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Electronic Supplementary Material

Oligonucleotide delivery by chitosan-functionalized porous silicon nanoparticles

Morteza Hasanzadeh Kafshgari1,§, Bahman Delalat1,§, Wing Yin Tong1, Frances J. Harding1, Martti Kaasalainen2,

Jarno Salonen2, and Nicolas H. Voelcker1 ()

1 ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Mawson Institute, University of South Australia, GPO

Box 2471, Adelaide SA 5001, Australia 2 Department of Physics and Astronomy, University of Turku, FI-20014 Turku, Finland §These authors contributed equally to this work.

Supporting information to DOI 10.1007/s12274-015-0715-0

Calculation of degree of deacetylation of chitosan

Broussignac’s method

Linear potentiometric titration based on Broussignac’s method [S1, S2] determines the degree of deacetylation

(DD% = 71) of chitosan based on Eq. (S1) through identification of inflexion points in the titration curve (see

Fig. S1). Chitosan dissolved in an excess of hydrochloric acid becomes deprotonated during titration with

NaOH. The first and second inflexion points are equivalence points for titration of excess HCl and the titration

of protonated chitosan, respectively.

The degree of deacetylation (DD%) can thus be calculated using the equation

DD% = ∆V × CNaOH × 10–3 × 161/(Mchitosan × 0.0994) (S1)

where ∆V, CNaOH (0.1 M), and Mchitosan (0.15 g) are the volume of the added NaOH titrant (mL) between two

inflexion points, concentration of NaOH solution (g/mL), and the weight of chitosan (g), respectively. In addition,

161 and 0.0994 are constant.

Infrared spectroscopy

Diffuse reflectance infrared Fourier transform (DRIFT) spectroscopic methods were also used to calculate DD%

for the low molecular weight chitosan used. Spectra were obtained using a Thermo Nicolet Avatar 370MCT

(Thermo Electron Corporation, Waltham, MA, USA) instrument. A smart diffuse reflectance accessory was

used for the dried chitosan sample embedded within KBr pellets. The spectrum obtained was analyzed using

OMNIC version 7.3 software (Thermo Electron Corp., Waltham, MA, USA). For the spectrum analysis, 128 scans

were averaged in the range of 4,000 to 700 cm–1 with a resolution of 4 cm–1. The mixture of chitosan and KBr

was run in dry air to remove noise from CO2 and water vapor.

Address correspondence to nico.voelcker@unisa.edu.au

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Figure S1 Titration curve of chitosan solution (1% w/v) based on Broussignac’s method.

DD% of the low molecular weight chitosan was calculated based on different baselines (see Fig. S2). These

baselines are based on the determination of the intensity of a probe band corresponding to N-acetyl or amine

content and the intensity of a reference constant band [S3].

Figure S2 DRIFT spectrum of chitosan presenting the baseline--a and baseline--b for calculating degree of deacetylation (DD%).

(1) Baseline--a

Baseline--a, which was proposed by Domszy and Robertsis [S3, S4] is a simple theoretical method to determine

DD% by following equation

DD% = 100 – [(A1655 / A3450) × 100 / 1.33] (S2)

where A1655 and A3450 are the absorbance at 1,655 cm–1 corresponding to the primary amide band, which is the

N-acetyl group content representative, and the absorbance at 3,450 cm–1 is the hydroxyl band as an internal

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standard reference band. The factor 1.33 describes the ratio of A1655 /A3450 for the fully N-acetylated chitosan

[S3, S4].

(2) Baseline--b

This is a modified form of the baseline reported by Domszy and Roberts. The baseline--b proposed by Baxter

et al. [S3, S4] can be estimated by Eq. (S3)

DD% = 100 – [(A1655 / A3450) × 115] (S3)

where A1655 and A3450 are the absorbance at 1,655 and 3,450 cm–1. The factor 115 is the reciprocal value of the

slopes of the linear curves (the absorption ratio versus degree of N-acetylation) [S3, S4].

The absorbances of amide and hydroxyl bands can be calculated by the following mathematical expressions

proposed by Sabnis and Block [S4]

A1655 = log10 (DG/DE) (S4)

A1655 = log10 (DF/DE) (S5)

A3450 = log10 (AC/AB) (S6)

where DG (corresponds to the baseline--a) and DF (corresponds to the baseline--b), DE, AC, and AB are

depicting the absolute heights of the absorption bands of the functional groups at their respective wavelengths.

All three methods of calculation of DD% produced a similar result for the low-molecular weight chitosan

used to coat THCpSiNP (Table S1).

Table S1 Calculated degree of deacetylation (DD%) of the low molecular weight chitosan using the different methods

Method DD%

Broussignac’s method 71

Infrared spectroscopy-Baseline--a 72

Infrared spectroscopy-Baseline--b 75

Molecular weight and gyration radius of chitosan

Poly-(β-1→4)-2-amino-2-deoxy-d-glucopyranose, chitosan, is a linear polysaccharide, so molecular weight (MW)

and gyration radius (Rg) are estimated by means of Eq. (S7) and Eq. (S8) [S5].

Figure S3 Viscosity at different shear stresses of 1% w/v aqueous solution of chitosan at 25 °C.

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Viscosity of chitosan (Pa·s, see Fig. S3) was measured using rheometer/viscometer (RA 1000, TA Instruments

Rheometers, Rydalmere, NSW). The gyration radius (Rg = 46 nm) of chitosan estimated is based on the intrinsic

viscosity ([η] = 3,933 mL/g) and average molecular weight (MW = 119 kDa).

[η] = 8.43 × 10−2 MW0.92 (S7)

Rg = 7.5 × 10−2 MW0.55 (S8)

Characterization of THCpSiNPs before and after chitosan capping

Figure S4 Representative average size of (a) THCpSiNPs and (b) CS/THCpSiNPs studied by means of SEM, and respective size distribution of (c) THCpSiNPs, (d) CS/THCpSiNPs, (e) Oligo/THCpSiNPs, and (f) CS/oligo/THCpSiNPs measured by means of DLS. Pores of THCpSiNPs before coating by using chitosan are indicated by red arrows (representative pores).

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Figure S5 Morphology and surface roughness of (a) THCpSiNPs and (b) CS/THCpSiNPs by means of representative SEM images. Pores of THCpSiNPs are indicated by blue arrows (representative pores).

Figure S6 Representative TEM images of (a) THCpSiNPs, (b) oligo/THCpSiNPs, and (c) and (d) CS/oligo/THCpSiNPs.

Figure S7 Measurement of IEP by pH titration for THCpSiNPs (□) and CS/THCpSiNPs (∆). Concentration of chitosan coating solution 0.1% w/v (n = 3; mean ± standard deviation shown).

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THCpSiNP degradation

Degradation of pSiNPs produces silicic acid measured by means of an ammonium molybdate colorimetric

assay. Unmodified pSiNPs and THCpSiNPs (0.1 mg) were placed into separate 1 mL solutions of 0.25 m

Tris–HCl (pH = 7.2). Each of these individual containers (1 mL) was allocated to a predetermined measurement

time over seven days. Supernatant to be assayed for silicic acid content was acidified with 0.3 M HCl at a ratio

of 5 :2 (100 μL:40 μL). Subsequently, 20 μL of a 42 mM ammonium molybdate solution was added to the acidified

supernatant, and incubated for 10 min. Finally, 20 μL of 27 mM EDTA and 1.35 M sodium sulphite was added

to each sample solution and the sample held for 1 h incubation at room temperature. Spectrophotometric

analysis at 650 nm was performed to measure the amount of the silicic acid released into solution. A silicic acid

concentration standard curve was prepared with sodium metasilicate pentahydrate.

Biocompatibility, cell uptake, and oligonucleotide release from chitosan capped THCpSiNPs

Figure S8 Measurement of degradation for unmodified (a) pSiNPs and (b) THCpSiNPs by means of a colorimetric ammonium

molybdate assay. THCpSiNPs were placed into 1 mL of Tris buffer (pH = 7.2). Concentration of THCpSiNPs 0.1 mg/mL (n = 2; mean ±

standard deviation shown).

Figure S9 Cell viability of BSR cells incubated with CS/oligo/THCpSiNPs (+CS) and oligo/THCpSiNPs (–CS) compared to BSR cell

sample incubated without the NP (control) as determined by LDH assay after 48 h exposure to pSiNPs (n = 3; mean ± standard deviation

shown).

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Figure S10 Viability of BSR cells incubated with (a) CS/oligo/THCpSiNPs, (b) oligo/THCpSiNPs, and (c) no treatment after 24 h towards BSR cells as measured by Live/Dead assay after 48 h (NP concentration: 0.1 mg/mL).

Figure S11 Progressive Z-stack laser scanning confocal microscopy image series for BSR cells incubated with CS/FAM-oligo/ THCpSiNPs (0.1 mg/mL). Cell nuclei were stained with Hoechst 33342 (blue), the CS/cell membranes were stained with phalloidin- TRITC (red) and FAM-oligo/THCpSiNPs emit green fluorescence. The roman numbers correspond to images at different planes (height interval: 250 nm; down to up). I and V are representative of the bottom and center plane of the central BSR cell, respectively.

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Figure S12 Time-lapse fluorescence microscopy images of BSR cells after incubation with CS/FAM-oligo/THCpSiNPs (0.1 mg/mL) for (A) 0, (B) 1, (C) 3, (D) 5, (E) 7, (F) 16, and (G) 24 h. The cell nuclei were stained with Hoechst 33342 (blue), the CS/FAM-oligo/THCpSiNPs emit green fluorescence and cell membranes were stained with phalloidin-TRITC (red). The roman numbers represent (I) merged images of nuclear and FAM labeling, and (II) merged images of nuclear, FAM, and cell membrane labeling.

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Oligonucleotide release in HS

To simulate an environment closer to in vivo release conditions, CS/FAM-labeled oligo (FAM-oligo)/THCpSiNPs

coated with chitosan solution of concentration of 0.1% w/v were suspended at a concentration of 0.1 mg/mL

in HS at 37 ± 0.5 °C. Retention of FAM-labelled oligonucleotide within the NP was monitored through the

extinguishing of green fluorescence during release an inverted fluorescence microscope Eclipse Ti-S (Nikon Inc.,

Rhodes, NSW, Australia) for 48 h. Prior to imaging, CS/FAM-oligo/THCpSiNPs were centrifuged and washed

briefly with MilliQ water.

Figure S13 Release study of FAM-oligo from CS/FAM-oligo/THCpSiNPs (0.1 mg/mL) by time-lapse fluorescence microscopy imaging of CS/FAM-oligo/THCpSiNPs after incubation with HS derived from male AB plasma, T = 37 ± 0.2 °C for (a) 0, (b) 6, (c) 12, (d) 24, and (e) 48 h. The CS/FAM-oligo/THCpSiNPs emit green fluorescence because of FAM labeled oligonucleotide. The excitation 490 nm and emission 520 nm were used.

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Figure S14 TEM images of BSR cells after exposure to CS/FAM-oligo/THCpSiNPs (0.1 mg/mL) for 24 h.

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In vivo compatibility of oligonucleotide loaded chitosan capped THCpSiNPs

Figure S15 Fluorescence microscopy images of the skin tissue site after the subcutaneous injection of CS/FAM-oligo/THCpSiNPs (700 µg/kg, over the right flank) and saline (control, over the left flank). The skin tissues were stained with Hoechst 33342 (blue emission), and CS/FAM-oligo/THCpSiNPs emit green fluorescence. Accumulation of the NPs is indicated by white arrows.

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