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Complex Assembly of Polymer Conjugated Mesoporous Silica Nanoparticles for Intracellular pH-Responsive Drug Delivery Yang Yang, Katharina Achazi, Yi Jia, Qiang Wei, Rainer Haag, and Junbai Li Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b01845 • Publication Date (Web): 28 Jul 2016 Downloaded from http://pubs.acs.org on July 30, 2016

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Complex Assembly of Polymer Conjugated Mesoporous Silica Nanoparticles for Intracellular pH-Responsive Drug Delivery Yang Yang, † Katharina Achazi, § Yi Jia, ‡ Qiang Wei, § Rainer Haag, § and Junbai Li*,†,‡ †

CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National

Center for Nanoscience and Technology, Beijing 100190, China ‡

Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Colloid

and Interface Science, Institute of Chemistry, Chinese Academy of Sciences (CAS), Beijing 100190, China. §

Department of Chemistry and Biochemistry, Freie Universität Berlin, Takustrasse 3, 14195

Berlin, Germany.

KEYWORDS: hyperbranched polyglycerol, mesoporous silica nanoparticles, pH response, drug delivery system

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ABSTRACT: There is a great challenge in constructing pH-responsive drug delivery systems in the research of the biomedical application. Many nanocomposites are intended to be pH-responsive as drug carriers due to tumorous or intracellular mild acidic environment. However, it is always difficult to find an appropriate system for quick response and release before the carrier is excreted from the living system. In this work, hyperbranched polymer, hyperbranched polyglycerol (hPG), conjugated mesoporous silica nanoparticles (MSNs) were assembled as complexes to serve as drug carriers. Herein, the conjugated polymer-MSNs were interacted through Schiff base bond, which possessed mild acid responsive property. Interestingly, the assembled system could rapidly respond and release guest molecules inside cancer cells. This would make the entrapped drug released before the carriers escape from the endosome counterpart. The results show that the assembled composite complexes can be considered as a drug delivery system for cancer therapy.

Introduction

In the past decade, mesoporous silica nanoparticles (MSNs) based nanocomposites have gained much attention for their potential biomedical applications.1-7 MSNs are considered to be suitable as drug delivery systems due to their unique characteristics, such as uniform and tunable particle/pore size, extremely high surface areas/pore volume as well as good biocompatibility. More importantly, there are abundant silanol groups (Si-OH) on the surfaces of MSN, which enable an easy post-modification of the MSN surfaces with various organic linkers, thereby simplifying the design of drug delivery systems.8-15 Therefore, MSN


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based nanomaterials are promising to be well designed as smart responsive nano-devices that deliver drugs into their destination due to a change in biological environment. Within the environment responsive MSN systems, pH responsiveness is particularly important because it can be used for cancer therapy or intracellular analysis. As we know, tumor tissue (pH 6.8)16 and intracellular endosomes (pH 5.5)17 are more acidic than normal tissues (pH 7.4), which enables pH responsive carriers to release their anticancer drug at the desired place.

Several techniques have been used to design pH responsive MSN based drug delivery systems. Zink et al. developed a “gate” controlled MSN system, in which pH responsive supramolecules were used as nanovalves attached on the surface of MSN.18-20 Radical polymerization and in situ polymerization were applied for conjugating pH sensitive polymer on the surfaces or into pores of MSN.21-25 Layer-by-layer (LbL) technique was used to coat pH sensitive polymer on the surfaces of MSN.26 Even though many reports about pH responsive MSN systems exist, researchers are still continually looking for a simple method to prepare a controllable and rapid pH responsive system to enable drug release before clearance of the carrier from the body.27-29 It has been reported that internalized MSNs are gradually excreted from the cells after uptake (within 4-6 hours).28 Therefore, it is important to develop a rapid intracellular responsive system to realize a maximum of drug efficacy.

In this work, biocompatible polymer, hyperbranched polyglycerol (hPG), conjugated MSNs (hPG-MSNs) were used as pH responsive drug carriers. HPG is a biocompatible, low-cost and water-soluble macromolecule that has been investigated as biomaterial for biomedical applications.30,31 Herein, hPGs were coated on the surfaces of MSNs through


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Schiff-base bond combined with LbL method. The Schiff-base bond is biodegradable via hydrolysis, and this process can be accelerated at low weak acidic conditions.32,33 Therefore, hPG-MSNs were pH sensitive for the cleavable Schiff-base bond between hPG and MSNs in low pH environment. Interestingly, this system could respond quickly to pH change of intracellular environment, which makes it a promising drug delivery system for cancer therapy.

Materials and Methods

Materials. HPG (Mw 10 kDa, structural formula as structure a in Scheme S1) was synthesized through anionic, ring-opening multi-branching polymerization of glycidol with slow

addition.34,35

monomer

3-aminopropyltriethoxysilane

Tetraethylorthosilicate

(APTEOS)

were

(TEOS) from

and Acros.

3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Amresco. Antibody for staining of early endosomes (Purified Mouse Anti-EEA1, Clone 14/EEA1) was obtained from BD Bioscience, 4’,6-diamidino-2-phenylindole (DAPI) for staining of the nucleus was obtained from life technologies, and Atto 647N-Phalloidin for staining

of

actin

was

obtained

from

Sigma-Alderich.

Rhodamine

B

(RhB),

Cetyltrimethylammonium bromide (CTAB), glutaraldehyde (GA) and other reagents were purchased from Beijing Chemical Reagent Co. without further purification. The millipore water (Milli-Q integral A10 system) was used in all needed experiments.

Preparation of surface-functionalized MSNs. Firstly, MSNs were synthesized using a base-catalyzed sol-gel method according to the published work.14,36 0.25 g CTAB and 0.87 4 ACS Paragon Plus Environment

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ml 2 M NaOH were stirred in 120 ml water. Then the mixture was heated to 80 oC. 1.25 ml TEOS was introduced dropwise to the surfactant solution. The mixed solution was stirred for 2 hours at 80 oC. Then, the product was washed repeatedly with methanol and dried under vacuum at room temperature. Furthermore, the particle surface was modified with -NH2 groups. Typically, 0.28 ml APTEOS was used to react with 0.1 g MSNs. The mixture was refluxed for 20 h in 30 ml toluene for avoiding APTEOS hydrolysis before reacting with MSNs. Then, the products were washed with methanol and dried under vacuum. Finally, the MSN-NH2 nanoparticles were refluxed in acidic ethanol solution with 0.37% HCl for removing the surfactant template.

Preparation of hPG coated MSNs for loading guest molecules. Firstly, hPGs were functionalized with -NH2 groups (hPG-NH2, structural formula as structure b in Scheme S1) before coating on the surfaces of MSN. HPG-NH2 (with 10% and 30% amino functionality, marked as hPG-L-NH2 and hPG-H-NH2, respectively) was synthesized according to our published work.37,38 The brief illustration and results of synthesizing hPG-NH2 were listed in supporting information (Scheme S1c and Figure S1). In this work, typical dyes, RhB, were selected as drug model molecules loaded into hPG-MSNs. Both adsorbing RhB and coating hPG proceeded simultaneously as follows: (1) ~5 mg MSN-NH2 were dispersed in 1 ml RhB solution (in pH 7.4 phosphate buffer saline (PBS), 1 mg/ml ) for 6 h adsorption; (2) after that, 0.3 ml GA aqueous solution (25wt%) was added into above mixture for another 12 h adsorption; (3) then, the sample was centrifuged and re-dispersed into 1 ml hPG-NH2 solution (in pH7.4 PBS buffer, 2 mg/ml) for 12 h reaction, (4) procedure 2 and 3 were repeated again for assembly another PG layer. The RhB loading efficiency was calculated according to the 5 ACS Paragon Plus Environment

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change of characteristic absorption of RhB (553 nm) before and after adsorption. Herein, hPG-H-MSNs represented MSNs coated by one layer of hPG-H-NH2, and hPG-L-MSNs represented MSNs coated by one layer of hPG-L-NH2, respectively. While, (hPG-H)2-MSNs represent MSNs coated by two layers of hPG-H-NH2. Preparation of GA cross-linked hPG. Firstly, hPG gels were prepared and researched their pH responsive property. Typically, 1 ml high concentration hPG-H-NH2 aqueous solution (300 mg/ml) was mixed with 500 µl GA aqueous solution (25 wt%) for 12 h reaction. After that, 200 µl pH 7.4 PBS buffer or pH 5.5 buffer (acetate buffer) was added into the prepared gel for observing its morphological change. As comparison experiment, hPG with no amino group also was used to repeat above experiment. Similarly, hPG-H-NH2 with low concentration (5 mg/ml) also was coss-linked by GA, and then was put into different pH buffer for DLS analysis.

Guest molecules were released from hPG conjugated MSNs in response to pH. The samples of hPG-L-MSNs or hPG-H-MSNs loaded with RhB (denoted as hPG-L-MSNs-RhB and hPG-H-MSNs-RhB) were centrifuged and washed thoroughly until no absorbance for supernatant.

Then samples were equally split into three parts and re-dispersed into different

pH buffer for shaking. (pH 8.0 and pH 7.4 PBS buffer, pH 5.5 acetate buffer respectively). Therefore, the concentrations of hPG-MSNs-RhB in different buffer were same (~4 mg/ml). At a pre-set time point, the sample suspension was centrifuged and measured the absorbance (at 553 nm) of supernatant using a UV-Vis spectrometer. The supernatant were then poured back into the tubes. This process was repeated until no further release was observed. As


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comparison, bare MSNs adsorbed RhB (MSNs-RhB) also was researched with the same assay.

Cellular uptake and imaging. For the uptake study, 50.000 A549 cells (adenocarcinoma human alveolar basal epithelial cells) cultured in DMEM supplemented with 10% FCS, penicillin, and streptomycin at 37°C and 5 % CO2, were seeded on 9 mm glass coverslips in each well of a 24-well plate and incubated for 24 h at 37°C before adding the hPG-L-MSN-RhB nanoparticles for 1.5h, 6h, or 24h, respectively. Cells treated with free RhB served as controls. For qualitative analysis by confocal laser scanning microscopy (CLSM), cells were washed 3 times with PBS and fixed with 4 % paraformaldehyde for 20 min. Afterwards, cells were permeabilized with 0.1 % TritonX for 5 min. To detect co-localization with early endosomes, samples were stained with FITC labeled Early Endosome Antibody EEA1. Cell nuclei and were stained with DAPI and f-actin (cytoskeleton) with Atto 647N labeled phalloidin. Cells were observed and imaged using a confocal laser scanning microscope (CLSM).

Cryogenic transmission electron microscopy (Cryo-TEM). A drop of sample solution was placed on the perforated carbon supported film. After removing the excess fluid, an ultra-thin layer of the solution was created spanned the holes of the supported film. Then, the samples were immediately vitrified by putting the grids into liquid ethane (-184 oC). After that, the sample grids were transferred with cryo-holder into TEM (FEI, Tecnai F20). Measurement was carried out at -175 oC sample temperature for observation.


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Real-time cell analysis (RTCA) for cytotoxicity. Cytotoxicity of nanoparticles was analyzed by using a xCELLigence real-time cell analyzer (RTCA). In short, A549 cells (adenocarcinoma human alveolar basal epithelial cells) cultured in DMEM supplemented with 10% FCS, penicillin, and streptomycin at 37°C and 5 % CO2, were seeded in a 96-well E-plate (10.000 cells/well). The plate was placed in the RTCA device and impedance was measured at least every 15 minutes. After 24h, the plate was removed from the RTCA and hPG-L-MSN-RhB nanoparticles were added in different concentrations in triplicates. Untreated cells and cells treated with free Rhodamin B (RhB) or MSN served as controls. The real-time impedance measurement was continued for another 24h after treatment and end point data analysis was performed after 12h and 24h.

Characterization. CLSM were carried out using LSM 510 (Carl Zeiss Jena GmbH, Jena, Germany). Toxicity measurements were performed using a xCELLigence real-time cell analyzer (RTCA) from Roche Applied Science (Mannheim, Germany). Dynamic light scattering (DLS) data were obtained from Malvern Instruments (Nano-ZS) equipped with a 633 nm He-Ne laser. Transmission electron microscopy (TEM, Philips CM12) with Cryo-TEM techniques was used for observing MSNs and hPG coated MSNs. 1H NMR spectra (400 MHz) were obtained with Bruker ECX 400 at room temperature.

UV-Vis were

recorded on Jasco FT/IR-4100. Micromeritics TriStarII 3020 (Micromeritics, USA) used to measure Brunauer-Emmett-Teller (BET) surface area and porosity of MSNs and their composites. The samples were degassed for 12 h under vacuum at room temperature before measurement. Thermogravimetric analysis (TGA) was carried out from room temperature to


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800 oC at a heating rate 10 oC/min in nitrogen atmosphere using Perkin Elmer Diamond TG/DTA instrument.

Scheme 1. Uptake of hPG-MSNs and intracellular release of guest molecules. Results and Discussion Fabrication of and characterization of hPG conjugated MSNs. In this work, both hPG and MSNs were functionalized with amino group for synthesizing hPG conjugated MSNs. Both MSNs and MSN-NH2 were synthesized according to a published method.14,36 It is notable that amino groups were only modified on the surface of MSN, rather than in the inner pores, which is significant for coupling with hPG only on the surface of MSN in next procedure. This is a common method to modify the surfaces of MSN instead of inside the pores. HPG and hPG-NH2 was also synthesized according to our published work.37,39 Herein, 10 kDa hPG were selected due to their better dispersity. More importantly, the hydrate diameter of 10 kDa hPG is about 3~4 nm,40 which is suitable for taking them as the cap of pores of MSN (~2 nm). 10% and 30% amino group functionalized hPG were synthesized as 9 ACS Paragon Plus Environment

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hPG-L-NH2 and hPG-H-NH2, respectively. Then, hPG-NH2 was coupled onto the surface of MSN-NH2 with GA crosslinker and LbL method (two layers hPG except mentioned especially). The whole process of preparing hPG coated MSNs is shown in Scheme 1. Typically, TEMs of two layers hPG coated MSNs synthesized with hPG-L-NH2 were shown in Figure 1B. Compared with uncoated MSNs (Figure 1A), it is difficult to observe the hPG layer directly from room temperature TEM (RT-TEM) due to small sized and flexible hPG structure. Furthermore, the hPG layer could be presented obviously when the sample was measured with Cryo-TEM (Figure 1C). The hPG layer could be more obvious when hPG-MSNs synthesized with hPG-H-NH2 (Figure 2). Moreover, the thickness of hPG layers seemed increased with the number of hPG layer. However, there was serious aggregated phenomenon for hPG-H-MSNs with two layers of hPG or more. It was indicated that inter-particles crosslinking of hPG-H-MSNs happened if using high amino group functionalized hPGs or more layers. Therefore, hPG-L-MSNs were used for further cell experiments except particular mentioned. Surface analysis of materials showed that the BET surface area and pore volume significantly decreased after MSNs were coated with one or more layers of hPG (Figure S2 and Table S1). TGA result of MSNs and hPG-MSN nanocomposties was shown in Figure S3. The content of hPG in hPG-MSN nanocomposties was 6.43% for hPG-L-MSN, 6.64% for hPG-H-MSN and 8.03% for (hPG-H)2-MSN, respectively, compared with bare MSN sample.


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Figure 1. (A)TEMs of MSNs, (B)TEMs of hPG-L-MSNs and (C) Cryo-TEMs of hPG-L-MSNs. The arrows in the images indicate the existed hPG layers.

Figure 2. TEMs of hPG-H-MSNs with (A) one layer of hPG and (B) two layers of hPG.


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Figure 3. In vitro RhB-release profiles of different drug delivery systems. (A) release from hPG-H-MSNs in different buffer, (B) release from unmodified MSNs in different buffer, (C) release from hPG-H-MSNs in pH 7.4 buffer firstly, then keep release in pH 5.5 buffer, and (D) comparison RhB release from hPG-H-MSNs with hPG-L-MSNs in different buffer. HPG conjugated MSNs release guest molecules in different pH buffer. After loading guest molecules RhB, the controlled release ability of hPG conjugated MSNs were investigated through several different methods. The controlled release property of hPG-H-MSNs-RhB were firstly researched. As shown in Figure 3A, obviously, RhB was released little from hPG-H-MSNs in pH 7.4 or 8.0 buffer due to the thicker hPG layer on the surface of MSNs. However, hPG-H-MSNs released RhB quickly in pH 5.5 buffer. The supernatant of hPG-H-MSNs-RhB in different buffer also showed different color after finishing measurement. For hPG-L-MSNs, they also presented more quickly release ratio in pH 5.5 buffer than in pH 7.4 buffer (Figure 3D). Compared with hPG-H-MSNs, hPG-L-MSNs released RhB more quickly in corresponding buffer due to the thinner polymer layers. For more directly observing pH responsive release behavior, hPG-H-MSNs-RhB were


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put into pH 7.4 buffer firstly for releasing RhB 400 min, after that, the sample was centrifuged and pH 7.4 buffer was replaced with pH 5.5 one. Then, the sample kept releasing RhB for another 400 min. As shown in Figure 3C, the sample did release little RhB in pH 7.4 buffer, but release them quickly after the buffer was replaced by acid one. As comparison, bared MSNs did not present pH sensitivity and released RhB with almost same rate in different buffer (Figure 3B). All these results proved that hPG conjugated MSNs is pH sensitive drug carriers. It is noticeable that their pH response speed was very rapid. They almost reached saturated release dose within 6 hours.

As we known that hPG coated on the MSNs surfaces through Schiff base bonds between aldehyde group of GA and amino group of functionalized hPG. Schiff base bonds have been widely applied in biomedical research because they are pH sensitive. They are stable in high pH solution, while easy to be cleaved in low pH environment.13,41-43 For this reason, the chemical bonds between hPG and MSN begun to be cleaved and RhB was released from the pores of MSNs in acid buffer. However, there would be still some polymer coated on the MSNs through physical adsorption, therefore, the release speed of RhB from hPG-L-MSNs/ hPG-H-MSNs was still lower than from bare MSNs.

Figure 4. Illustrations of fabricating GA cross-linked hPG gel and its pH-responsive property. 13 ACS Paragon Plus Environment

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Figure 5. DLS analysis for hydrate diameters of hPG (A) , GA cross-linked hPG in dilute solution and re-dispersed in pH 7.4 buffer (B) and in pH 5.5 buffer (C). GA cross-linked hPG gel was prepared for understanding the process of hPG cross-linking with GA in solution. After GA added into high concentration hPG-NH2 solution, stable hPG gel formed. The gel was still stable when it was put into pH 7.4 buffer (Figure 4C). However, the gel was immediately decomposed when it was put into pH 5.5 buffer for 10 min (Figure 4D). As a compared experiment, the same concentration of hPG with no amino group was mixed with GA solution. However, there was no hPG gel formation (Figure S4).

On the

other hand, hPG-H-NH2 with low concentration also was coss-linked by GA. Then different pH buffers were added into coss-linked hPG, respectively. DLS analysis showed that coss-linked hPG in pH 7.4 buffer kept aggregated state (~11 nm, Figure 5B), while the one in pH 5.5 buffer decomposed, in which the size of hPG come back the value before corss-linking(~ 3 nm, Figure 5C). These phenomenon validated amino functionalized hPG


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could be cross-linked by GA. The GA cross-linked hPG also presented pH sensitivity. They could be decomposed rapidly in acid environment and kept stable in neutral solution.

Cytotoxicity analysis. The cytotoxicity of MSNs and hPG-L-MSNs to human tumor cells (A549) were investigated by real-time cell analysis (RTCA). For RTCA, cells were cultured in microelectrode-coupled microtiter plates and were monitored via the electrode impedance that reflects the cell number, cell morphology, and degree of cell adhesion.44,45 In this work, cells were treated with different concentrations of MSN and hPG-L-MSNs, respectively, and cell viability was determined 12 or 24 h post treatment. The cell viability was calculated relative to untreated control cells. As shown in Figure 6, hPG-L-MSNs exhibited no cytotoxicity up to 0.5 mg/ml indicating their potential to serve as drug carrier in biomedical applications.

Figure 6. A549 cells relative viability determined by RTCA at different MSN or hPG-L-MSN concentrations. The cell viability of untreated control cells was set to 100%. The solid bars and the shadowed bars represented cells were cultured for 12 h and 24 h, respectively. Bars representing the mean of triplicate measurements with the standard deviation.


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Guest molecules release in microenvironment of cells.

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For investigating the release

process of guest molecules from hPG-L-MSNs, hPG-L-MSNs loaded with RhB (hPG-L-MSNs-RhB) were co-cultured with A549 cells up to 24 h and observed under a CLSM. As shown in Figure 7B, already after 1.5 h uptake of hPG-L-MSNs (red spots in channel III) by endocytosis could be detected as shown by co-localization of the red fluorescence of RhB with early endosome staining inside the cells. In addition, some red fluorescence was visible in the cytoplasm most probably from free RhB that was released from hPG-L-MSNs (area in yellow circle in channel III and IV). After 6 h, the amount of internalized hPG-L-MSNs increased and also a higher red fluorescence was visible in the cytoplasm (area in yellow circle in channel III and IV of Figure 7C). After 24 h, a decrease of the red fluorescence inside the cells was observed indicating a rapid release of RhB from the pH responsive hPG-L-MSNs and a probable exocytosis of hPG-L-MSNs after release.27,28 There would be some residual RhB in hPG-L-MSNs due to physical adsorption, which made hPG-L-MSNs still present red fluorescence after release RhB. According to our observations we suppose that hPG-L-MSNs-RhB were internalized by cells through endocytosis in endosome. The Schiff base bond of the hPG-L-MSNs-RhB would be cleaved in mild acid environment as known for endomsomes (pH 5.5),17 and hPG-L-MSNs would begin to release RhB. Due to its small size, RhB can escape from the endosome in the cytoplasm. The amount of released RhB should increase over time as more hPG-L-MSNs are internalized. After that, the materials would go through exocytosis process and RhB release was also reached maximum.


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Figure 7. CLSM images of A549 cells treated with free RhB for 6 h (A); and with hPG-L-MSN loaded RhB for 1.5 h (B), 6 h (C), 24 h (D), respectively. Rhodamin B is shown in red (III); early endosomes were stained with FITC labeled Early Endosome Antibody EEA1 (green; I); cell nuclei were stained with DAPI (blue); f-actin (cytoskeleton) was stained with Atto 647N labeld phalloidin (white; II); IV shows an overly image of all channels recorded. Conclusions In this work, a rapid pH responsive drug delivery system was designed and synthesized. The system consists of a hyperbranched polymer, hPG, conjugated to mesoporous silica nanoparticles (MSNs) which were synthesized with a GA cross-linker combined with the LbL method. The existing Schiff base bonds between hPG and MSNs could be cleaved in mild acid environment, which enables the release of entrpped drug from MSNs in acidic conditions. The results show that the system presented rapid pH response in different pH solution. The cell-based experiments showed that the materials have low cytotoxicity. More


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interestingly, the materials also presented rapid intracellular release behavior before they were excreted from the cells. In conclusion, this system provided a potential for future in vivo intracellular delivery and controlled release applications.

ASSOCIATED CONTENT

Supporting Information Available: Structure of hPG and hPG-NH2, procedure of synthesizing hPG-NH2 and its 1H NMR spectra, BET and TGA data of MSNs and hPG-MSN nanocomposties, the image illustration of hPG was mixed with GA, a 3-D movie of a single cell co-cultured with hPG-L-MSNs-RhB and observed by CLSM.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work is financially supported by the National Nature Science Foundation of China (21273055, 21433010, 21320102004 and 21321063), National Basic Research Program of China (2013CB932800), National key foundation for exploring scientific instrument (2013YQ16055108) and visiting scholar project of Chinese Academy of Sciences. We thank Florian Paulus and Cathleen Schlesener for synthesizing hPG/hPG-NH2, Andrea Schulz for TEM and Christoph Böttcher for cryo-TEM measurement. 18 ACS Paragon Plus Environment

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Table of contents Complex Assembly of Polymer Conjugated Mesoporous Silica Nanoparticles for Intracellular pH-Responsive Drug Delivery Yang Yang, Katharina Achazi, Yi Jia, Qiang Wei, Rainer Haag, and Junbai Li


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Scheme 1 and TOC 478x295mm (150 x 150 DPI)

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Figure 1 238x360mm (150 x 150 DPI)


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Figure 2 277x281mm (150 x 150 DPI)


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Figure 3 1181x886mm (72 x 72 DPI)


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Figure 4 225x141mm (150 x 150 DPI)


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Figure 5 202x354mm (72 x 72 DPI)


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Figure 6 248x173mm (150 x 150 DPI)


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Figure 7a 257x233mm (150 x 150 DPI)


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Figure 7b 257x233mm (150 x 150 DPI)


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Figure 7c 257x232mm (150 x 150 DPI)


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Figure 7d 258x232mm (150 x 150 DPI)


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Scheme S1 277x146mm (150 x 150 DPI)


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Figure S1a 107x74mm (220 x 220 DPI)


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Figure S1b 115x79mm (220 x 220 DPI)


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Figure S2a 297x204mm (150 x 150 DPI)


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Figure S2b 297x207mm (150 x 150 DPI)


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Figure S3 297x207mm (150 x 150 DPI)


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Figure S4 140x67mm (150 x 150 DPI)


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