Silica Composite

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Intracellularly Biodegradable Polyelectrolyte/Silica Composite Microcapsules as Carrier for Small Molecules Hui Gao, Olga Goriacheva, Nadezda V. Tarakina, and Gleb B. Sukhorukov ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01921 • Publication Date (Web): 23 Mar 2016 Downloaded from http://pubs.acs.org on March 28, 2016

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ACS Applied Materials & Interfaces

Intracellularly Biodegradable Polyelectrolyte/Silica Composite Microcapsules as Carrier for Small Molecules Hui Gao †, Olga A. Goriacheva ‡, Nadezda V. Tarakina † and Gleb B. Sukhorukov †, * †

School of Engineering and Materials Science, Queen Mary University of London, Mile End

Road, London, E1 4NS, United Kingdom ‡

Saratov State University, 83 Astrakhanskaya Street, Saratov, 410012, Russia.

KEYWORDS: biodegradable, polyelectrolyte, SiO2, composite capsules, small molecule delivery ABSTRACT: Microcapsules that can be efficiently loaded with small molecules and effectively release them at the target area through the degradation of the capsule shells hold great potential for treating diseases. Traditional biodegradable polyelectrolyte (PE) capsules can be degraded by cells and eliminated from the body but fail to encapsulate drugs with small molecular weight. Here, we report a poly-L-arginine hydrochloride (PARG) / dextran sulfate sodium salt (DEXS) / silica (SiO2) composite capsule that can be destructed in cells and of which the in situ formed inorganic SiO2 enables loading of small model molecules, Rhodamine B (Rh-B). The composite capsules were fabricated based on the layer-by-layer (LbL) technique and the hydrolysis of tetraethoxysilane (TEOS). Capsules composed of non-degradable PEs and SiO2, polyllamine

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hydrochloride (PAH) / poly(sodium 4-styrenesulfonate) (PSS) /silica (the control sample), were prepared and briefly compared with the degradable composite capsules. Intracellular degradation study of both types of composite capsules revealed that PARG/DEXS/silica capsules were degraded into fragments and lead to the release of model molecules in a relatively short time (2 hours) while the structure of PAH/PSS/silica capsules remained intact even after 3 days incubation with B50 cells. Such results indicated that the polymer components played a significant role in the degradability of the SiO2. Specifically, PAH/PSS scaffolds blocked the degradation of SiO2. For PARG/DEXS/silica capsules, we proposed the effects of both hydrolytic degradation of amorphous silica and enzymatic degradation of PARG/DEXS polymers as a cell degradation mechanism. All the results demonstrated a new type of functional composite microcapsule with low permeability, good biocompatibility and biodegradability for potential medical applications.

1. Introduction Most drugs being developed and marketed by the pharmaceutical industry are small molecules under 1000 Da.

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Small molecules, although in routine use as chemotherapeutic agents for

cancer treatment, have characteristics that limit their therapeutic efficacy, including a lack of water solubility, suboptimal biodistribution, and nonspecific targeting as well as low intracellular absorption.3 An effective method to circumvent these limitations is to encapsulate small drugs in proper carriers which are able to protect and deliver the drugs to the target areas and finally release them to realize their particular functions.1,4 Hence, over the past years, using capsules to load and deliver small molecules for application in anticancer therapy has been a rapidly growing area of research.4-6


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Polyelectrolyte (PE) microcapsules are widely studied and used as a drug delivery system for potential biomedical applications such as magnetic resonance imaging (MRI), due to their versatility with respect to payload, targeted delivery, and controlled release options.7-12 By using the electrostatic interactions between oppositely charged polyelectrolytes (PEs), diversified PE capsules with different sizes (20nm-5µm) were facilely fabricated with controllable architectures and properties by the layer-by-layer (LbL) assembling technique.13-16 A great advantage of these capsules is that they can be modified with different functionalities; for example, they can encapsulate a variety of active compounds and other payload with different water solubility and perform controllable release triggered by external activation with light, magnetic field, ultrasound, temperature, pH, salinity and chemicals.13,14,17,18 These excellent merits facilitate their applications in medical areas. However, the encapsulation of small molecules within the PE-shelled capsules still presents a significant challenge because of the shell’s high permeability.19 Over the past few years, various methods to reduce the PE shell’s permeability have been reported. One approach is to conjugate the small molecules such as cancer therapeutic doxobicin hydrochoride (DOX) to the assembly material so as to bypass their diffusion through the multilayers.20 Christopher et al. reported the LbL assembly and click stabilization of poly (Lglutamic acid) capsules to which the anticancer drug DOX was covalently conjugated through an amide bond. The release of DOX occurred through enzymatic degradation of the polypeptide backbone.20 The major problem of this method is that the loading efficiency is limited. Using an additional lipid coating on the outer surfaces of PE-shelled capsules is another way to reduce the shell permeability, but lipids are unstable under high temperature situations, restricting them to only low temperature usage. Very recently, we reported an innovative way to seal small molecules efficiently by modifying PE capsules with in situ formed inorganic nanoparticles, the


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as-prepared composite shells showing an effective interception of small molecules and a strong ultrasound sensitivity.19 Besides the ability of loading small molecules (≤1000 Da), another issue of carriers that must be considered for biomedicine usage is their biocompatibility. Ideally, the employed capsules should be not toxic to cells and their fragments can be safely removed from the body after they release the drugs.6,21-23 Using drug carriers composed of biocompatible and biodegradable materials are a promising choice. Indeed, the development of LbL microcapsules that could be spontaneously decomposed by cells to let the drugs released is still under-researched, even through various stimuli-responsive microcapsules have already been widely reported.24-25 Biodegradable polyelectrolytes such as oppositely charged polysaccharide-coupled polyamino acids, polypeptides, or polysaccharose were used to synthesize enzymatically degradable PE capsules which could be spontaneously decomposed and gradually eliminated by living cells.26-29 This kind of PE capsules have been extensively investigated in recent years with the scope of degradation of shells and release of water-soluble macromolecules from them; a typical example is their employment for the delivery of genes, growth factors, and vaccines.29-33 However, as we mentioned above, these LbL PE capsules failed to encapsulate small molecules due to the intrinsic high permeability of the PE multilayers, which greatly limited their practical application in medical fields.19,34 Hence, in order to reduce shell permeability for loading small molecules and synchronously remain biodegradation property, a further modification of these biodegradable PE capsules with other biodegradable materials is of particular significance. Inorganic nanoparticles (NPs) can be used as modifying agent to effectively tune the physical and chemical properties of PE capsules at the nanoscale.19,35-36 SiO2 NPs are among the most abundant and widely used synthetic materials applied in drug delivery systems, as they are


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generally accepted as to be biocompatible and nontoxic to cells.22,23,37,38 Our previous work demonstrated the in situ formation and assembly of silica NPs inside or on the non-degradable PE shell surfaces based on the hydrolysis of TEOS produced innovative PE/SiO2 composite capsules with lower permeability.19 Such reduced permeability arises from the consumption of water in polymer shells and the formation of robust SiO2 nanoparticles to block the pores during the hydrolysis of TEOS. Furthermore, interfacial complexation of polyelectrolytes and the amorphous silica via electrical interaction, hydrolysable hydrogen bonding, and van der Walls forces also enables the produced composite capsules with reduced permeability.19,39-41 After the modification of in situ formed silica NPs, the obtained composite capsules were confirmed to be able to load small molecules inside their cavities with high efficiency. In addition, as reported before, amorphous silica can be dissolved and degraded in biological systems and the biodegradation by-product of silica is the nontoxic silicic acid that is naturally found in numerous tissues.22,23,38 Silicic acid can act as a source of Si and is useful for the formation of connective tissue such as bones.38 The excess silicic acid in the human body can be efficiently excreted from the body through the urine.42 It is also worth mentioning that the hydrophilic surface of the SiO2 NPs is favourable for cellular uptake. Therefore, microcapsules composed of biocompatible and biodegradable PEs with a properly engineered silica coating are expected to combine the merits of biodegradability and small molecule encapsulation together, and such composite capsule is promising for the encapsulation and delivery of small drugs into cells. Here, we explore the possibility of fabricating biodegradable composite polyelectrolytes/silica capsules with potential for the encapsulation of cargos with small molecular weight (≤1000 Da). Enzymatically degradable LbL capsules were made with positively charged poly-L-arginine hydrochloride (PARG) and negatively charged dextran sulfate sodium salt (DEXS), as already


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reported previously.17,32,33 For comparison, we also prepared non-degradable ‘control capsules’ by coating CaCO3 particles with polyallylamine hydrochloride (PAH) and poly(sodium 4styrenesulfonate) (PSS).19 An additional in situ silica coating is applied to both type of PE capsules to seal a small water-soluble model drug, Rodamine B (Rh-B). Then the morphology and structure of the obtained polymer-based silica capsules are investigated by means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). To confirm that the biopolymer/silica capsules are suitable for bio-related applications, firstly their cytotoxicity is studied and benchmarked against the pure PARG/DEXS multilayer assembly which was previously investigated for intracellular drug delivery.26 Their degradation in B50 cells was monitored by the confocal laser scanning microscopy (CLSM). We believe that, after internalization by cells, capsule degradation may lead to the release of the embedded molecular cargo to the cytosol and consequently stain the cells. In order to demonstrate this proposed ideas powerfully, we studied two types of model drugs, small molecules (Rh-B) and large molecules (TRITC-dextran). 2 Experimental Section 2.1 Materials. Tetraethyl orthosilicate (TEOS, Si-(OC2H5)4), ammonium hydroxide solution (NH4OH), poly(allylamineydrochloride) (PAH, Mw = 56 kDa), poly(sodium 4-styrenesulfonate) (PSS, Mw = 70 kDa), poly-L-arginine hydrochloride (PARG, Mw = 15-700 kDa), dextran sulfate sodium salt (DEXS, Mw = 10 kDa), dextran-tetramethylrhodamine isothiocyanate (TRITC-dextran, Mw = 500 kDa), rhodamine B (Rh-B, Mw = 479), ethylenediamine-tetraacetic acid disodium salt (EDTA) and other salts were purchased from Sigma-Aldrich. Materials used for cell culture and cell viability studies including Dulbecco’s modified eagles media (DMEM),


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fetal bovine serum (FBS), penicillin, streptomycin, trypsin, thiazolyl blue tetrazolium bromide (MTT) and methyl sulfoxide (DMSO) were also supplied by Sigma. 2.2 Polyelectrolyte capsule synthesis. Calcium carbonate (CaCO3) cores and polyelectrolyte microcapsules were prepared as reported elsewhere.19 Briefly, a 0.33 M CaCl2 solution was mixed under magnetic stirring with the same volume of a 0.33 M Na2CO3 solution. After 30 s, the CaCO3 particles were then collected and washed three times with deionized water (resistivity 18.2 M Ω cm). The encapsulation of TRITC-dextran was carried out similarly as described above for the synthesis of plain CaCO3 particles by coprecipitation of them with the CaCO3 during the particle formation. Polyelectrolyte microcapsules were assembled on sacriﬁcial CaCO3 templates via the layer-by-layer method. As reported before, enzymatically degradable LbL capsules were made with PARG (positively charged, 1mg/mL in 0.5 M NaCl) and DEXS (negatively charged 1mg/mL in 0.5 M NaCl.26 Four bilayers of polyelectrolytes were assembled, resulting in (PARG/DEXS)4 microcapsules. Non-degradable capsules (PAH/PSS)4 were also made by assembling four bilayers of PAH (positively charged) and PSS (negatively charged) using as-prepared PAH and PSS solution with a concentration of 2 mg·mL−1 in 0.2 M NaCl. After that, hollow microcapsules were obtained by dissolving the core templates in 0.2 M aqueous EDTA. Then they were washed 3 times with pure water (resistivity 18.2 M Ω·cm) and 3 times with ethanol and finally dispersed in ethanol. 2.3 In situ synthesis and incorporation of silica. Similar to the method we reported previously[19], the PE capsules were dispersed in 6 mL of ethanol and 330 µL of ammonium hydroxide solution together with 1660 µL of H2O, keeping them in a water bath at 50 oC. Under magnetic stirring at 700 rpm, 46 µL of tetraethyl orthosilicate was added. The nucleation and growth of the silica nanoparticles were accelerated at 50 oC for 30 minutes. After this, the


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solution was stirred at a steady speed of 500 rpm for 20 h at a lower temperature (25 oC). Finally the suspensions were centrifuged and washed by ethanol and distilled water for several times. Two different PE/silica composite shell had formed, i.e., (PAH/PSS)4-SiO2 and PARG/DEXS)4-SiO2. The encapsulation of Rh-B was realized during the synthesis, assembly and condense process of silica NPs. 400 µg Rh-B were firstly mixed with 6 mL ethanol and a certain amount of polyelectrolyte capsules. After 1 hour stirring at a steady speed of 500 rpm, the deposition of silica was carried out in the same procedure as described above. 2.4 Cell Culture and Cell Viability Test. Rat neuroblastoma cells B50 Cells were cultured in the DMEM supplemented with 10% FBS and penicillin-streptomycin (1%) containing 5% CO2 / 95% air at 37 °C. For cell viability studies, B50 cells were plated at 20000 cells per well on 96 well plates. The next day microcapsules were added at ratios of 10, 50 and 100 capsules per cell to triplicate wells. The experiment was terminated later when total incubation times reached 24, 48 and 72 hours for respective wells. Then 100 µl MTT solution (5 mg·mL−1 in cell culture medium) was added to each well, plates were briefly shaken and then incubated for 3 hours before DMSO was added. Finally cell viability after co-culturing with capsules for 24-72 hours was assessed (compared with the control) and read by a BMG Fluostar Galaxy plate reader. It is worth mentioning that hemocytometer (a counting chamber) was used for determining the number of cells or microcapsules per unit volume of a suspension. The original cell and microcapsule suspensions were diluted low enough for counting on hemocytometer under optical microscopy, and the concentration of these original suspensions were carefully calculated based on the dilution factor and the obtained number of cells or capsules per unit volume. Then the cell/capsule ratios can be easily adjusted and controlled in the experiment.


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2.5 Instrument and Measurement Scanning Electron Microscopy (SEM). The capsule morphologies were observed by SEM (FEI Inspect-F) using an accelerating voltage of 10 kV. The dried microcapsules were coated with gold before observation. Energy dispersive X-ray spectroscopy (EDX). Elemental analysis was performed by an EDX Oxford INCA X-ray detector attached to the SEM, operating at an accelerating voltage of 20 kV. Transmission electron microscopy (TEM). TEM measurement were carried out by using a JEOL 2010 transmission electron microscope with LaB6 filament, operated at 200 kV. Samples were prepared by dropping the diluted microcapsule suspension on a copper grid with holey carbon film and letting it dry for 5 minutes. Confocal laser scanning microscopy (CLSM). For the study of the biodegradable behavior of capsules, confocal laser scanning microscopy (CLSM) images were recorded with a Leica TS confocal scanning system (Leica, Germany) equipped with a 63 × /1.4 oil immersion objective. BMG Fluostar Galaxy. Cell viability was assessed via a standard MTT assay and examined by a BMG Fluostar Galaxy plate reader. 3 Results and Discussion 3.1 Silica capsule formation and characterization


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Figure 1 Schematic representation of polyelectrolyte/silica microcapsules’ encapsulation and release of small molecules. Grey and black lines represent biodegradable PEs, the red represents in situ formed silica, and the pink represents small molecules. The freshly synthesized CaCO3 microparticles were used as templates for the formation of PE microcapsules. The prefabricated PE capsules were washed with ethanol and then transferred into a mixed solution of ethanol/ammonia hydroxide/H2O. In this solution, under continuous magnetic stirring, PE capsules could be directly coated with silica upon addition of TEOS which then hydrolyzed and condensed, forming a concrete-like and strengthened shell. The structural and schematic diagram is shown in Figure 1. In fact, the PE capsules dispersed in the precursor solution acted as foreign substrates, which may well promote the heterogeneous nucleation on their surfaces.19,43 Upon adding TEOS to the system, SiO2 nucleus would initially form within the flexible PE layers as well as their surfaces through heterogeneous nucleation (Figure 1), especially at the positions with low energy, for example, the concave points and pores inside the


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shells.19 Once the capsule pores and surfaces were completely covered by silica NPs, further nucleation and growth could continue on the formed composite shell surfaces if both TEOS and H2O still exist in the solution, because the formed shell surface with a nanoscale roughness is available for further material deposition.19,43 As a consequence, the silica NPs were firmly mixed, combined and concreted together with PEs. However, without this typical Stöber silica coating, soft PEs are entangled together forming a soft shell with no ability to intercept small molecules.19,44 They can be easily washed out. In contrast, the composite shells with silica coating are denser and thick enough to seal the small payloads. Such points were demonstrated previously for a non-degradable PE capsule system.19 In addition, if all the used building materials are biodegradable, the formed capsules should be able to be spontaneously degraded by the living cells to let the cargo release and their fragments could be removed from the body. Here, biodegradable PEs, PARG and DXES, were used to assemble into (PARG/DXES)4 capsules, and then coated them with silica NPs.


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Figure 2 SEM and TEM images of (PARG/DEXS)4 capsules before (A, B) and after silica coating (C-F). Firstly, we studied whether the silica NPs were in situ synthesized and embedded into (PARG/DEXS)4 capsules. Figure 2 shows the SEM and TEM images of (PARG/DEXS)4 capsules without and with silica coating. It is clearly seen from the SEM data that silica coating has a great impact on PE capsule morphology. The collapsed flat (PARG/DEXS)4 capsules were strengthened into free-standing composite capsules, and the obtained composite capsules displayed relatively rough surfaces with loads of NPs, Figure 2D and F. Energy dispersive X-ray (EDX) spectrum of the as-prepared composite capsules (Figure S1, Supporting Information) confirmed the formation of SiO2 in PARG/DEXS multilayers, which also indicates that the morphology change was originated from the additional silica incorporation. The amount of SiO2 in the resulted composite capsules is around 40 wt%, which was estimated from the thermogravimetric analysis (TGA) data (Figure S1 B, Supporting Information). Specifically, in contrast to the rather thin pancake-like structure of the pure PE capsules (Figure 2A and B), the composite capsules showed a robust and free-standing structure in the absence of a huge amount of silica NPs (Figure 2C and D), indicating that their mechanical strength increased significantly by incorporation with the in situ formed SiO2 NPs.45 Besides, the images in Figure 2C, D and E also verified that, by the well-controlled hydrolysis of TEOS, the PARG/DEXS multilayers were fully covered by silica NPs and no excess free silica particles was observed along with the composite capsules. And all of the SiO2 NPs were compactly concrete together with the soft PE multilayers with a uniform distribution, Figure 2D and F. Furthermore, TEM data in Figure 2E and F confirmed their hollow structures and an increased shell thickness reaching around 128 nm making capsules strong enough to support themselves upon drying. The inset diffraction pattern


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revealed that the silica NPs are amorphous, as no bright diffraction spots or rings were observed.46 These results are consistent well with our previous results for the PAH/PSS capsule systems. 3.2 Encapsulation of cargos The encapsulation of various molecules in the inner cavity of the composite capsules was successfully carried out in a similar way as loading them in LbL PE capsules. Large molecules such as TRITC-dextran (500 kDa) can be easily encapsulated via coprecipitation with CaCO3 cores. Due to the large size of such molecules, most of them can not diffuse out of the capsules in the CaCO3 dissolution process by EDTA. So there is no doubt that TRITC-dextran can be sealed inside the capsules with an additional silica coating. The red fluorescent signal was observed with a main distribution around the inner shell of the composite capsules, Figure 3B, due to the attachment of dextran to the amino-terminated inner surface of the microcapsules.47 Actually this type of attachment can be facilitated by both hydrogen bonds and hydrolyzable covalent cross-links resulting from aldehydes and primary amines coupling.47 In order to investigate whether it is possible to encapsulate small drugs, Rh-B was used as a model molecule. In this case, it was necessary to load them inside the capsules during the silica synthesis and assembly process rather than during the precipitation process of CaCO3 cores.19 Because Rh-B inside CaCO3 cores diffused to the outside during the subsequent washing processes, demonstrated by the obtained pink templates changing to white before assembling the PEs layers (data not given). Without silica sealing, it was impossible to contain the water soluble Rh-B inside the (PARD/DEXS)4 capsules for a reasonable time due to the intrinsic high permeability of PE multilayers. This was also observed for another PE capsule system.19 Attributed to the compression and condensation effects arising from the in situ formation of


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silica NPs, the composite shell became more dense and compact, showing a reduced size and increased shell thickness (from a few nanometers to more than 100 nm). The formation, growth and assembly of rigid silica inside the PE multilayers can block the possible diffusion or escape path for the cargos.19 Thus, with proper control of the in situ silica formation and incorporation processes, Rh-B molecules were sealed inside the obtained composite capsules, as shown in Figure 3A. These two ways to encapsulate cargo show that it is possible to load the composite capsules with a library of molecules with different molecular weights. With silica on surfaces, it is worth mentioning that some composite capsules appeared slight aggregation (Figure 3A), which might be due to hydrogen bonding effect between silica nanoparticles.48

Figure 3 Confocal laser scanning microscopic (CLSM) images of PARG/DEXS /silica composite capsules loading with cargos: (A) Rh-B; (B) TRITC-dextran. The inset are line scan images On the other hand, the encapsulation of payload in PARG/DEXS /silica capsules was compared in our work with the encapsulation by another well studied non-degradable polyelectrolyte system (PAH/PSS) which is commonly investigated for encapsulation applications. The PAH/PSS capsules and their silica coating were prepared following the same procedure. The corresponding SEM and TEM images are given in Figure S2 (Supporting


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Information), showing a similar morphology and structure with that of the PARG/DEXS capsule system. The images also indicated that the PAH/PSS/silica capsules were rigid while the pure PE capsules folded and collapsed. The successful encapsulation of TRITC-dextran and Rh-B inside PAH/PSS/silica was demonstrated by CLSM measurement, seen images in the Figure S3 (Supporting Information). 3.3 In vitro cytotoxicity test.

Figure 4 Cell viability of B50 cell line mixed with different concentration of PAH/PSS, PAH/PSS/silica, PARG/DEXS, PARG/DEXS /silica capsules for (A) 24 h, (B) 48h and (C) 72 h at 37°C respectively as measured by an MTT assay compared with the control. The error bars show the standard deviations.


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Before studying the biodegradation behaviour of the four different capsules, i.e. PAH/PSS, PAH/PSS/silica, PARG/DEXS, PARG/DEXS/silica, an in vitro cytotoxicity study was carried out for B50 cells at different concentrations of capsules/cell (10, 50, 100) for 24 h, 48h, and 72h as shown in Figure 4. The pre-fabricated microcapsules with various compositions were dispersed in cell culture medium and cocultured with the B50 cells, after which the cell viability was measured by MTT colorimetric assay at different selected time point.49 The results indicated that all the investigated compositions evidently possess low cytotoxicity at concentrations equal or below 50 capsules/cell during the first 24h, showing the cell viability more than 80% (Figure 4A). For both the normal and the degradable PE systems, capsules with silica coating presented a higher cell viability than that without silica incorporation, indicating that amorphous silica is not toxic to cells. With the concentration at 100 capsules/cell, the cell toxicity of PAH/PSS capsules was significantly higher than other 3 types of capsules, displaying a value of 66%, Figure 4A. This might due to a few reasons: (1) compared with PARG/DEXS multilayers, cells are in less favour of PAH/PSS ones; (2) for capsules with same polymeric scaffold, silica coating improves their biocompatibility effectively. Nevertheless, the concentration-dependent toxicity behavior of PAH/PSS/silica was different to that of plain PAH/PSS capsules. The cell viability of PAH/PSS/silica increased when increasing the ratio of capsules/cell, suggesting that the observed toxicity is only due to the presence of non-degradable capsules in solution, which further confirms the stability and excellent biocompatibility of capsules with silica coating in cell systems. After the next 24 h, the MTT assay revealed that the cell viability increased by a great amount (≥90%) for all cells except that cocultured with PAH/PSS capsules at high ratio (100). Practically, various factors including the composition of capsules, capsule concentration and the obtained debris degraded or decomposed by cells determined the final proliferation and death


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rate of cells. These factors interacted among each other and leaded to such specific phenomenon. Cells prefer to reject any external materials initially and they need time to get used to the exotic. It is worth mentioning that PAH/PSS/silica capsules displayed the best cell viability, which means that the B50 cells already get used to these capsules with silica coating and show good cell proliferation. Cell viability of biodegradable PE capsules with silica was not as good as that even though it still remained around 100%, which might because a certain amount of nontoxic silicic acid produced by the hydrolysis of silica NPs slightly inhibited the cell proliferation process by decreasing local extracellular or intracellular pH.23 Indeed the amount of silicic acid in the system would affect the pH value so as to the final cell viability. Capsules with silica of 48h showed lower toxicity than that of 24h might because that the outmost silica layer on the composite capsules was hydrolyzed quickly in the first 24h and consequently produced more silicic acid, while the silica concreted and entangled with soft polymers was more difficult to be hydrolyzed and resulted in less acid products. Interestingly, after 72h of incubation the cell viability of different capsules dropped below 70% for all concentrations except that of the biodegradable PARG/DEXS/silica composite capsules (around 80% or above), revealing that PARG/DEXS/silica capsules were the most biocompatible. Therefore, the above data and analysis clearly point out that microcapsules composed of biodegradable PEs and amorphous silica have no major negative effect on cell viability. 3.4 Degradation of PE/silica composite capsules.


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Figure 5 CLSM images of B50 cells cocultured with the PARG/DEXS/silica composite capsules with Rh-B sealed inside their cavities. (A) 2h; (B) 7h; (C) 32h; (D) 72h. When the capsule shell is raptured during in vitro degradation processes, the fluorescent payload molecules should be released into the cytosol and thusly dye the cells. In view of the above observation on cell viability, the subsequent in vitro cell degradation experiments utilized a microcapsule : cell ratio of 20 : 1. Rh-B was employed as a model small molecule cargo which


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was sealed inside the composite capsules with both types of PE building blocks. The CLSM images in Figure 5 revealed a gradual degradation of PARG/DEXS/silica capsules loaded with Rh-B, which confirmed that the PARG/DEXS/silica capsules are intrinsically degradable. The B50 cells were stained into red within only two hours, revealing that Rh-B molecules released outside quickly from the composite capsules as a consequence of the possible intracellular degradation of silica composite shells. More capsules were degraded into very small fragments (as indicated by the arrows) and the fluorescence intensity of the cells increased gradually with prolonging the incubation time. When time reached 72 hours, no intact composite capsules could be found, but only small red dots were observed. In addition, we studied whether PARG/DSS/silica capsules degraded when incubated them at 37oC in the cell culture medium. CLSM images in Figure S4 (Supporting Information) demonstrated a good stability of PARG/DSS/silica capsules, as they still showed the Rh-B filled and intact balls after 24h incubation. Such data revealed that the PARG/DEXS/silica capsules were not destructed by the cell culture media but probably by the intracellular effects.


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Figure 6 CLSM images of B50 cells cocultured with the PAH/PSS/silica composite capsules with Rh-B sealed inside their cavities. (A) 2h; (B) 8h; (C) 33h; (D) 72h. Many previous reports revealed that amorphous silica could be degraded by cells and finally removed from the body through liver and spleen (particle size above 10 nm) into bile and feces or via renal clearance (

Silica Composite

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