The impact of alkyl chain length

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

A concentration-dependent insulin immobilization behavior of alkyl modified silica vesicles: The impact of alkyl chain length Jun Zhang, Long Zhang, Chang Lei, Xiaodan Huang, Yannan Yang, and Chengzhong Yu Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00377 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

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A concentration-dependent insulin immobilization behavior of alkyl modified silica vesicles: The impact of alkyl chain length Jun Zhang,† Long Zhang,† Chang Lei, Xiaodan Huang, Yannan Yang and Chengzhong Yu* Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia. KEYWORDS: silica vesicles, hydrophobic modification, alkyl chain length, protein enrichment, mass spectrometry

ABSTRACT: In this study, the insulin immobilization behaviors of silica vesicles (SV) before and after modification with hydrophobic alkyl –C8 and -C18 groups have been studied and correlated to the grafted alkyl chain length. In order to minimize the influence from the other structural parameters, monolayered -C8 or -C18 groups are grafted onto SV with controlled density. The insulin immobilization capacity of SV is dependent on the initial insulin concentrations (IIC). At high IIC (2.6-3.0 mg/ml), the trend of insulin immobilization capacity of SV is SV-OH > SV-C8 > SV-C18, which is determined mainly by the surface area of SV. At medium IIC (0.6-1.9 mg/ml), the trend changes to SV-C8 ≥ SV-C18 > SV-OH as both the surface

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area and alkyl chain length contribute to the insulin immobilization. At an extremely low IIC, the hydrophobic-hydrophobic interaction between the alkyl group and insulin molecules plays the most significant role. Consequently, SV-C18 with longer alkyl groups and the highest hydrophobicity show the best insulin enrichment performance compared to SV-C8 and SV-OH, as evidenced by an insulin detection limit of 0.001 ng/ml in phosphate buffered saline (PBS) and 0.05 ng/ml in artficial urine determined by mass spectrometry (MS).

Introduction Hydrophobically modified silica nanomaterials have tremendous applications in catalysis,1-2 separation,3 nanomedicine4-7 and biomolecule sensing/detection.4, 8-9 The hydrophobic moieties modified onto hydrophilic siliceous frameworks enhance the hydrophobic/adhesive interactions towards hydrophobic guest molecules, showing great potential especially in the immobilization and controlled release of hydrophobic organic or bio-molecules.10-11 The so-called postsynthesis, or grafting method, is usually selected as a facile strategy to modify the surface of silica nanomaterials with alkyl groups.12 Compared to small organic molecules, the immobilization of biological macromolecules by hydrophobic mesoporous silica nanoparticles (MSNs) has drawn much attention with promising application potential in cancer therapy,13-14 gene delivery/transfection,6,

14-15

vaccination16-17 and biomolecule detection.4,

8

For example,

MSNs modified with octadecyl (-C18) groups show much higher loading capacity of Ribonuclease A and survivin siRNA, as well as higher intracellular delivery efficacy to cancer cells.13, 18 It is also shown that the antigen uptake and presentation is significantly enhanced by C18 modification of MSNs with high adjuvant potency.16 The outstanding biomolecule


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immobilization capacity of alkyl group modified siliceous nanomaterials has also been utilized for sensitive detection. Lv et al. has reported octyl (-C8) modified CeO2/SiO2 fibers for the selective capture and sensitive detection of low-abundance peptide.19 Thus, understanding of the fundamental relationship between alkyl group modification and immobilization performance of host materials is of importance for various applications. To date, many research efforts have been devoted to identifying the optimized alkyl group (e.g., from different chain length) with the best immobilization capacity. In a chromatography study, silica capillaries were modified with methyl (-C1), -C8 and -C18 groups, and -C18 with the longest chain showed the best extraction efficiency due to strongest interaction with nonpolar compounds.20 In another study, MCM-41 modified with a shorter alkyl chain (-C4) showed better chromatographic performance than –C8 modified counterparts when the surface coverage (or density) of alkyl groups was considered.21 In a drug loading study, SBA-15 materials modified with hydrophobic -C8 or -C18 groups have been used to adsorb ester molecules.22 It is revealed that the longer -C18 groups are beneficial for enrichment of esters. In comparison, Doadrio et al. find out SBA-15 materials modified with longer -C18 groups in toluene show no significant difference in loading while slightly faster release of a macrolide-type antibiotic than the -C8 modified counterparts.23 The contradictory findings in the above two studies might be attributed to their experimental designs where only the impact of alkyl chain length was investigated. The influence of alkyl chain length on biomacromolecule immobilization has also been reported for various host materials. Wang et al. modified magnetic Fe3O4 nanoparticle with different alkyl length (C3-C18) for lipase immobilization.24 Shakeri has reported a similar study using mesocellular foam.25 However, no significant difference in lipase immobilization capacity can be observed for various alkyl chain length. This is because in addition to the alkyl chain


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length, there are other structural parameters that contribute to the immobilization behaviors of guest molecules, such as the surface area and grafted alkyl density.21 The hydrophobicity of alkyl modified silica nanomaterials also depends on the number of organosilane units per reacted silanol group.26 To investigate solely the contribution from the modified alkyl chain length, the other structural parameters should be controlled and comparable. In this regard, there are few reports that can meet this criterion in studying the impact of alkyl chain length on protein immobilization behaviors. Both alkyl chlorosilanes and alkyl alkoxysilanes have been widely used to modify alkyl groups to the surface of MSNs.12 By using trimethylchlorosilane, trimethylsilyl groups can be grafted on the surface of MCM-41.27 It is revealed that a dehydration process increases the grafted density. Furthermore, for trimethylsilyl groups with a projected area of 0.43 nm2, the maximum grafted density achieved is 1.9 per nm2. When using alkyl chlorosilane to modify the surface of MSNs, a monolayer of alkyl groups can be easily obtained.28-30 The grafted density of alkyl groups can be controlled by synthetic parameters, such as the amount of alkyl chlorosilane and the nature of solvent. Yasmin and coauthors report on the surface modification of hydrated MCM-41 with alkyltrimethoxysilane. With abundant amount of alkyltrimethoxysilane in the modification process, –C4 and –C8 groups can be modified with the density of 0.19-2.06 µmol m-2 (equals to 0.11-1.24 per nm2).21 Based on DFT calculations, some researchers believe that the presence of hydration pre-organizes the alkoxysilane molecule to build homogeneous monolayers on the silica surface.31 In Garcia's study, 2-4 alkyltrimethoxysilane molecules can react with one surface silanol group in hydrated aqueous layers of the silica surface, resulting in multilayered alkyl modification.26 So far, a technique to finely control the chain length, grafted density of the alkyl


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group and the alkyl alkoxysilane condensation behavior on the surface of MSNs is still a challenge. In the present work, SV with a hollow spherical morphology are selected as a simple model to investigate the influence of alkyl chain length on protein immobilization. Using a modified grafting method, hydrophobic alkyl –C8 or –C18 have been modified onto the surface of SV. By pretreating SV with moderate dehydration process and selecting non-polar toluene as the grafting medium, the grafted alkyl groups are controlled to be in a monolayer as the alkyltrimethoxysilane molecules react only to the surface silanol groups (Scheme 1A). With abundant amount of alkyltrimethoxysilane, the maximum grafted alkyl density is ~1.2 nm-2, which is determined by the projection area of alkyltrimethoxysilane molecules (Scheme 1B). SV with the same density of monolayer –C8 and -C18 are selected for immobilization of insulin in comparison with SV without any modification (named as SV-OH). At a high IIC (2.6-3.0 mg/ml), the surface area of SV dominates their insulin immobilization capacity, leading to a trend of SV-OH > SV-C8 > SV-C18. At medium IIC (0.6-1.9 mg/ml), the immobilization trend changes to SV-C8 ≥ SV-C18 > SV-OH as both of SV surface area and alkyl chain length contribute to the insulin immobilization. At an extremely low IIC of 0.001-0.05 ng/ml, the contribution of the alkyl chain length to insulin immobilization is dominant, when the influence from the surface area is minimized. SV-C18 with a longer alkyl chain length and the highest hydrophobicity show the best insulin enrichment performance with the insulin detection limit of 0.001 ng/ml in PBS and 0.05 ng/ml in artficial urine by MS. The knowledge gained from this work is essential for designing advantageous nanomaterials with the optimized protein immobilization capability for various applications.


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Scheme

1.

Post-modification

of

silica

vesicles

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(SV)

with

alkyl

group

using

alkyltrimethoxysilane (A). Monolayer grafting of alkyltrimethoxysilane leads to alkyl group grafted on the surface with a fixed density determined by the projected area of alkyltrimethoxysilane (B).

Experimental Section Chemicals. Block copolymer EO39BO47EO39 [commercial name B50-6600, EO = poly(ethylene oxide), BO = poly(butylene oxide)] was received from Dow Company. Human insulin (Mw 5807.6), tetraethyl orthosilicate (TEOS, ≥98%), sodium sulfate (Na2SO4, anhydrous), acetonitrile (ACN, anhydrous), acetic acid glacial (HAc, ≥ 99.85%) noctadecyltrimethoxysilane (ODMS) and n-Octyltrimethoxysilane (OTMS) were purchased from Sigma-Aldrich. Sodium hydroxide (NaOH), hydrochloric acid (HCl), sodium acetate (NaAc, anhydrous) and ethanol were received from ChemSupply. Anhydrous toluene was purchased from Merck. Deionized water (DI water, 18.2 mΩ cm) used for all experiments was obtained from a Milli-Q system (Millipore, Bedford, MA). The other reagents were of analytical reagent grade. Synthesis and alkyl modification of silica vesicles. The synthesis procedure of SV is reported in literation.34 0.5 g of surfactant EO39BO47EO39 was dissolved in 30 g of pH = 4.7 NaAc-HAc


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buffer solution ([NaAc] = [HAc] = 0.40 M) as the structure directing agent. 0.852 g of Na2SO4 (0.20 M) was added to the above solution under stirring at 10 °C overnight to form a homogenous solution. To the above mixture, 3.33 g of TEOS was added with continuously stirring for 24 h. The reaction mixture was then removed to an autoclave for hydrothermal treatment at 100 °C for another 24 h. The precipitates were filtered, repeatedly washed with DI water to remove the added salts, and then dried in air. The as-synthesized product was treated in calcination at 550 °C in air for 5 h to remove the surfactant and stored in the open air. Before the alkyl modification, the SV samples were treated with a moderate dehydration process by baking at 50 °C in a vacuum oven for 7 h. SV were then modified with either hydrophobic –C8 or –C18 groups by a grafting method using OTMS or ODMS as the hydrophobic silica source. In a typical procedure of grafting, 48 mg of calcined SV and 6 ml of the reaction medium were added to a 50 ml three-neck flask. The mixture was then stirred for 6 h before adding OTMS or ODMS. In this experiment, a series of alkyltrimethoxysilanes:silica molar ratio (φ) has been selected in order to investigate the influence of OTMS/ODMS amount on the final grafting density. Two kinds of organic solvents have been selected as the reaction medium – anhydrous toluene and ACN. When toluene is used as the reaction medium, the reaction mixture was refluxed with stirring at 110 °C for 12 h. When using ACN, the reaction mixture was refluxed with stirring at 80 °C for 12 h. The SV after modifications were then centrifuged, extensively washed with selected solvent and ethanol, and dried in a fume-hood at room temperature. The modified products are denoted as SV-Cn-A/T-φ, where n indicates the carbon chain length of the grafted alkyl groups, A or T indicates the usage of ACN or toluene as reaction medium.


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In order to compare the self-hydrolysis and condensation of alkyl alkoxysilane in different solvent, another series of experiments were carried out. 0.12 ml (0.2 v/v%) of ODMS was added to 6 ml of toluene or ACN, respectively. The mixture was refluxed with stirring at 110 or 80 °C for 24 h, respectively. Characterization. The morphologies of SV before and after alkyl modifications were observed using and JEOL JSM 7800 field-emission scanning electron microscopy (FE-SEM) operated at 0.8–1.5 kV. For FE-SEM observations, the samples were prepared by dispersing the SV in ethanol, after which they were dried on the aluminum foil pieces and attached to conductive carbon film on SEM mounts. Transmission electron microscopy (TEM) images were obtained using JEOL 1010 operated at 100 kV. For TEM measurements, the samples were prepared by dispersing and drying the SV-ethanol dispersions on carbon film on Cu grids. Nitrogen adsorption-desorption isotherms were measured at 196 °C by using a Micromeritics Tristar II system. Before the measurement the samples were degassed at either 180 °C (calcined SV samples) or 100 °C (SV after alkyl modification) overnight on a vacuum line. The total pore volume was calculated from the amount adsorbed at a maximum relative pressure (P/P0) of 0.99. The Barrett–Joyner–Halanda (BJH) method was utilized to calculate the entrance size from the desorption branches of the isotherms and the Brunauer–Emmett–Teller (BET) method was utilized to calculate the specific surface areas. Fourier transform infrared (FTIR) spectra were collected on a ThermoNicolet Nexus 6700 FTIR spectrometer. For each spectrum, 64 scans were collected at resolution of 2 cm-1 over the range 500-4000 cm-1. ζ potential measurements were carried out at 25 °C using a Zetasizer Nano-ZS from Malvern Instruments. During the ζ potential measurements, the samples were tested in dispersion with Milli-Q water.


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Alkyl group density evaluation. After hydrophobic modification, the density of alkyl groups grafted onto siliceous surface can be evaluated by difference techniques, including FTIR21, thermogravimetry27, water contact angle32 and elemental analysis.26, 33 In this work, the surface grafting density of alkyl groups was measured and calculated by an elemental analysis method.26 The carbon weight percentages of the –C8 and –C18 modified SV were determined by a Thermo Scientific FLASH 2000 CHNS/O Analyzer. The density of –C8 and –C18 group modified on the siliceous surface was calculated using the carbon weight percentage with the following equation: =

% × . × ×

× 10 ( ) (Equation 1),

where C% is the weight percentage of carbon determined by elemental analysis, NA is Avogadro's constant, NC is the number of carbon atoms per alkyl group, SBET is the BET surface area of SV-OH. Loading of insulin by hydrophobically modified silica vesicles. First of all, the insulin stock solution was prepared by dissolving insulin powder in 0.01 M HCl solution, and then the pH was adjusted to 7.0 with 5 M NaOH solution. The final concentration of the insulin stock solution was adjusted and diluted to 6.0, 5.2, 3.8, 2.8, 2.2 and 1.2 mg/ml. The loading of insulin by SV was tested by an immersion method. The SV samples (SV-OH, SV-C8-T-2, or SV-C18-T-2) were dispersed in PBS (pH 7.4) with a silica concentration of 2 mg/ml with ultrasonic treatment. 0.5 ml of SV in PBS solution containing 1 mg of SV was mixed with 0.5 ml of insulin stock solutions. As a result, the IIC in the loading experiments are 0.6, 1.1, 1.4, 1.9, 2.6 and 3.0 mg/ml, respectively. After shaking in dark at 200 rpm at 10 °C for 12 h, the mixtures were centrifuged at 20,000 rpm. To evaluate the insulin loading efficiency, the supernatant were collected and the residual insulin content was measured by using a Shimadzu UV-2450 double beam


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spectrophotometer at a wavelength of 276 nm in a 1 mm Quartz cell. The loading amount of insulin can be calculated based on the original and residual insulin concentrations and volumes. Enrichment of human insulin. Before every experiment the human insulin/PBS solution was prepared by stepwise dilution freshly. For the analysis in standard solution, 1 µl of SV (SV-OH, SV-C8-T-2, or SV-C18-T-2, 10 mg/ml) suspension was prepared and added into the 500 µl of insulin solution. The mixture was then stirred for 5 min followed by centrifugation for 10 min and the supernatant was removed. Then 1 µl of Α-Cyano-4-hydroxycinnamic acid (CHCA) matrix solution (10 mg/ml, in ACN/water/TFA, 50: 49.9: 0.1%, v/v/v) was added to the precipitate to elute the human insulin, and the mixtures were deposited on a Matrix Assisted Laser Desorption/Ionization (MALDI) MTP 384 plate. Another series of enrichment tests was performed using freshly made human insulin/artificial urine solution and SV. All the samples were analyzed on a Bruker Autoflex TOF/TOF III Smart beam. The mass spectra were obtained in the LP-PepMix mode via an accumulation of 200 laser shots at ten different sites under a laser intensity of 39 % for data collection and calibrated. Three standard peptides, Angiotensin II (Mw = 1046.5 Da), ACTH-Clip (Mw = 2465.2 Da), and somatostatin 28 (Mw 3147.5 Da) were used for calibration purposes to reduce variability. Circular Dichroism spectrum measurements. Electronic circular dichroism (CD) spectra were recorded on a Jasco J-815 spectropolarimeter (Jasco Inc., Easton, MD) at 20 °C in a 1 mm quartz cell. Raw ellipticities were converted to molar ellipticities using Equation 2: =

(deg cm2 dmol-1)

(Equation 2),

where [θ] is the molar ellipticity, θ is raw ellipticity in mdeg, C is the insulin concentration in M and l is the path length in cm. Results and discussion


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SV with a thin shell, hollow cavity and uniform structure are considered as advantageous nano-carriers for immobilization of various proteins, such as cytochrom c, ribonulcease A and antigen proteins.34-36 In this work, SV were synthesized utilizing a cooperative vesicle templating method34 and selected as a simple model to study the influence of modified surface alkyl group on insulin immobilization. The FE-SEM image of SV-OH (Figure 1A) show uniform spherical nanoparticles with the particles size of less than 100 nm. TEM image shows that SV-OH possesse hollow structure with the cavity size of ~ 40 nm and a thin wall of ~ 6 nm (Figure 1D), which is in accordance with previous report.34

Figure 1. SEM (A-C) and TEM (D-F) images of silica vesicles. (A, D) SV-OH; (B, E) SV-C8T-2 and (C, F) SV-C18-T-2.

The nitrogen sorption analysis was further utilized to measure pore structures of SV-OH. Figure 2A shows SV-OH have type IV isotherms with type H3 hysteresis loops. The adsorption branch shows a major capillary condensation step at a high relative pressure (P/P0) of ≈ 0.9.


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These phenomena show that SV-OH possess well-defined mesopores and the existence of entrance on the shells.34 The BJH pore size distribution of SV-OH derived from the desorption branch shows a peak centered at 6.7 nm (Figure 2B), indicating SV-OH have openings (also called entrances) on the shells with the size of 6.7 nm. SV-OH show BET surface area of 299 m2/g and total pore volume of 1.7 cm3/g (Table 1).

Figure 2. (A) Nitrogen adsorption isotherm plots of SV before (SV-OH) and after alkyl group modification (SV-C8 and SV-C18) in toluene; (B) BJH pore size distribution calculated from the desorption branches.

Table 1. Physicochemical properties of SV before and after alkyl modification in toluene. Particle

SBET (m2/g)

Vp (cm3/g)

PE (nm)

ζ potential (mV)

SV-OH

299

1.7

6.7

-25.0 ± 7.0

SV-C8-T-2

253

1.3

5.6

-16.8 ± 4.5

SV-C18-T-2

213

1.2

5.2

-13.7 ± 4.4

Note: SBET is the BET surface area; Vp is the total pore volume; PE is the entrance size calculated from the desorption branch; each ζ potential data represents the mean value ± SD (n=3).


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In this study, the influence of the alkyltrimethoxysilane amount to the alkyl group grafting density is investigated by tuning the alkyltrimethoxysilane:silica molar ratio (φ) in the grafting process. The 6.7 nm-sized entrances on the shells of SV-OH allow the access of alkyl alkoxysilane to the cavity, thus the internal surface of SV cavity can also be modified with alkyl groups. Table 2 shows the carbon weight percentage (C%) of SV detected by elemental analysis and the average density (S) of alkyl chain grafted on the silica surface calculated using Equation 1. SV-OH without hydrophobic modification show C% = 0, indicating the surfactant has been completely removed by calcination. When non-polar organic solvent - anhydrous toluene was used as the reaction medium for the –C8 modification, the C% and S of SV-C8-T-1 are 2.0 ± 0.1% and 0.36 nm-2 (φ= 1:7.18). When the OTMS amount increased (φ = 1:5.52), the C% and S increased to 6.1 ± 0.1% and 1.26 nm-2, respectively. However, futher increasing the amount of OTMS to φ = 1:4.31 did not increase the C% and S, resulting in similiar C% and S values of SVC8-T-3 (6.2 ± 0.2% and 1.28 nm-2) to SV-C8-T-2. A similar trend has been observed in the modification of SV with –C18 groups using ODMS. When φ = 1:5.89, the C% and S of SV-C18-T-1 were 8.9 ± 0.1% and 0.83 nm-2, respectively. If the ODMS amount increased (φ = 1:2.95), the C% and S changed to 12.1 ± 1.9% and 1.13 nm-2, respectively. Futher increasing the amount of ODMS to φ = 1:1.83 did not increase the C% and S with a maximum value of 12.3 ± 0.4% and 1.15 nm-2, respectively. Table 2. The carbon weight percentage from elemental analysis results and the average density of alkyl chain of a series of SV after alkyl modification. Sample

φ

C% (%)

S (number·nm-2)

SV-OH

-

0

0

SV-C8-T-1

1:7.18

2.0 ± 0.1

0.41


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SV-C8-T-2

1:5.52

6.1 ± 0.1

1.26

SV-C8-T-3

1:4.31

6.2 ± 0.2

1.28

SV-C18-T-1

1:5.89

8.9 ± 0.1

0.83

SV-C18-T-2

1:2.95

12.1 ± 1.9

1.13

SV-C18-T-3

1:1.83

12.3 ± 0.4

1.15

SV-C8-A-1

1:28.8

1.7 ± 0.0

0.36

SV-C8-A-2

1:14.4

6.8 ± 0.3

1.18

SV-C8-A-3

1:7.18

8.5 ± 1.2

1.76

SV-C18-A-1

1:23.5

4.3 ± 0.2

0.40

SV-C18-A-2

1:11.8

12.9 ± 0.7

1.21

SV-C18-A-3

1:5.89

18.7 ± 1.6

1.75

Note: φ is the molar ratio of silane to silica; C% is carbon weight percentage detected by elemental analysis; S is the alkyl group density.

Another series of alkyl group grafting experiments were conducted in ACN - a polar organic solvent. When ACN was used as the –C8 modification medium, the C% of SV and grafted density of –C8 increased continuously with increasing OTMS amount. When φ = 1:28.8, 1:14.4 and 1:7.18, the C% of SV-C8-As increased from 1.73 ± 0.0% to 6.8 ± 0.3% and 8.5± 1.2%, respectively. Accordingly, S changed from 0.36 to 1.18 and 1.76 nm-2. Similarly, the C% values (4.3 ± 0.2%, 12.9 ± 0.7% and 18.7 ± 1.6%) and S values (0.40, 1.21 and 1.75 nm-2) of SV-C18-As increased with the increasing of φ in the -C18 modification in ACN (Table 2). A lower alkyltrimethoxysilane amount is needed to achived comparible alkyl group density of SV in ACN than in toluene. The density difference of grafted alkyl group in toluene and ACN can be explained by the hydrolysis and condensation behavior of the alkyltrimethoxysilanes. Figure S1 shows the digital


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images of ODMS being refluxed for 24 h in toluene at 110 °C and in ACN at 80 °C, respectively. After reaction at 110 °C for 24 h, ODMS dissolved in toluene is in a transparent form, indicating there is no obvious hydrolysis and condensation among ODMS molecules. It is revealed that in non-polar toluene the self-reaction between alkyltrimethoxysilane molecules is quite slow without the existence of aqueous layer or the addition of catalysis.26 In this work, the moderate dehydration of SV before modification and the utilization of anhydrous toluene minimize the existence of aqueous layer in the alkyl modification process, thus, the self-hydrolysis and condensation rate of alkyltrimethoxysilane is low in toluene. As a result, the surface modification of SV is based on the reaction between alkyltrimethoxysilane molecules and surface silanol groups only (as shown in Scheme 1A), leading to a monolayered alkyl group grafting on the silica surface.37-38 The density of silanol groups on MSN surfaces after ~ 550 °C calcination is reported to be 2.5 nm-1.27 The maximum grafting density for both –C8 and –C18 groups is ~ 1.2 nm-1 in toluene, indicating not all surface silanol groups have been silylated by alkyl groups. The maximum alkyl grafting density is determined by the projection area of the alkyltrimethoxysilane molecule (Scheme 1B), which is a result of silylation process in non-polar toluene and steric hindrance of silane molecules. In contrast, ODMS formed white sand-like sediments after stirring in ACN at 80 °C for 24 h, indicating self-hydrolysis and condensation among the ODMS molecules. As a polar solvent, the nitrogen atoms of ACN will bond to the hydrogen sites of silica surface, which decreases the formation of hydrolysed silanols.39 Both the protic and polar characters of the solvent molecules affect the hydrolysis and condensation reactions of the alkyltrimethoxysilane molecules, resulting in multilayered alkyl group grafted on the surface of SV (Scheme S1). As a result, SVC18-A-3 with multilayered grafting show a much higher –C18 density (1.75 cm-1) than SV-C18-T-


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1 (0.85 cm-1) when φ = 1:5.89. Such a difference can also be found in –C8 modification (when φ = 1:7.18, SSV-C8-A-3 = 1.76 and SSV-C8-T-1 = 0.41). These phenomena can also be observed in the grafting of other functional groups40 that a grafting approach in ACN can achieve a much higher grafting density. SV-C8-T-2 and SV-C18-T-2 with a similar density of monolayered -C8 or -C18 groups are potential candidates for investigation of their biological macromolecule immobilization behaviors. Their detailed structural parameters are also characterized by a series of techniques. The FE-SEM (Figure 1B and C) and TEM image (Figure 1E and F) of SV-C8–T-2 and SV-C18– T-2 show similar structures as SV-OH, indicating SV samples maintain their typical morphology after alkyl modification. The nitrogen adsorption-desorption isotherms of SV-C8–T-2 and SVC18–T-2 (Figure 2A) show also similar isotherms and hysteresis loop types. After –C8 and –C18 modification, the entrance size of SV (Figure 2B) decreases to 5.6 and 5.2 nm, respectively, due to the modification with varied alkyl chain length.41 All the other structural parameters are listed in Table 1. After the modification of alkyl groups, the BET surface area and total pore volume of SV decreased. SV-C8-T-2 and SV-C18-T-2 show BET surface areas of 253 and 213 m2/g and total pore volumes of 1.3 and 1.2 cm3/g, respectively. The ζ potential of SV before and after the hydrophobic modification in toluene were measured and illustrated in Table 1. For all SV samples, the ζ potentials are negatively charged. Before the modification, the surface charge of SV-OH is -25.0 ± 7.0 mV in PBS buffer (pH 7.4) due to the existence of unmodified silanol groups. After –C8 or –C18 modification, the ζ potentials of SV slightly increase but remain negative. The ζ potential values of SV-C8-T-2 and SV-C18-T-2 in PBS are -16.8 ± 4.45 and -13.7 ± 4.4 mV, respectively.


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FTIR technique was used to characterize the surface chemistry of SV before and after alkyl modification in toluene. Figure 3 shows the FTIR spectrum of SV-OH, where a characteristic peak at 808 cm-1 (ν(Si-O)) and a broad band in the range of 1050-1200 cm-1 with the highest intensity at 1074 cm-1(–Si-O-Si bonding) can be observed.42 A shoulder peak can also been observed at 960 cm-1, which is associated with the Si-O stretching of Si-OH groups.43 Beside the characteristic peaks of the siliceous framework, both SV-C8-T-2 and SV-C18-T-2 show three extra characteristic peaks at 2854, 2926 and 2962 cm-1, which can be attributed to symmetric and antisymmetric -CH2- stretching of the alkyl chain and ν(C-H) from methyl groups, respectively.22, 37 The FTIR spectra indicate the successful attachment of octyl and octadecyl groups. A shoulder peak at 960 cm-1 can still been observed in the FTIR spectra of both SV-C8T-2 and SV-C18-T-2, supporting the existence of non-reacted silanol groups in these two samples after the grafting process.

Figure 3. FTIR spectra of SV-OH, SV-C8-T-2 and SV-C18-T-2.


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Compared to multilayered functionalization, a monofunctional group which promotes monolayer formation onto the silica surface is preferred for the investigation of cargo molecule immobilization.33 Therefore, SV-C8-T-2 and SV-C18-T-2 with similar structures and surface density of monolayered -C8 and -C18 groups have been chosen as the model materials to investigate the insulin immobilization capacity. As both the internal and outer surfaces of SV-C8T-2 and SV-C18-T-2 have been grafted with alkyl groups, the insulin can directly interact with the surface alkyl groups during the immobilization process. Figure 4A and Table S1 summarize the loading amount of insulin by three types of SV at different IIC. Overall, all of three particles show an increasing trend of insulin loading amount with the increasing IIC. At a high IIC of 3.0 mg/ml, the insulin loading amounts of SV-OH, SVC8-T-2 and SV-C18-T-2 are 933±98, 854±92 and 771±46 mg/g, respectively. The insulin immobilization capacity is SV-OH > SV-C8-T-2 > SV-C18-T-2, which is the same trend with the BET surface area of the three sample. At an IIC of 2.6 mg/ml, all three SV samples show a lower insulin loading amount but the trend maintains the same. However, the insulin loading amount of SV-OH dramatically decreases when the IIC further decreases. At a lower IIC of 1.9 mg/ml, the insulin loading amounts of SV-OH, SV-C8-T-2 and SV-C18-T-2 are 587±24, 646±57 and 595±78 mg/g, respectively. When the IIC is in the range of 0.6-1.9 mg/ml, the insulin immobilization capacity changes to SV-C8-T-2 ≥ SV-C18-T-2 > SV-OH. At the IIC of 0.6 mg/ml, SV-C8-T-2 and SV-C18-T-2 show the insulin loading amount of ~ 300 mg/g, which is higher than that of SV-OH (207±9 mg/g).


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Figure 4. (A) Insulin loading amount by three SV and (B) the calculated surface coverage (K) of SV by insulin.

FTIR technique has been used to confirm the loading of insulin (Figure S2). The FTIR spectrum (green) of pure insulin shows several characteristic peaks at the wavelength of 3288 (amide A band), 1644 (amide I), 1514 (amide II) and 1236 cm−1 (amide III).44-45 After insulin loading at an IIC of 3.0 mg/ml, the FTIR spectra of all SV samples show characteristic peaks of insulin at 1644 and 1514 cm−1, indicating the successful loading of insulin. The insulin characteristic peak at 1236 cm−1 has overlapped with the broad characteristic peak at 1050-1200 cm-1, which is contributed to the –Si-O-Si bonding of silica framework. In order to explain the IIC-dependent insulin immobilization behavior, the surface coverage of SV by insulin (K) is calculated. With the existance of zinc ion and HCl, the insulin involved in this study is in a hexamer form when the concentraiton is higher than 0.6 mg/ml at pH = 7.4.46 The SV surface coverage percentage by insulin hexamers can be estimated by Equation 3, if considering insulin hexamer as a plate with a diameter of 5.6 nm47 and considering only surface adsorption onto the SV.


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! × " × #$ %

= & × '

( ×

× 10) (%)

Page 20 of 33

Equation 3

where K is the surface coverage of SV by insulin hexamers, M is the loading amount insulin by SV; NA is Avogadro's constant, r is the radius of insulin hexamer, Mw is the molecular weight of insulin; SBET is the BET surface area of individual SV. The K values of three SV samples at different IIC have been summarized in Figure 4B and Table S1, which increase with the increasing IIC for all SV samples. At high IIC of 2.6 and 3.0 mg/ml, both SV-C8-T-2 and SV-C18-T-2 show the K values of > 100 %. This indicates that insulin hexamers are immobilized onto the hydrophobic surface of SV through multilayer adsorption. Although the hydrophobicity of alkyl groups enhances the interaction with insulin, further interaction of insulin from the outer layers and alkyl groups is hindered. In contrast, SVOH shows the highest loading amount of insulin but only 84.8 and 91.8% surface coverage due to its higher surface area. It is revealed that the surface area of SV dominate their insulin immobilization capacity at high IIC. At a medium IIC of 1.9 mg/ml, the K values of SV-OH, SVC8-T-2 and SV-C18-T-2 are 57.8, 86.6 and 94.6 %, respectively, indicating insulin hexamers could directly attached onto the surface of SV through single layer adsorption. The insulin loading amount has changed to SV-C8-T-2 > SV-C18-T-2 > SV-OH at this IIC, which is dominated by the combination of the surface area and hydrophobic interaction of SV. At relatively lower IIC of 0.6-1.4 mg/ml, all SV samples show decreased K values. Although the trend of K is SV-C18-T-2 > SV-C8-T-2 > SV-OH, both of the –C8 and –C18 modified SV show similar insulin loading amount (higher than that of SV-OH). The alkyl chain length does not show a significant role, as surface area still contribute to the insulin immobilization at this IIC. In order to evaluate the contribution of grafted alkyl chain length to the insulin immobilization, the insulin enrichment capacity by SV at an extremely low IIC was investigated. MS has been


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utilized to detect the immobilization of insulin by SV at low concentrations. Figure 5A-C display the MS spectra obtained from the SV elution after enrichment of insulin from PBS buffer solution. When SV-OH were used for insulin enrichment, MS spectrum of eluted buffer showed no significant signal at an IIC of 0.1 ng/ml (A). When SV-C8-T-2 or SV-C18-T-2 were used, their MS spectra showed a small single-charge peak for insulin (B and C, m/z 5808, indicated by black star), indicating a low limit of detection at an IIC of 0.001 ng/ml. A higher insulin peak intensity of SV-C18-T-2 indicates a better insulin enrichment performance of –C18 with longer chain length. When the concentration of insulin solution is less than 0.6 mg/ml, the insulin involved in this study is in a monomer form with a much smaller size than the hexamer.46 At a low IIC of 0.001 ng/ml, the maximum theoretical surface coverage of SV by insulin is only 7.1 × 10-6 %, thus the contribution from surface area and its difference to insulin immobilization is minimized. Consequently, the contribution from the alkyl chain length is dominant. Our results have shown that at extremely low IIC, longer alkyl chain surface modification with higher hydrophobicity is beneficial for insulin immobilization than shorter alkyl chain. The insulin enrichment behaviors of the three SV samples were also tested in a more complex enrichment medium - artificial urine. When SV-OH and SV-C8-T-2 with short alkyl chain length were used to enrich insulin at an IIC of 1 ng/ml, no significant characteristic peaks can be observed in the MS spectra (Figure 5D and E). Figure 5F demonstrated the limit of detection of insulin from artficial urine is only 0.05 ng/ml when SV-C18-T-2 was used for enrichment. This value is much lower than the literature reports.48


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Figure 5. MS spectra of (A) 0.1 ng/ml human insulin/PBS solution after enrichment by SVOH; (B, C) 0.001 ng/ml human insulin/PBS solution enriched by (B) SV-C8-T-2 and (C) SV-C18T-2; (D, E) 1 ng/ml human insulin/artificial urine solution enriched by (D) SV-OH and (E) SVC8-T-2, (F) 0.05 ng/ml human insulin/artificial urine solution enriched by SV-C18-T-2.

Surface functional groups may have influence on the conformation and potentially functions of immobilized molecules. In order to evaluate the conformation of insulin before and after immobilization on three SV materials, its secondary structure was investigated by far-UV CD spectrum (Figure 6). The CD spectrum of 1 mg/ml free insulin/PBS solution (black line) shows strong negative peaks in the range of 208-223 nm, indicating the presence of secondary strcuture


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of α-helix and β-sheet.49 After insulin was loaded by SV samples at the IIC of 3.0 mg/ml, the CD spectra were also collected. The CD spectrum of insulin/SV-OH (red) shows decreasing peak intensity in the range of 208-223 nm, indicating the deconformation of the insulin secondary structure. The CD spectra of both insulin/SV-C8-T-2 (blue) and insulin/SV-C18-T-2 (green) show slightly decreasing peak intensity at 208 nm, indicating the conformation change of α-helix. It is demonstrated that insulin loaded in SV modified with alkyl groups shows less insulin secondary structure deconformation after loading compared to SV without any modification.

Figure 6. Far-UV CD spectrum of free insulin, and insulin loaded by a series of SV.

Conclusions In conclusion, a monolayer of either –C8 or –C18 groups has been modified onto moderately dehydrated SV in anhydrous toluene. The grafted alkyl density increases with the alkyltrimethoxysilane to silica ratio added until reaching a maximum value of ~ 1.2 nm-1, which is determined by the projection area of alkyltrimethoxylsilane. To minimize the influence of


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grafted alkyl group density, SV with the same density of monolayered –C8 and -C18 are selected for immobilization of insulin in comparison with SV-OH. An IIC-dependent trend of insulin immobilization capacities of SV has been observed. It is revealed that both the surface area and alkyl chain length of SV contribute to insulin immobilization at medium to high IIC. The influence of surface area of SV is not significant for insulin immobilization test at an extremely low IIC (0.001-0.05 ng/ml). SV-C18 with longer alkyl group length and the highest hydrophobicity show an outstanding insulin enrichment behavior from both of the PBS (0.001 ng/ml) and complex artficial urine (0.05 ng/ml). It is also shown that insulin loaded by alkyl modified SV shows less conformation change of secondary structure than that of hydrophilic SV. These findings provide useful information for the rational choice of supporting materials as biomolecule enrichment and carriers in various applications.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org AUTHOR INFORMATION Corresponding Author * Email [email protected] Author Contributions †

These authors contribute equally to this work. All authors have given approval to the final

version of the manuscript.


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Notes Any additional relevant notes should be placed here. ACKNOWLEDGMENT The authors acknowledge the financial support from the Australian Research Council and the Queensland Government. We also thank the Australian Microscopy and Microanalysis Research Facility at the Centre for Microscopy and Microanalysis for the technical assistance. ABBREVIATIONS SV, silica vesicles; OTMS , n-octyltrimethoxylsilane; ODMS, n-octadecyltrimethoxylsilane; ACN, acetonitrile; IIC, initial insulin concentrations; MSNs, mesoporous silica nanoparticles; MS, mass spectrometry; CD, circular dichroism; PBS, phosphate buffered saline; TEOS, tetraethyl orthosilicate; FE-SEM, field-emission scanning electron microscopy; TEM, transmission electron microscopy; BJH, Barrett–Joyner–Halanda; BET, Brunauer–Emmett– Teller; FTIR, Fourier transform infrared; CHCA, Α-Cyano-4-hydroxycinnamic acid; MALDI, Matrix Assisted Laser Desorption/Ionization. REFERENCES 1. Somorjai, G. A.; Frei, H.; Park, J. Y., Advancing the Frontiers in Nanocatalysis, Biointerfaces, and Renewable Energy Conversion by Innovations of Surface Techniques. J. Am. Chem. Soc. 2009, 131 (46), 16589-16605. 2. Yue, Q.; Li, J. L.; Luo, W.; Zhang, Y.; Elzatahry, A. A.; Wang, X. Q.; Wang, C.; Li, W.; Cheng, X. W.; Alghamdi, A.; Abdullah, A. M.; Deng, Y. H.; Zhao, D. Y., An Interface Coassembly in Biliquid Phase: Toward Core-Shell Magnetic Mesoporous Silica Microspheres with Tunable Pore Size. J. Am. Chem. Soc. 2015, 137 (41), 13282-13289.


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3. Sayari, A.; Hamoudi, S., Periodic mesoporous silica-based organic - Inorganic nanocomposite materials. Chem. Mater. 2001, 13 (10), 3151-3168. 4. Biju, V., Chemical modifications and bioconjugate reactions of nanomaterials for sensing, imaging, drug delivery and therapy. Chem. Soc. Rev. 2014, 43 (3), 744-764. 5. Xie, J.; Lee, S.; Chen, X. Y., Nanoparticle-based theranostic agents. Adv. Drug Deliv. Rev. 2010, 62 (11), 1064-1079. 6. Slowing, II; Vivero-Escoto, J. L.; Wu, C. W.; Lin, V. S. Y., Mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers. Adv. Drug Deliv. Rev. 2008, 60 (11), 1278-1288. 7. Wang, X. Q.; Zhang, Y.; Luo, W.; Elzatahry, A. A.; Cheng, X. W.; Alghamdi, A.; Abdullah, A. M.; Deng, Y. H.; Zhao, D. Y., Synthesis of Ordered Mesoporous Silica with Tunable Morphologies and Pore Sizes via a Nonpolar Solvent-Assisted Stober Method. Chem. Mater. 2016, 28 (7), 2356-2362. 8. Melde, B. J.; Johnson, B. J.; Charles, P. T., Mesoporous silicate materials in sensing. Sensors 2008, 8 (8), 5202-5228. 9. Sun, Z. K.; Zhou, X. R.; Luo, W.; Yue, Q.; Zhang, Y.; Cheng, X. W.; Li, W.; Kong, B.; Deng, Y. H.; Zhao, D. Y., Interfacial engineering of magnetic particles with porous shells: Towards magnetic core - Porous shell microparticles. Nano Today 2016, 11 (4), 464-482. 10. Vallet-Regi, M.; Balas, F.; Arcos, D., Mesoporous materials for drug delivery. Angew. Chem.-Int. Edit. 2007, 46 (40), 7548-7558.


26

Page 27 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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11. Vinu, A.; Hossain, K. Z.; Ariga, K., Recent advances in functionalization of mesoporous silica. J. Nanosci. Nanotechnol. 2005, 5 (3), 347-371. 12. Descalzo, A. B.; Martinez-Manez, R.; Sancenon, R.; Hoffmann, K.; Rurack, K., The supramolecular chemistry of organic-inorganic hybrid materials. Angew. Chem.-Int. Edit. 2006, 45 (36), 5924-5948. 13. Bale, S. S.; Kwon, S. J.; Shah, D. A.; Banerjee, A.; Dordick, J. S.; Kane, R. S., NanoparticleMediated Cytoplasmic Delivery of Proteins To Target Cellular Machinery. ACS Nano 2010, 4 (3), 1493-1500. 14. Wu, M. Y.; Meng, Q. S.; Chen, Y.; Zhang, L. X.; Li, M. L.; Cai, X. J.; Li, Y. P.; Yu, P. C.; Zhang, L. L.; Shi, J. L., Large Pore-Sized Hollow Mesoporous Organosilica for RedoxResponsive Gene Delivery and Synergistic Cancer Chemotherapy. Adv. Mater. 2016, 28 (10), 1963-1969. 15. Wu, M. Y.; Meng, Q. S.; Chen, Y.; Du, Y. Y.; Zhang, L. X.; Li, Y. P.; Zhang, L. L.; Shi, J. L., Large-Pore Ultrasmall Mesoporous Organosilica Nanoparticles: Micelle/Precursor Cotemplating Assembly and Nuclear-Targeted Gene Delivery. Adv. Mater. 2015, 27 (2), 215-222. 16. Yang, Y. N.; Lu, Y.; Abbaraju, P. L.; Zhang, J.; Zhang, M.; Xiang, G. Y.; Yu, C. Z., Multishelled Dendritic Mesoporous Organosilica Hollow Spheres: Roles of Composition and Architecture in Cancer Immunotherapy. Angew. Chem.-Int. Edit. 2017, 56 (29), 8446-8450. 17. Scheiblhofer, S.; Machado, Y.; Feinle, A.; Thalhamer, J.; Husing, N.; Weiss, R., Potential of nanoparticles for allergen-specific immunotherapy - use of silica nanoparticles as vaccination platform. Expert Opin. Drug Deliv. 2016, 13 (12), 1777-1788.


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Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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18. Meka, A. K.; Niu, Y. T.; Karmakar, S.; Hartono, S. B.; Zhang, J.; Lin, C. X. C.; Zhang, H. W.; Whittaker, A.; Jack, K.; Yu, M. H.; Yu, C. Z., Facile Synthesis of Large-Pore Bicontinuous Cubic Mesoporous Silica Nanoparticles for Intracellular Gene Delivery. ChemNanoMat 2016, 2 (3), 220-225. 19. Lv, N.; Wang, Z. G.; Bi, W. Z.; Li, G. M.; Zhang, J. L.; Ni, J. Z., C8-modified CeO2//SiO2 Janus fibers for selective capture and individual MS detection of low-abundance peptides and phosphopeptides. J Mater. Chem. B 2016, 4 (25), 4402-4409. 20. Segro, S. S.; Malik, A., Sol-gel coatings with covalently attached methyl, octyl, and octadecyl ligands for capillary microextraction. Effects of alkyl chain length and sol-gel precursor concentration on extraction behavior. J. Chromatogr. A 2009, 1216 (45), 7677-7686. 21. Yasmin, T.; Muller, K., Synthesis and surface modification of mesoporous mcm-41 silica materials. J. Chromatogr. A 2010, 1217 (20), 3362-3374. 22. Huang, H. Y.; Yang, C. L.; Zhang, H. X.; Liu, M. C., Preparation and characterization of octyl and octadecyl-modified mesoporous SBA-15 silica molecular sieves for adsorption of dimethyl phthalate and diethyl phthalate. Microporous Mesoporous Mater. 2008, 111 (1-3), 254259. 23. Doadrio, J. C.; Sousa, E. M. B.; Izquierdo-Barba, I.; Doadrio, A. L.; Perez-Pariente, J.; Vallet-Regi, M., Functionalization of mesoporous materials with long alkyl chains as a strategy for controlling drug delivery pattern. J. Mater. Chem. 2006, 16 (5), 462-466.


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Page 29 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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24. Wang, J. Q.; Meng, G.; Tao, K.; Feng, M.; Zhao, X. B.; Li, Z.; Xu, H.; Xia, D. H.; Lu, J. R., Immobilization of Lipases on Alkyl Silane Modified Magnetic Nanoparticles: Effect of Alkyl Chain Length on Enzyme Activity. PLoS One 2012, 7 (8). 25. Shakeri, M.; Kawakami, K., Enhancement of Rhizopus oryzae lipase activity immobilized on alkyl-functionalized spherical mesocellular foam: Influence of alkyl chain length. Microporous Mesoporous Mater. 2009, 118 (1-3), 115-120. 26. Garcia, N.; Benito, E.; Guzman, J.; Tiemblo, P., Use of p-toluenesulfonic acid for the controlled grafting of alkoxysilanes onto silanol containing surfaces: Preparation of tunable hydrophilic, hydrophobic, and super-hydrophobic silica. J. Am. Chem. Soc. 2007, 129 (16), 5052-5060. 27. Zhao, X. S.; Lu, G. Q., Modification of MCM-41 by surface silylation with trimethylchlorosilane and adsorption study. J. Phys. Chem. B 1998, 102 (9), 1556-1561. 28. Kulkami, S. A.; Ogale, S. B.; Vijayamohanan, K. P., Tuning the hydrophobic properties of silica particles by surface silanization using mixed self-assembled monolayers. J. Colloid Interface Sci. 2008, 318 (2), 372-379. 29. Han, X. L.; Wang, L.; Li, J. D.; Zhan, X.; Chen, J.; Yang, J. C., Tuning the hydrophobicity of ZSM-5 zeolites by surface silanization using alkyltrichlorosilane. Appl. Surf. Sci. 2011, 257 (22), 9525-9531. 30. Duchet, J.; Gerard, J. F.; Chapel, J. P.; Chabert, B., Grafting of alkylchlorosilanes onto silica from solution for adhesion enhancement. J. Adhes. Sci. Technol. 2000, 14 (5), 691-718.


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Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 33

31. Dkhissi, A.; Esteve, A.; Jeloaica, L.; Esteve, D.; Rouhani, M. D., Self-assembled monolayers and preorganization of organosilanes prior to surface grafting onto silica: A quantum mechanical study. J. Am. Chem. Soc. 2005, 127 (27), 9776-9780. 32. Ji, T.; Ma, C.; Brisbin, L.; Mu, L. W.; Robertson, C. G.; Dong, Y. L.; Zhu, J. H., Organosilane grafted silica: Quantitative correlation of microscopic surface characters and macroscopic surface properties. Appl. Surf. Sci. 2017, 399, 565-572. 33. Nechifor, A. M.; Philipse, A. P.; deJong, F.; vanDuynhoven, J. P. M.; Egberink, R. J. M.; Reinhoudt, D. N., Preparation and properties of organic dispersions of monodisperse silica receptor colloids grafted with calixarene derivatives or alkyl chains. Langmuir 1996, 12 (16), 3844-3854. 34. Zhang, J.; Karmakar, S.; Yu, M. H.; Mitter, N.; Zou, J.; Yu, C. Z., Synthesis of Silica Vesicles with Controlled Entrance Size for High Loading, Sustained Release, and Cellular Delivery of Therapeutical Proteins. Small 2014, 10 (24), 5068-5076. 35. Dai, J. T.; Zhang, Y.; Li, H. C.; Deng, Y. H.; Elzatahry, A. A.; Alghamdi, A.; Fu, D. L.; Jiang, Y. J.; Zhao, D. Y., Enhancement of gemcitabine against pancreatic cancer by loading in mesoporous silica vesicles. Chin. Chem. Lett. 2017, 28 (3), 531-536. 36. Mody, K. T.; Mahony, D.; Zhang, J.; Cavallaro, A. S.; Zhang, B.; Popat, A.; Mahony, T. J.; Yu, C. Z.; Mitter, N., Silica vesicles as nanocarriers and adjuvants for generating both antibody and T-cell mediated immune resposes to Bovine Viral Diarrhoea Virus E2 protein. Biomaterials 2014, 35 (37), 9972-9983.


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37. Kailasam, K.; Muller, K., Physico-chemical characterization of MCM-41 silica spheres made by the pseudomorphic route and grafted with octadecyl chains. J. Chromatogr. A 2008, 1191 (12), 125-135. 38.

Rustamov, I.; Farcas, T.; Ahmed, F.; Chan, F.; LoBrutto, R.; McNair, H. M.; Kazakevich,

Y. V., Geometry of chemically modified silica. J. Chromatogr. A 2001, 913 (1-2), 49-63. 39. Hu, Z.; Weeks, J. D., Acetonitrile on silica surfaces and at its liquid-vapor interface: structural correlations and collective dynamics. J. Phys. Chem. C, 2010, 114 (22), 10202–10211. 40. Bissadi, G.; Weberskirch, R., Efficient synthesis of polyoxazoline-silica hybrid nanoparticles by using the "grafting-onto" approach. Polym. Chem. 2016, 7 (6), 1271-1280. 41. Sun, L.; Siepmann, J. I.; Schure, M. R., Monte Carlo simulations of an isolated n-octadecane chain solvated in water - Acetonitrile mixtures. J. Chem. Theory Comput. 2007, 3 (2), 350-357. 42. Tejedor-Tejedor, M. I.; Paredes, L.; Anderson, M. A., Evaluation of ATR-FTIR spectroscopy as an "in situ" tool for following the hydrolysis and condensation of alkoxysilanes under rich H2O conditions. Chem. Mater. 1998, 10 (11), 3410-3421. 43. Zhang, J.; Yu, M. H.; Yuan, P.; Wang, H. N.; Qian, K.; Tan, L.; Wang, Y. H.; Yu, C. Z., Tuning cooperative vesicle templating and liquid crystal templating simply by varying silica source. J. Mater. Res. 2010, 25 (4), 648-657. 44. Marcano, A.; Markushin, Y.; Melikechi, N.; Connolly, D., FOURIER TRANSFORM INFRARED SPECTROSCOPY OF DEUTERATED PROTEINS. In Linear and Nonlinear


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Optics of Organic Materials Viii, Jakubiak, R., Ed. Spie-Int Soc Optical Engineering: Bellingham, 2008; Vol. 7049. 45. Nielsen, L.; Frokjaer, S.; Carpenter, J. F.; Brange, J., Studies of the structure of insulin fibrils by Fourier transform infrared (FTIR) spectroscopy and electron microscopy. J. Pharm. Sci. 2001, 90 (1), 29-37. 46. Carpenter, M. C.; Wilcox, D. E., Thermodynamics of Formation of the Insulin Hexamer: Metal-Stabilized Proton-Coupled Assembly of Quaternary Structure. Biochemistry 2014, 53 (8), 1296-1301. 47. Hvidt, S., INSULIN ASSOCIATION IN NEUTRAL SOLUTIONS STUDIED BY LIGHTSCATTERING. Biophys. Chem. 1991, 39 (2), 205-213. 48. Lei, C.; Noonan, O.; Jambhrunkar, S.; Qian, K.; Xu, C.; Zhang, J.; Nouwens, A.; Yu, C. Z., Sensitive Detection of Human Insulin Using a Designed Combined Pore Approach. Small 2014, 10 (12), 2413-2418. 49. Melberg, S. G.; Johnson, W. C., CHANGES IN SECONDARY STRUCTURE FOLLOW THE DISSOCIATION OF HUMAN INSULIN HEXAMERS - A CIRCULAR-DICHROISM STUDY. Proteins 1990, 8 (3), 280-286.


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85x43mm (300 x 300 DPI)


The impact of alkyl chain length

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