was siological conditions. Then, we report the biocompatibility apparently lower than LbL-Dox-treated cells. The images using call viability measurements and Hemolysis assay indicate that the fluorescence intensity of Dox is in proximity using fresh RBCs. Eventually, the anticancer evaluation to the nuclei in both the treatment elucidating the increased studies were performed using a CCK-8 assay for the intracellular Dox concentration. This result confirms the LbL determination of cell viability, the cell apoptosis determi- MPs have significantly enhanced drug delivery efficacy. nation using FITC annexin V and PI stains. Fig. 5 CLSM images of MCF-7 cells treated with a fresh medium (control), b pure Dox and c LbL-Dox (5 μg/mL Dox concentration) 68 Page 8 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:68 Fig. 6 Confocal images of MCF-7 cells after being treated with a pure Dox, b LbL-Dox for 4 h. Cell nuclei were stained with DAPI (blue-stained objects) After entering the tumor cells, the drug would undergo quick release triggered by acidic pH and a high con- centration of enzymes in the intracellular circumstance. In this study, the CaCO MPs were prepared by a very simple and efficient method based on the coprecipitation of CaCO and CMC. Further, these CaCO MPs were coated 3 3 with PLO and fucoidan by LbL technique. The experi- mental results demonstrated that LbL MPs worked as an efficient drug delivery micro-platform for breast cancer therapy. TEM image (Fig. 7c) indicated that the polymers were successfully assembled on the surface of the CaCO MPs and resulted in core-shell structure. The zeta-potential values confirmed the alternative deposition of different polyelectrolyte layers on the outermost surface of the MPs, demonstrating the successful formation of the LbL film coatings on the surface of the MPs and these layers were arranged through electrostatic attractions [38]. The results of FT-IR (Fig. 2a) indicated that there were no significant Fig. 7 Schematic representation showing the outline of the LbL design changes or result in hybrids through chemical reactions and the effect of MPs in the tumor environment between polymers. Dox immobilized in the LbL MPs resulted in slow release elucidating the interactions between For effective tumor therapy, a few factors such as the the drug and polyelectrolyte layers (Fig. 8). Eventually, the stability and tumor-specific drug release of carriers play a antitumor effects were performed to investigate the effi- crucial role. The formulations utilizing carriers for drug ciency of Dox, which corresponds to its release and effec- delivery could avoid degradation of drugs in an enzyme- tiveness against the tumor. rich plasma environment, in addition, the polyelectrolyte CaCO Mps are widely used to study phagocytosis layers coated over carriers provide an extra-barrier against because they possess narrow particle size distribution, leakage, excessive dilution of Dox. Most importantly, they highly stable in biological fluids, ease of labeling fluor- are internalized into cells by “stealth” endocytosis effect to escent moieties via modifying various functional groups on overcome multidrug resistance, i.e., the microparticles can the MPs [39, 40]. Dox, an anthracycline anticancer drug, prevent drug molecules from being recognized by cancer acts against various cancers through the interaction with cells because of the charged surface of polyelectrolyte [37]. DNA base pairs, resulting in its fragmentation, thus Journal of Materials Science: Materials in Medicine (2018) 29:68 Page 9 of 10 68 system using CaCO MPs. The drug delivery platform based on natural polymers coating over inorganic MPs with excellent biocompatibility and biodegradability is more promising and can emerge as an efficient drug delivery system. Acknowledgements This work was supported by Financial support from National marine economic innovation and development project (16PYY007SF17), the Science Research Foundation of National Health and Family Planning Commission of PRC & United Fujian Provincial Health and Education Project for Tacking the Key Research (WKJ2016-2-22), the Program for New Century Excellent Talents in Fujian Province University (2014FJ-NCET-ZR01), the Promotion Program for Young and Middle-aged Teachers in Science and Tech- nology Research of Huaqiao University (ZQN-PY108) and Subsidized Project for Postgraduates’ Innovative Fund in Scientific Research of Fig. 8 In vitro Dox release from LbL MPs at various intervals in Huaqiao University. simulated physiological fluids (PBS, pH 7.4) Compliance with ethical standards inducing apoptosis. The CLSM observation is in good Conflict of interest The authors declare that they have no conflict of agreement with the cell cytotoxicity results determined by interest. 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Journal of Materials Science: Materials in Medicine – Springer Journals

Published: May 10, 2018

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