Letter www.acsami.org
Enhanced Cellular Uptake of Bowl-like Microcapsules Huiying Li, Wenbo Zhang, Weijun Tong,* and Changyou Gao* MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027China S Supporting Information *
ABSTRACT: Among several properties of colloidal particles, shape is emerging as an important parameter for tailoring the interactions between particles and cells. In this study, bowl-like multilayer microcapsules were prepared by osmotic-induced invagination of their spherical counterparts in a concentrated polyelectrolyte solution. The internalization behaviors of bowl-like and spherical microcapsules were compared by coincubation with smooth muscle cells (SMCs) and macrophages. The bowl-like capsules tended to attach onto the cell membranes from the bend side and could be enwrapped by the membranes of SMCs, leading to a faster uptake rate and larger accumulation inside cells than those of their spherical counterparts. These results are important for understanding the shape-dependent internalization behavior, providing useful guidance for further materials design especially in biomedical applications. KEYWORDS: bowl-like, microcapsules, polyelectrolyte, shape effect, cellular uptake, osmotic pressure
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Because of the strong impact of the particle shape on the carrier performance, various methods, including microfluidics, dry etching techniques, soft lithography, and thermal− mechanical deformation, have been employed to fabricate particles with diverse shapes.15,16 Nonspherical structure can also be obtained because replicas of biological structures of anisotropic inorganic microparticles and such methods usually result in rod, cubic, or discoidal shapes.8,17,18 Among diverse colloidal particles, polyelectrolyte microcapsules with tailored structures and properties have gained much interest in the biomedical field especially for drug loading and release.19,20 However, researches about the interactions between nonspherical polyelectrolyte microcapsules and cells are rare,21,22 and the mechanism of shape-induced different interactions between capsules and cells needs further investigation. For instance, Caruso and co-workers21 reported the fabrication of rod-shaped hydrogel capsules with tunable aspect ratios by a templating method. With increasing aspect ratios, slower and less cellular internalization of capsules was observed. Kharlampieva and co-workers22 obtained polymer capsules with hemispherical geometry by drying poly(N-vinylpyrrolidone)/tannic acid multilayer capsules and found that, compared with their spherical and cubic counterparts, the hemispherical capsules are taken up to a greater extent. However, the mechanism behind it is not very clear. Herein, a simple and facile osmotic pressure method to fabricate bowl-like polyelectrolyte microcapsules is reported. This method was originally developed by our group to measure
olloidal particles have been widely used as carriers in biomedical fields including drug delivery, imaging, and vaccination.1,2 The physicochemical properties of colloidal particles can strongly influence their interactions with biological systems.3,4 In particular, slight changes in the physicochemical characteristics of particles may strongly influence the interactions between particles and cells and further affect the cellular uptake, intracellular distribution, and ultimate cellular fate.5 So far, parameters that can affect the performance of carriers such as the surface chemistry and size have been well recognized.6 In recent years, shape has been found to play a crucial role in the interactions between cells and particles and is regarded as a new important parameter for designing materials to realize specific biological functions.7,8 For example, Smith and coworkers9 reported that polystyrene particles with three shapes (spheres, prolate ellipsoids, and oblate ellipsoids) could manipulate different attachment and internalization of macrophages. Kolhar and Mitragotri10 found that, with identical total volumes, polystyrene particles with spherical shape exhibited significant perinuclear accumulation compared to rods, while such a result may be affected by the size as well. They also demonstrated that regulation of the immune response could be achieved by changing the sizes and shapes of nanoparticles when ovalbumin is used as a model antigen conjugated to particle surfaces.11 Moreover, in a model microvascular network, elongated particles exhibited higher adhesion and binding probability than spheres, and the extent was dependent on the aspect ratio of the particles.12,13 In vivo experiments also showed that hydrogel microparticles mimicking the shape of red blood cells could possess increased blood circulation and enhanced adhesion ability.13,14 © 2016 American Chemical Society
Received: March 9, 2016 Accepted: April 27, 2016 Published: April 27, 2016 11210
DOI: 10.1021/acsami.6b02965 ACS Appl. Mater. Interfaces 2016, 8, 11210−11214
Letter
ACS Applied Materials & Interfaces the mechanical properties of polyelectrolyte microcapsules.23 In this study, it is first used to fabricate bowl-like microcapsules. Because of the effect of osmotic pressure, incubation of the multilayer microcapsules in a concentrated poly(styrenesulfonate) (PSS) solution results in shape transformation from spherical to bowl-like (Scheme 1). The
in a PSS solution of high concentration (30 wt %). The coating of BSA can increase the compactness of the shell, which can prevent the permeation of PSS into the microcapsules and ensure invagination of microcapsules under osmotic pressure. The BSA coating also can improve the biocompatibility of the microcapsules. The PSS counterions and polyelectrolytes thereby create an osmotic pressure difference across the capsule wall to induce capsule deformation.23 The deformed capsules were still dispersed well in water, and their size had no significant change, whereas their shape was transformed from spherical to bowl-like (Figure 1D−F). The bowl-like structure was very stable and could be maintained even after being incubated in the culture medium for 7 days (Figure 1F). The surface properties of objects have been known to affect the cellular uptake behavior.6 Osmotic deformation is likely accompanied by the adsorption of a part of PSS onto the capsule surface [about 20% calculated from the X-ray photoelectron spectroscopy results (Table S1)]; thus, the bowl-like capsules had more negative charges (−41.9 ± 2.7 mV) than their spherical counterparts (−17.6 ± 1.7 mV). After being incubated in the cell culture medium for 12 h, the difference of the surface charge was much smaller because of the adsorption of serum proteins (bowl-like, −22.7 ± 1.6 mV; spherical, −13.3 ± 1.9 mV). When particles come into contact with the physiological environment, proteins will adsorb on their surface to form protein coronas, which then influence the following particle/cell interaction behaviors.24 Thus, protein adsorption on the surface of microcapsules was further measured. The amounts of protein adsorption on bowl-like and spherical microcapsules were similar (Table S2). Because the major protein in fetal bovine serum is BSA, the result was further compared with the adsorption in the cell culture medium with only BSA; no obvious difference was found. So, the bowl-like and spherical microcapsules show similar protein adsorptions during cell culture, and the adsorbed protein was mainly BSA. Thus, the original slight differences of the chemical nature were covered up by the adsorbed proteins, and the capsules had similar surface properties in the cell culture media. The influence of the capsule shape on internalization was investigated by using two cell lines, SMCs and RAW 264.7, both having important physiological functions and strong internalization ability. After coculture of the microcapsules with cells, the cells were treated by Trypan blue to quench the fluorescence of capsules adhered on the cell surface.25 As shown in Figure 2A, internalization of both capsules was a timedependent process regardless of their shape. At 1 h, the cellular uptake ratio of both capsules was very low. Afterward, the bowllike capsules were preferentially internalized with a significantly larger percentage than the spherical ones. A total of 24 h later, over 80% of cells had internalized the bowl-like capsules, whereas it was only about 56% for the spherical ones. Moreover, at the same incubation time, each cell internalized a significantly larger number of bowl-like capsules than spherical ones. For example, at 12 h the number of bowl-like capsules per cell was 3.8-fold larger than that of their spherical counterparts. This fact was further substantiated by coincubation of both types of capsules with SMCs simultaneously (Figure 2B). Colocalization with the cell nucleus showed that the microcapsules have been successfully internalized by SMCs and distributed around the cell nucleus (Figure S1). A similar phenomenon was observed for RAW 264.7 cells (Figure S2), but the internalization ratios for both types of capsules were synchronously smaller than those of SMCs likely because of the
Scheme 1. Fabrication Process of Spherical and Bowl-like (PAH/PSS)10/BSA Microcapsules and Their Cellular Uptake Behaviors
structure and stability of the capsules in the cell culture medium are characterized, and the uptake behaviors of the bowl-like and spherical microcapsules by human vascular smooth muscle cells (SMCs) and typical macrophage cells RAW 264.7 are compared. The mechanism for different uptake behaviors is discussed. The spherical microcapsules were fabricated as hollow replicas of spherical MnCO3 templates via layer-by-layer assembly (Scheme 1). As shown in Figure 1A−C, after core
Figure 1. SEM (A and D), cross-sectional (ultramicrotomy) transmission electron microscopy (B and E), and confocal laser scanning microscopy (C and F) images of spherical (A−C) and bowllike (D−F) (PAH/PSS)10/BSA microcapsules. The samples for SEM observation were prepared by critical point drying. The capsules for confocal laser scanning microscopy observation were incubated in a cell culture medium for 7 days, and the spherical and bowl-like microcapsules were labeled by fluorescein isothiocyanate and Rhodamine B isothiocyanate, respectively.
removal, the capsules maintained the spherical morphology of the templates with an average diameter of 2.0 ± 0.1 μm. These capsules could be well dispersed in water. To induce deformation of the capsules, an osmotic pressure technique was applied.23 For this to occur, the spherical capsules were coated with bovine serum albumin (BSA) and then immersed 11211
DOI: 10.1021/acsami.6b02965 ACS Appl. Mater. Interfaces 2016, 8, 11210−11214
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ACS Applied Materials & Interfaces
microcapsules was an energy-dependent process. Although with similar diameter, the volume of bowl-like capsules was smaller than that of spherical capsules, which means that during internalization less energy is needed.27 To further elucidate the detailed interactions between the capsules and cells, the cells were incubated with the capsules for a certain time, and then the samples were fixed and observed by scanning electron microscopy (SEM). As shown in Figure 3A,B, both the bowl-like and spherical capsules were found on the cell surfaces after incubation with SMCs for 3 h. However, the interaction manners were very different. The cells already expanded their membranes around the bowl-like capsules and engulfed some of the capsules (Figure 3A). By contrast, at the same incubation time, no obvious structure change of the cell membrane near the contact area with the spherical capsules was found, and the capsules were merely adsorbed onto the cell membranes (Figure 3B). A statistical study found that about 80% of the bowl-like capsules tended to attach to the cell membrane from the bend side (Figure 3C). When the bowl-like capsule was fitted with a circle, it was found that the apparent diameter (2.3 ± 0.2 μm) was slightly larger than that of the spherical capsules (2.0 ± 0.2 μm), conveying that their bottom side becomes flatter and thereby facilitating the attachment.9,28 Serda and co-workers29 found that pseudopodia projection from the surface of the endothelial cells was seen “looping” around porous silicon microparticles, and the membrane eventually spread outward to engulf the microparticles. However, for RAW 264.7 cells, membrane expansion around the capsules was not seen for both capsules, although better attachment of the bowl-like capsules to the cells was observed (Figure S4A, B). The difference may result from the different internalization mechanism of SMCs and RAW 264.7 cells. It is known that the macrophages usually take up particles by the mechanism of actin-dependent phagocytosis,30 and thus membrane expansion is not indispensable. It is worth mentioning that more internalization of the bowl-like capsules resulted in a decrease of the cell viability (Figure S5A,B) without obvious differences in the cell cytoskeleton (Figure S5C,D). With these results, the mechanism is proposed in Scheme 2. During coculture with cells, both the bowl-like and spherical capsules can attach to the cell membrane. With an asymmetrical structure, the bowl-like capsules mainly attach from the bend side, which may facilitate the interactions and further induce the enwrapping of capsules by the cell membrane. On the contrary, their spherical counterparts interact significantly less with the cell membrane. Moreover, the energy cost for cell membrane deformation of the bowl-like capsules is less than that of the spherical ones because of the smaller actual
Figure 2. (A) Cellular uptake of bowl-like and spherical (PAH/ PSS)10/BSA microcapsules as a function of the incubation time. The microcapsule-to-SMC ratio was 40:1. (B) Confocal laser scanning microscopy image of SMCs after being coincubated with fluorescein isothiocyanate-labeled bowl-like capsules and Rhodamine B isothiocyanate-labeled spherical capsules for 12 h. The dashed lines indicate cell contours. The feeding ratio bowl-like capsules/spherical capsules/ SMCs was 40:40:1.
smaller size of the RAW 264.7 cells. Hence, one can safely conclude that the bowl-like shape promotes cellular internalization more than the spherical one. This observation correlates well with the earlier study that hemispherical capsules were more efficiently taken up than the spherical counterparts.22 Extracellular substances can be transported into cells through different pathways.26 So, the uptake mechanism of microcapsules was studied. As shown in Figure S3, for both bowl-like and spherical microcapsules, the uptake efficiency was significantly blocked by sodium azide, suggesting that the uptake was an energy-dependent process. For bowl-like microcapsules, the uptake was also mediated by clathrin- and caveolae-mediated endocytosis, while for spherical microcapsules, clathrin-mediated endocytosis and cytoskeleton also played an important role during internalization. During the internalization process, the particles need to attach onto the cell membrane first, whose stability and interaction with the cells are significantly influenced by the particle curvature. First, because of the movement of the particles and shear stress induced by fluid, the particles could be detached again. As reported, nonspherical particles usually show better attachment ability than spheres.8,12,13 Second, it is not the overall surface but the local curvature that affects the interactions between the cells and particles. For the spheres, attachment from any point makes no difference because of their symmetric character. However, the nonspherical particles may contact cells with different angles. Another reason for the favorable internalization of bowl-like capsules may be the different energy needed for internalization. As indicated in Figure S3, internalization of both bowl-like and spherical
Figure 3. SEM images of SMCs after being incubated with bowl-like (A) and spherical (B) (PAH/PSS)10/BSA microcapsules for 3 h. The insets in A and B are the corresponding magnified pseudocolored SEM images showing details of the interaction between cells and capsules. (C) SEM image of SMCs after being incubated with bowl-like (PAH/PSS)10/BSA microcapsules for 1 h. The capsule-to-cell ratio was 40:1. The samples were prepared by critical point drying. 11212
DOI: 10.1021/acsami.6b02965 ACS Appl. Mater. Interfaces 2016, 8, 11210−11214
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ACS Applied Materials & Interfaces
The Key Science Technology Innovation Team of Zhejiang Province (Grant 2013TD02), and the Fundamental Research Funds for the Central Universities (Grant 2016QNA4033).
Scheme 2. Cartoon Depicting the Hypothesis That Different Contact Modes of Bowl-Like and Spherical (PAH/PSS)10/ BSA Microcapsules Govern the Cellular Internalization and Thereby the Uptake
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volume.27 Thus, the different orientations of capsules finally result in the shape-dependent internalization. In summary, the bowl-like microcapsules were fabricated by osmotic-induced invagination of microcapsules in a concentrated PSS solution. Both the bowl-like and spherical capsules maintained their colloidal stability and shape in the cell culture medium up to 7 days. The bowl-like microcapsules were internalized with a faster rate and a higher number by SMCs and macrophages than their spherical counterparts. Preferential attachment onto the cell membrane from the bend side and easier enwrapping by cell membranes are likely the major reasons enabling the uptake of bowl-like capsules over their spherical counterparts. Such results may help people to understand the role of capsule shape in the interaction with cells and provide useful guidance for the further design of more efficient carriers.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b02965. Experimental and measurement details, protein adsorption, cellular uptake kinetics, and mechanism of bowl-like and spherical capsules for RAW 264.7 cells, SEM images of interactions between capsules and RAW 264.7 cells, and cell viability and cytoskeleton organization of SMCs (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (W.T.). *E-mail:
[email protected] (C.G.). Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study is financially supported by the Natural Science Foundation of China (Grants 21374101 and 51120135001), 11213
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