One-Step Synthesis of Cagelike Hollow Silica Spheres with Large

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One-step Synthesis of Cage-like Hollow Silica Spheres with Large-through-holes for Macromolecule Delivery Shengnan Wang, Min Chen, and Limin Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11639 • Publication Date (Web): 15 Nov 2016 Downloaded from http://pubs.acs.org on November 18, 2016

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ACS Applied Materials & Interfaces

One-step Synthesis of Cage-like Hollow Silica Spheres with Large-through-holes for Macromolecule Delivery Shengnan Wang, Min Chen* and Limin Wu

Department of Materials Science and State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, People’s Republic of China

ABSTRACT: A facile, one-step method to prepare cage-like hollow silica nanospheres with large-through-holes (HSNLs) using a lysozyme-assisted O/W miniemulsion technique is presented in this paper. Tetraethoxysilane (TEOS)-xylene mixture forms oil droplets which are stabilized by cationic surfactant Cetyl trimethyl ammonium bromide (CTAB), co-surfactant hexadecane (HD) and protein lysozyme. HSNLs (with diameter of 300-460nm) with large-through-holes (10-30nm) were obtained directly after ultrasonic treatment and aging. Lysozyme can not only stabilize oil/water interface, assist the hydrolysis of TEOS and interact with silica particles to assemble into silica-lysozyme clusters, but also contribute to the formation of through-holes due to its hydrophilicity variation at different pH conditions. A possible new mechanism called interface-desorption method is proposed to explain the formation of the through-holes. To confirm the effectiveness of large-through-holes in delivering large molecules, Bovine Serum Albumin (BSA, 21×4×14 nm3) was chosen as a model guest molecule, HSNLs showed much higher loading capacity compared with common 1 ACS Paragon Plus Environment


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hollow mesoporous silica nanospheres (HMSNs). The release of BSA can be well controlled by wrapping HSNLs with a heat-sensitive phase change material (1-tetradecanol). Cell toxicity was also conducted with Cell Counting Kit-8 (CCK-8) assay to roughly evaluate the feasibility of HSNLs in biomedical applications.

KEYWORDS: hollow, Large-through-holes, silica, lysozyme, oil-in-water miniemulsion

1. INTRODUCTION Hollow mesoporous nanospheres (HMNs) have drawn particular attention in drug delivery recently for their outstanding merits,1-2 such as large specific surface area for absorption,3 large void interior for higher loading capability,4 lower dosage and toxicity due to decreased amount of carrier materials entering the body, 5 pore channels for guest materials to penetrate6 and stimuli-response drug release.7-9 However, typical HMNs usually have small pores (2-5nm in diameter), effective loading and release are hindered for large-size molecules, such as DNA, nucleic acids and proteins, since the diffusion process through pores (50nm) caused by breakage of hollow spheres. On the other hand, the N2 adsorption-desorption method and BJH calculating method are more suitable for mesopores (2-50nm), when applied in calculating macropores (>50nm), big deviation may occur. The surface area and pore volume of HSNLs are 352.8 m2g-1 and 0.71 cm3g-1, respectively.

Figure 1. N2 adsorption-desorption isotherm and pore diameter distribution curve from adsorption branch (inset picture) of HSNL with typical formulation. 10 ACS Paragon Plus Environment

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3.1. Effect of Lysozyme. Lysozyme is a hydrophilic and positively charged chain protein ((MW ≈14 000, pI≈10.8) with large amount of –NH2 and –OH functional groups, it is flexible and stable over wide temperature and pH ranges. It has 129 amino acid residues, including 8 cysteine residues, forming 4 disulfide bonds.42-43 Many researches confirmed it could stabilize buffer–TEOS interface, assist the hydrolysis of TEOS and interact with silica particles to assemble into lysozyme-silica clusters (3.3-8.5 nm), which were primary building blocks of the spherical shell.44-46 This owed to its increasing hydrophobicity with increasing pH, at values below its pI, so that it could increase access to TEOS through hydrophobic interactions and further interact with silicate species through electrostatic interactions during the growth of silica structures.47

Figure 2. TEM and SEM images of HSNLs with various amounts of lysozyme (a) 0g (b) 0.035g (c) 0.067g (d) 0.1g. 11 ACS Paragon Plus Environment


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Distortional hollow spheres with broken-like large holes on the surface were obtained in the absence of lysozyme (Figure 2a), which was similar to the phenomenon Peng et al.48 reported previously. It isn’t hard to understand because TEOS hydrolyzes fast in water and resulted silica particles are quite hydrophilic with hydroxyls, which may easily dissociate from O/W interface to water phase and lead to defects of the spherical shell since their affinity with the water/oil interfaces is determined by their surfaces’ hydrophilic/lipophilic character.49 Addition of lysozyme could obviously improve the morphology of the hollow spheres and largely reduce the large broken-like holes (Figure 2b). As mentioned previously, lysozyme is a flexible chain protein including large amount of –NH2 and –OH functional groups which can interact with silica and form lysozyme-silica building blocks in-situ. So on one hand, a bit of lysozyme could function as glue between growing structures to effectively prevent silica dissociation to water phase and facilitate integrity of the shell.47 On the other hand, it acted as skeleton of the shell, improving its flexibility to avoid deformation and breaking. However, increasing the amount of lysozyme to 0.067g, cage-like large-through-holes appeared on the surface (Figure 2c). Pores trended to be larger with further increased concentration of lysozyme, meanwhile, conglutination of HSNLs turned to be more serious (Figure 2d). When the concentration was high enough, no hollow spheres would be obtained, fragment aggregation forms instead. The large-through-hole formation mechanism and role of lysozyme are explained in detail in section 4.1.2. FT-IR and EDS mapping were employed to confirm the existence of lysozyme in the shell of the spheres. In Figure 3, the IR spectrum of lysozyme (Figure 3a) shows characteristic 12 ACS Paragon Plus Environment

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peaks of vibrations of polypeptide chain at 1655cm-1 (amide Ⅰ:C=O/C-N stretch) and 1530cm-1(amideⅡ:N-H bend/C-N stretch).47 The same peaks can be observed in HSNLs (Figure 3b) as well, but not in the sample in the absence of lysozyme (Figure 3c), while it, together with HSNLs, have the typical bands of silica at 1080 (Si-O-Si antisymmetric stretches), 970(Si-OH stretches) 780(Si-O-Si symmetric stretches) and 460 cm-1(Si-O-Si bend).50 The peak at 1469 cm-1 (CH2 bend) appears in Figure 3c due to the long aliphatic chains in CTAB, while 1633 cm-1 is most likely to be the signal of water since potassium bromide can easily absorb water when grinded with samples. This peak also exists in the IR spectrum of silica gel.47 In addition, the sulfur signal in EDS analysis displayed in Figure 4a is a diagnosis for lysozyme. The S element is distributed homogeneously in the spheres (Figure 4f), indicating the existence of lysozyme, and has a uniform distribution in the shell of HSNLs.

Figure 3. FT-IR spectra of (a) lysozyme powder (b) washed HSNLs (c) products obtained without lysozyme 13 ACS Paragon Plus Environment


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Figure 4. (a) representative results of EDS analysis of HSNLs (b) Electron image of HSNLs (c-f) Si, O, N and S elemental maps, respectively, scale bar: 500 nm.

3.2. Effect of CTAB. In this miniemulsion system, CTAB/HD and lysozyme worked together to stabilize the oil droplets and facilitate the hydrolysis and condensation of TEOS at the oil-water interface.51 Besides, hydrolyzed negative charged silica particles were attracted by CTAB molecules through electrostatic interaction to condensate and aggregate on the surface of the oil droplets. In a control experiment without CTAB, a mass of solid silica nanoparticles (around 15nm) were obtained, while a few hollow spheres appeared due to the weak emulsifying ability of lysozyme. What’s more, the concentration of CTAB had great influence on the size of through-holes. Almost no visible holes were observed at low concentration of CTAB (Figure 5a). However, obvious through-holes started to show up and became larger with increased amount of CTAB, 14 ACS Paragon Plus Environment

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simultaneously the size of the spheres trended to be smaller (Figure 5b-d, Table 1). Nevertheless, high enough concentration would destroy the sphere formation, leaving debris stacking. We explain the changes in section 4.1.1.

Figure 5. Typical SEM images for products synthesized in different amount of CTAB (a) 0.067g (b) 0.085g (c) 0.1g (d) 0.131g Table 1.

Particle size variation with different dosage of CTAB

Run

TEOS(mL)

Xylene(g)

CTAB(g)

Particle Size(d•nm)

1

5

3

0.06

396.6

2

5

3

0.085

350.7

3

5

3

0.1

319.8 15

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4

5

3

0.115

281.4

5

5

3

0.131

131.8

(Other parameters were the same as the typical formulation if unspecified. Particle size data were the peaks of number-average size distributions.) 3.3. Effect of pH. When lysozyme was added, pH had a big impact on the size of the holes. HSNLs with better morphology were obtained at pH 8.8-9, the lower of the pH, the larger of the holes. When the pH was below 8, holes were large enough to break down the shell, as a result, only fragments were observed. In contrast, visible holes disappeared as pH reached 9.5. To sum up, the size of the holes decreased with increasing pH in the presence of lysozyme, while no big difference of the morphology was found as pH changed in control experiments without lysozyme. The distinction was caused by the hydrophilicity variation of lysozyme with different pH conditions. It was closely related to the through-hole formation mechanism and their relationship was elaborated in section 4.1.3. 3.4. Effect of Xylene. Xylene served as the soft template in this O/W system, together with TEOS which was the silica precursor as well. With no or little xylene, the shells were more likely to deform resulted from the hydrolysis of TEOS. The more xylene was added, the larger the size of the HSNLs would be, along with smaller pore dimension (Figure 6).

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Figure 6. SEM and TEM images of HSNLs with various amount of xylene (a, b) 1g (c, d) 3g (e, f) 5g

3.5. Effect of TEOS and Silane Coupling Agents. As mentioned before, TEOS mainly functioned as silica precursor and being part of the soft template in the initial reaction stage. We found that increasing the amount of TEOS had little impact on the thickness of the shell



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(also proved by Peng49) but diminished the through-holes evidently (Figure 7), moreover, aging time increased.

Figure 7. TEM images of HSNLs with different volumes of TEOS (a) 5mL (b) 7.5mL (c) 10mL

Control experiments by addition of various silane coupling agents with different hydrophobicity-hydrophilicity were carried out to investigate the mechanism of through-hole formation. Notably, those (DTMS, KH-570, KH-560, PFTS) with more hydrophobic chains compared with TEOS, seem to be an obstacle of through-hole formation, while those (KH-550, APS) with more hydrophilic chains trend to enlarge the holes (Table 2). Table 2. Morphology variation with the additive of different silane coupling agents

Run

Silane coupling agent type

Structural formula

Dosage (mL)

1

DTMS

0.25

2

KH-560

0.4

3

KH-570

0.25

Morphology Hollow spheres without visible holes on surface Hollow spheres without visible holes on surface Cupped structure without visible holes on surface 18

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4

PFTS

0.1

Hollow spheres with smaller and less holes on surface

5

KH-550

0.05 (VNaOH=0)

Broken debris of hollow spheres

6

APS

0.1

Broken debris of hollow spheres

(VNaOH=0)

(VTEOS=5mL, other parameters were the same as the typical formulation if unspecified)

4. DISCUSSION 4.1. HSNLs Formation Mechanism Since cavity of silica hollow spheres are formed based on well-known soft-template mechanism, we here mainly focus on discussing the formation mechanism of large-through-holes on the shell. A possible mechanism called interface-desorption is briefly proposed. Silica precursor, TEOS, is initially mixed with xylene, forming oil droplets which are mainly stabilized by cationic surfactant CTAB, co-surfactant HD and protein lysozyme. When NaOH is added, lysozyme becomes more hydrophobic and adsorbs onto the surface of oil droplets due to its foaming effect.52 Simultaneously the hydrolysis of TEOS is accelerated by alkaline condition and lysozyme, forming silanol groups (Si-O- or Si-OH), which are more hydrophilic and tend to move to interface or the water phase to condense into polymer species.53 In the aging process, those silica nanoparticles absorbing on the interface form granular shell and becomes thicker and more complete as time consuming. With the addition 19 ACS Paragon Plus Environment


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of lysozyme, granular layers become much flexible since the lysozyme could be encapsulated into silica matrix by interacting with silica through electrostatic interaction and hydrogen bonding owing to its cationic charges and –OH groups of peptides,54 thus forming intact hollow spheres with very few breakages. For the large-through-hole formation, as mentioned above, hydrolyzed silicate species and primary silica nanoparticles are quite hydrophilic and easy to move into the water phase. There exists a competition between remaining on the interface and dissociating to the water phase, while the latter is the main cause of the formation of large-through-holes. We call it interface-desorption method. CTAB amount, lysozyme concentration, pH value and xylene dosage were verified to have great impact on the morphology of HSNLs, as revealed in Section 3, mainly due to their effects on the curvature of interface and hydrophilicity of hydrolyzed silica aggregates that led to variation of large-through-holes. Their specific roles in the formation of large-through-holes were investigated and elaborated as follows. 4.1.1. Role of CTAB. Recall the data in (Figure5 and Table 1), more CTAB means smaller oil droplets, as a result, the curvature of the surface increased. Silica granular layers were not so flexible to suit the large curvature to splice with each other, some might desorb from the surface to the water phase and leave holes on the shell. Moreover, excessive CTAB molecules on the surface could absorb on the silica aggregates and make them too compact and larger so that they were not able to stand on the interface steadily (proved by lots of researches55-58). Also those excessive free CTAB molecules in the water phase could enhance their hydrophobicity and electrical interaction to promote their desorption from the interface.59 20 ACS Paragon Plus Environment

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4.1.2. Role of Lysozyme. As described above, lysozyme is a flexible cationic chain protein which has strong interaction with silica through its functional groups (–NH2 and –OH) with silanols. When a small amount of lysozyme was introduced, lysozyme molecules were mainly encapsulated in silica matrix to function as skeleton of the shell, increasing the flexibility of silica granular layers so that they were much easier to fit the curvature of the surface and splice with each other, as a result, the morphology of HSNLs and the integrity of the shell were obviously improved by greatly diminishing the broken-like holes (Figure 2a-b). However, excessive lysozyme molecules might have counter effect. They would increase the hydrophilicity of lysozyme-silica building blocks on the interface thus some of them might be hydrophilic enough to desorb from the interface into the water phase, causing through-holes on the shell (Figure 2c). More lysozyme was added, more and larger holes would appear. On the other hand, they might connect with another HSNL and result in conglutination of HSNLs (Figure 2d). To a certain extent, conglutination would cause deformation and damage the HSNLs. The evolution with different amount of lysozyme is illustrated in Scheme 2.



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Scheme 2. Schematic illustration for different amount of lysozyme

4.1.3. Role of pH. pH mainly influences the hydrophilicity of lysozyme, which further affect the through-holes of the spheres, as explained in the role of lysozyme. When pH was low (

One-Step Synthesis of Cagelike Hollow Silica Spheres with Large

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