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The formation of polyethyleneimine–trimethoxymethylsilane organic–inorganic hybrid particles
Accepted Manuscript Title: The formation of polyethyleneimine-trimethoxymethylsilane organic-inorganic hybrid particles Author: Frances Neville Thomas Murphy Erica J. Wanless PII: DOI: Reference:
S0927-7757(13)00306-3 http://dx.doi.org/doi:10.1016/j.colsurfa.2013.04.022 COLSUA 18351
To appear in:
Colloids and Surfaces A: Physicochem. Eng. Aspects
Received date: Revised date: Accepted date:
21-1-2013 11-4-2013 13-4-2013
Please cite this article as: F. Neville, T. Murphy, E.J. Wanless, The formation of polyethyleneimine-trimethoxymethylsilane organic-inorganic hybrid particles, Colloids and Surfaces A: Physicochemical and Engineering Aspects (2013), http://dx.doi.org/10.1016/j.colsurfa.2013.04.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The formation of polyethyleneimine-trimethoxymethylsilane organic-inorganic hybrid particles
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Frances Neville a,*, Thomas Murphy a, Erica J. Wanless a
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School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia.
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* Corresponding author. Dr Frances Neville, Chemistry Building, University Drive,
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NSW 2308,
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Callaghan,
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Postal address:
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Australia
Telephone: +61 249216458; Fax: +61 249215472; Email: [email protected]
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Abstract Polyethyleneimine-silica (PEI-silica) organic-inorganic hybrid particles were formed in the presence of multivalent anions, using trimethoxymethylsilane as the silica source. Here we present new understanding on the difference in particle formation when multi- and monovalent anions were
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used as well as providing clear evidence that anions other than phosphate may be used to produce PEI-silica particles, unlike the phosphorylated peptides involved in silication in nature. We also
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show that PEI-silica particle size increases with ionic strength when monovalent anions were used,
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but that the diameter is not ionic strength dependent with the use of multivalent anions. Furthermore, our results suggest that PEI-silica particle production involves the formation of
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polyethyleneimine aggregates with multivalent anions due to electrostatic interactions, producing sites from which silica particles can grow. In addition, for the first time zeta potential measurement
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data show the surface charge of the PEI-silica particles, giving evidence of the presence of the polyethyleneimine at the surface of the particles, without the need of an additional surface
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modification step. The superior understanding of silica particle growth in order to control the
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parameters needed to refine and improve the production of silica particles made using less toxic
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reagents is of great significance in silica particle fabrication and processing. In addition, the potential application of the polyethyleneimine on the silica particle surface is of great benefit since hybrid organic-inorganic silica particles have several applications from carbon dioxide capture to drug delivery in many fields of science, engineering and medicine.
Keywords: polyethyleneimine; trimethoxymethylsilane; silica particles; hybrid particles.
Abbreviations: PEI, polyethyleneimine; TMOMS, trimethoxymethylsilane, TMOS, tetramethoxysilane; DLS, dynamic light scattering; SEM, scanning electron microscopy; ATRFTIR, attenuated total reflectance Fourier transform infrared spectroscopy; EDS, Energy-dispersive X-ray spectroscopy.
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1. Introduction
Spherical silica particles are used in a wide range of applications including coatings, cosmetics, catalysis, separation materials, enzyme immobilization and sensors, [1-4] and the current global
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demand for them is forecast to grow by more than 6 % per year [5]. For optimum use it is important to characterise the particles, in particular paying attention to particle size and morphology since
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physical, electrical, and optical properties of the silica particles vary with size. In addition, it is
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advantageous if the particles can be fabricated using neutral conditions and more environmentally
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friendly reagents than commonly used methods which often involve harsh reaction conditions [6-8].
Polyethyleneimine (PEI) is a polyamine which is very much in use at present for a number of
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important applications involving particles. For instance, PEI functionalized silica is used for carbon dioxide capture from the air [9] as well as for separation applications, [10] and PEI functionalized
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particles are also used for gene delivery [11]. Furthermore, there is great potential to produce
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improved functionalised silica particles for these applications as well as biomedical materials,
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sensors and composite materials [10, 12]. However, it is vital to know the parameters which have to be tuned to be able to control the formation of the silica with the desired properties, such as size, shape and surface charge.
Recent work [4, 6, 13-18] has demonstrated that polybasic peptide mimics such as polyethyleneimines (PEIs) and polyallylamine hydrochloride are efficient at directing silica deposition into nanoparticles in a similar manner to silaffin peptides involved in biomineralisation [2, 6, 19]. This is due to the multiple amine functional groups of PEI, as is found in nature where the silaffin peptides are highly decorated with polyaminated hydrocarbon chains [19]. The method of silica formation involves hydrolysis and condensation polymerisation of silane precursors to form silica nanostructures with different morphologies including particles, fibres and star-like
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architectures amongst others [1, 3, 14, 17]. However, there is very little work [6] on the use of PEI for controlled silica particle synthesis, or indeed on the effect of different anions on particle growth.
Phosphate is thought to act as a bridge between amine groups to form polyamine aggregates [13, 21,
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23-24, 25]. It is believed that the polyamines in solution associate with multivalent anions forming aggregates and that silicic acid derivatives are adsorbed on to the aggregate or are incorporated into
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the aggregate which then solidifies into silica [13]. It has been proposed that the silica particle
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formation occurs after the aggregation of polyamines associated with multivalent anions via electrostatic interactions [13, 20, 23-24, 26-29]. However, hydrogen bonding interactions between
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the hydrogen phosphate anion and PEI are also thought to be important [3, 13, 21, 23, 25] since hydrogen bonding may occur between the silanol groups of silicic acid and proton donors such as
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amines in polyethyleneimine and free hydroxyl groups in other additives [30] . However, there is no previous work published on the hypothesis that trimethoxymethylsilane (TMOMS) polymerises
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around PEI-multivalent anion aggregates to form PEI-silica particles. In addition, most studies to
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date have used tetraethoxysilane or tetramethoxysilane for the silicic acid precursor [3, 9, 13, 23,
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31]. Other studies have used sodium silicate [6, 32], silicon catecholate which dissociates to silicic acid [33-34] and glycol modified silanes [35], but in general these silica sources do not produce uniform sized spherical particles, which was our aim. The results presented here are for trimethoxymethylsilane which is substantially less toxic and harmful than TMOS [8].
This work presents the study of bioinspired silication of TMOMS using the polyamine PEI, to form PEI-silica hybrid particles. Zeta potential measurements were made to determine the surface charge of the PEI-silica particles. Furthermore, the effects of PEI concentration, sodium phosphate buffer conditioning prior to incubation and the presence of different anions during particle synthesis were examined in order to gain insight into the mechanism of PEI-silica particle formation. ATR-FTIR
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and EDS measurements were also used to characterise the PEI-silica hybrid particles formed in this one-pot method.
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2. Experimental Section
2.1. Materials
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Hydrochloric acid, polyethyleneimine (PEI) (molecular weight 25 kDa & 750 kDa), sodium
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dihydrogen phosphate, disodium hydrogen phosphate, sodium nitrate, sodium sulfate, trisodium citrate, sodium chloride and PIPES (Piperazine-N,N'-bis(2-ethanesulfonic acid) were purchased
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from Sigma-Aldrich. The alkoxysilane precursor was trimethoxymethylsilane (TMOMS) and was also supplied by Sigma-Aldrich. Unless otherwise noted, all reagent-grade chemicals were used as
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received and deionised water (18.2 MΩ) was used in the preparation of all aqueous solutions. In general the PEI-silica particles were produced in the presence of a mixture of NaH2PO4 and
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Na2HPO4, which from now on is referred to as sodium phosphate buffer, [36] for easy comparison
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of anions. In addition, trisodium citrate was combined with sodium chloride at ten times the
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trisodium citrate concentration and this will be referred to as sodium citrate buffer [36]. All salt solutions were adjusted to pH 7.0 before use in particle synthesis using 10 mM sodium hydroxide or hydrochloric acid.
2.2. Preparation of particles.
A summary of all the experimental conditions is given in Table S1 (supplementary data). In general, polyethyleneimine (2.5 g/L) of the desired molecular weight was added to the sodium phosphate buffer followed by TMOMS. Prior to use in the reaction 1000 mM TMOMS was hydrolysed for 15 min with 1 mM hydrochloric acid. It was then added to the reaction to give a final concentration of 100 mM TMOMS in all reactions. The molar ratio of sodium phosphate buffer concentration to the concentration of the ethyleneimine moiety ([Pi]/[r.u.]), where the molar sodium phosphate buffer
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concentration is represented as [Pi] and the ethyleneimine moiety, or repeat unit, molar concentration as [r.u.] [6, 18, 23, 31] was maintained, independent of the PEI molecular weight. For example, a sodium phosphate buffer concentration of 29 mM with a 25 kDa polyethyleneimine concentration of 2.5 g/L would give a [Pi]/[r.u] ratio of 0.5, where one ethyleneimine repeat unit is
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equal to C2H5N (molar mass = 43 g/mol) [6].
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The reaction proceeded as a condensation polymerization reaction with a cloudy precipitate of
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particles formed. In general, the PEI was added to the aqueous sodium salt and the hydrolysed TMOMS was added last at which point the solution was mixed by vortex mixing. The whole
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mixture was then left to incubate for 30 min. After this time, the precipitate was washed twice by resuspension in water followed by sonication and then centrifugation at 16493 g for 2 min to
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sediment the particles, before finally resuspending the particles in water.
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2.2. Effect of PEI concentration and molecular weight
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Previous studies used a fixed molar concentration of PEI for comparison of particle formation [6,
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17]. However, this approach leads to significant variation in the concentration of ethyleneimine repeat units, especially when using a range of polymer molecular weights (25 kDa to 750 kDa). Therefore, in this study the g/L concentration was fixed in order to maintain a constant ethyleneimine repeat unit concentration, independent of the PEI molecular weight. The final polymer concentrations ranged from 1.0 to 50 g/L. The range of polymer concentrations was used with final sodium phosphate buffer concentrations of 29 mM and 250 mM.
2.3. Conditioning of PEI with phosphate prior to silication To obtain further insight into the role of phosphate ions in particle growth, the PEI was conditioned with sodium phosphate buffer for different periods of time up to 120 min prior to the addition of hydrolysed TMOMS, instead of immediately combining all the reagents together (time 0 min).
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Apart from this factor, the reaction was carried out as described in the preparation of particles section above, where the final PEI concentration was 2.5 g/L and the sodium phosphate buffer concentration was 29 mM.
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2.4. Effect of different salts
Particles were prepared using the preparation of particles procedure described above (section 2.2),
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but using different salts (sodium nitrate, sodium sulfate, sodium citrate buffer). Particles were also
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made with 1, 4-piperazinediethanesulfonic acid (PIPES) [37] as it has been successfully used in a similar particle fabrication method [6, 17]. The non-phosphate salts were initially used at 29 mM in
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the absence of phosphate. In addition, they were used at a higher concentration (100 mM) both with
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and without 29 mM sodium phosphate buffer (see Table S2, supplementary data).
2.5. Particle Characterization
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The PEI-silica hybrid particles made in the presence of different salts and molecular weights of
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polyethyleneimine were characterised to determine particle diameter and morphology together with
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elemental composition and binding by using dynamic light scattering, scanning electron microscopy EDS and ATR-FTIR.
2.5.1. Scanning electron microscopy
The PEI-silica particles were analysed directly from the final suspension using scanning electron microscopy (SEM). A drop of a particle suspension was dried onto an aluminium stub before sputter-coating with gold. A Philips XL30 SEM was used for the characterisation. The SEM images were analysed for particle size using the freeware UTHSCSA Image Tool [38]. In general, 50 different particles were analysed. The standard deviation within each image was obtained.
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2.5.2. Dynamic light scattering Particle sizes were determined using a Malvern NanoZS ZetaSizer instrument. The intensity average (hydrodynamic diameter) size distributions were obtained. The approximate particle
runs were produced with the standard error of the mean value.
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2.5.3. Zeta potential measurements
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concentration was 0.5 g/L. The mean hydrodynamic values of triplicate measurements each of 15
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Zeta potential measurements were made on PEI-silica particle suspensions against a constant background ionic strength of 10 mM sodium nitrate. The pH was adjusted with 10 mM sodium
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hydroxide or hydrochloric acid. Measurements were performed using a Malvern NanoZS ZetaSizer instrument. The approximate particle concentration was 0.5 g/L. The mean zeta potential values
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from triplicate measurements each of 15 runs were produced with the standard deviation of the
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2.5.4. ATR-FTIR
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mean value.
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A Spectrum Two (Perkin Elmer) ATR-FTIR instrument was used for making measurements. The PEI-silica particle suspension, acid hydrolysed TMOMS, PEI and sodium phosphate buffer solutions were analysed directly using the viscous liquid mode. A 20 µL aliquot was used for each measurement and the data produced was the average of four runs for each sample. The background signal of water was subtracted from the data to facilitate comparison.
2.5.5. EDS The PEI-silica particles were analysed using the EDS detector of the Sigma Zeiss FEGSEM. This instrument is a high resolution scanning electron microscope which meant that the elemental composition of individual TMOMS-PEI particles could be ascertained. The particles were mounted onto clean silicon wafers, assuring that there was no contaminant signal from the underlying
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aluminium sample stub. In addition, sputter coating of the samples was not required with this instrument. The EDS data were gathered for a one minute period when the beam was focussed on a single particle.
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3. Results and Discussion
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The biomimetic silication of trimethoxymethylsilane (TMOMS) using polyethylenimine (PEI) was
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studied in order to gain insights into the mechanism of PEI-silica hybrid particle formation and to
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fully characterise the PEI-silica hybrid particles.
3.1. Effect of polymer molecular weight and sodium phosphate buffer concentration
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Figure 1 shows the particle diameters obtained for two different molecular weight PEIs and two different concentrations of sodium phosphate buffer. The most important feature in Figure 1 is that
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the particle diameters produced with TMOMS using 25 and 750 kDa PEI gave size results which
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were similar independent of the molecular weight of the polyethyleneimine. This result indicated
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that the concentration of ethyleneimine repeat units is more important in the formation of PEI-silica particle than the molecular weight of the PEI.
According to the results shown in Figure 1, the PEI-silica particle diameter increased with [Pi]/[r.u.] until reaching a plateau at a value of around 0.8. In terms of [Pi]/[r.u.] ratio, the maximum particle diameter was seen at the highest [Pi]/[r.u.] value and the smallest particle diameters were seen at the lowest [Pi]/[r.u.] values (Figure 1). The trend line of all the data in Figure 1 follows the dose response equation in the form y=a+(b-a)/1+ c•10-dx, where a is the mean minimum particle size (100 nm), b is mean maximum particle size (400 nm) and c and d are constants ( c= 14, d = 3.3). This is a similar trend to that observed by Neville et al., [6] even though the previous study used tetramethoxysilane (TMOS) rather than TMOMS which contains a methyl group rather than the
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fourth methoxy moiety. It is also important to note that the same trend was observed whether the fixed sodium phosphate buffer concentration was low (29 mM) or relatively high (250 mM). Therefore, it is very interesting to note that this trend of increasing particle size with [Pi]/[r.u.] ratio is valid for different alkoxysilanes and for a range of absolute PEI and sodium phosphate buffer
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concentrations. However, the results are different to polyallylamine hydrochloride-silica particles previously studied in this way [39] which required a minimum [Pi]/[r.u.] ratio of 0.3 for any
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significant particle formation. This difference may be due to the structure of the polyallylamine
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hydrochloride which only contains primary amines, rather than the primary, secondary and tertiary amines found in PEI, or that Brunner et al. used sodium dihydrogen phosphate as the phosphate
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source [39], rather than the predominantly higher anion valency disodium hydrogen phosphate used
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in this study.
Figure 2 shows selected SEM images of the PEI-silica hybrid particles formed at selected points
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throughout the range of [Pi]/[r.u] ratio values shown in Figure 1. The SEM images (Figure 2)
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clearly show the increase in size with [Pi]/[r.u.] ratio and also show that the surfaces of the particles produced are fairly smooth independent of the [Pi]/[r.u.] ratio. In addition, the PEI-silica particles
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are all spherical which was not the case when TMOS was used [17] rather than TMOMS.
3.2. Presence of PEI on the silica surface It has long been known that silica has a negative charge above an isoelectric point of around pH 2, which may be observed by zeta potential measurements [40-41]. It has also been demonstrated that PEI may be adsorbed onto the surface of silica particles resulting in a shift in the isoelectric point to pH 10 or above [40-41]. The zeta potential measurements given in Figure 3 indicate that the isoelectric point of the PEI-silica particles synthesised is around pH 11.5. This suggests that at least some of the PEI involved in particle formation is on the silica surface since the results are very similar to those of silica particles whose surfaces have had PEI adsorbed to them in a separate surface modification step [41-42]. We propose that our particles contain PEI around which the silica Page 10 of 37
forms as well as it being present at the surface although the exact mechanism of particle formation is not fully clear. However, it is possible that sections of the PEI chains are present at the surface of the particles whilst being permanently cemented [15] into the silica. This is proposed since zeta potential measurements on PEI-silica hybrid particles that had been sonicated in ethanol showed
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similar results to those in Figure 3 (Figure S1, supplementary data), suggesting that the PEI could not be easily removed. Our hypothesis is supported by work [31] which proposes that positively
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charged PAMAM and polypropyleneimine dendrimers attract negatively charged silanolate groups
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from silicic acid to the growing silica particle surface, accounting for the entrapment of the
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dendrimers within the silica as is the case for PEI.
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EDS analysis was carried out on the PEI-silica particles to see if any elemental information could be obtained on the PEI-silica particles. Figure 4 shows the EDS pattern for a single PEI-silica particle
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from sample made using PEI, TMOMS and sodium phosphate buffer. It is clear to see that the only
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elements visible are silicon, oxygen, carbon and nitrogen. Since the only source of nitrogen was from the PEI it is suggested that the PEI is on the outside of the particle due to the visible signal in
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the data (Figure 4). Furthermore, even though the beam may be powerful enough to penetrate the whole particle sample, no other elements were detected, such as the cations and anions from the buffer, suggesting that they are more internal in the particles, rather than at the surface or are present at levels below the detection limit.
To further support the hypothesis of the presence of PEI on the surface of the particles, ATR-FTIR analysis was carried out. The individual components of the particle synthesis (PEI, phosphate buffer, acid hydrolysed TMOMS) were analysed as well as the washed hybrid particles. Figure 5 shows the FTIR data from 1600-600 cm-1 which show some of the defining components of the different samples analysed. The PEI FTIR data show three peaks at approximately 1113, 1040 and 950 cm-1 which correspond to the primary, secondary, tertiary C-N stretch of the PEI molecule Page 11 of 37
respectively [43]. The hydrolysed TMOMS has major peaks at 1016 cm-1 and 1113 cm-1 which correspond to Si-O-Si bonds found during the acid catalysed hydrolysis step before the reagents are combined [44]. Weaker peaks at 1272 and 1410 cm-1 correspond to the Si-CH3 moiety of the TMOMS molecule [45-46]. The main peaks from the sodium phosphate buffer analysis are found at
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1153, 1077 and 941 cm-1 and correspond to the P=O stretch of the phosphate group [47].
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When the PEI-silica particles were analysed some similar peaks from the individual reagents were
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observed such as the 1272 Si-CH3 peak from the TMOMS. In addition, a moderately strong peak at around 780 cm-1 was visible which also corresponds to the Si-CH3 group of the TMOMS molecule
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[44, 46]. As well as this strong peaks at around 1039 and 1120 cm-1 and a weaker peak at ~950 cm-1 are present in the data. These three peaks correspond to those found in the PEI sample and
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correspond to the C-N bonds of the primary, secondary and tertiary amines in the PEI sample. However, this region also overlaps with the vibration signals for Si-O-Si, Si-O and [RSiO1.5]n and
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Si-NH-Si bonds [48] which mean it is difficult to definitely assign the peak to contributions from C-
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N or Si-O-Si bonds. Nevertheless, the broadening of the peaks around 1200-1000 cm-1 indicates the
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siloxane polymer is becoming longer and branched during particle growth as suggested in the literature [48].
3.3. Effect of sodium phosphate buffer incubation The role of phosphate anions in silica formation has been studied for a number of different bioinspired polyamine sources [7, 13, 23-24, 26]. However, this is the first study of PEI-silica formation using TMOMS and PEI with different anions other than phosphate. We hypothesise that the multivalent anions, such as phosphate, induce ionic cross-linked PEI-anion aggregates to form, which then act as the nucleation sites for silica particle formation.
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Firstly the effect of conditioning the PEI with sodium phosphate buffer for different time periods before the addition of hydrolysed alkoxysilane was studied. A comparison of the SEM images of the PEI-silica particles prepared by the standard procedure (Figure 6A-B, conditioning time 0 min) with the particles made by conditioning the PEI with sodium phosphate buffer for 120 min (Figure
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6C-D) was carried out. This conditioning step results in far greater efficiency of incorporation of material into the PEI-silica hybrid samples (Figure 6C-D) compared to the unconditioned samples
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(Figure 6A-B, shown by arrows). This is viewed as evidence of the importance of PEI-phosphate
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interactions in the subsequent condensation polymerisation process, as here associations between PEI amine groups and phosphate ions can be established before silica condensation polymerisation
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is initiated. This results in more efficient conversion of silane into silica particles.
In addition, the particle sizing data obtained using dynamic light scattering for the sodium
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phosphate buffer conditioned PEI-silica particles for different time periods between 0 and 120 min
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(Figure 7) show that in general the particles increase in size with conditioning time. Since we propose that silica formation occurs around the polyamine-phosphate aggregate, it seems logical
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that if this aggregation is allowed to proceed for longer, larger aggregates will be produced, explaining the trend observed in Figure 7. It should be noted however, that there was no benefit in phosphate conditioning in excess of 90 minutes.
Furthermore, the difference in PEI-silica particle size between the particles formed with 25 or 750 kDa PEI may be due to the molecular volume of the polyamine templates. The PEI molecules would occupy distinct volumes in solution depending on their molecular weight and therefore when associated with the negatively charged anions, form different sized aggregates around which the silica would condense into particles.
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3.4. Effect of different anions and salt concentration In order to gain more insight into the growth mechanism of the silica particles made with TMOMS, some variations were made to the standard procedure. Three possible factors affecting the growth of PEI-silica particles were examined: (1) the role of phosphate ions in particle growth, (2) the
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resulting particles formed when sodium phosphate buffer is either replaced or supplemented by
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another salt and (3) the effect of ionic strength.
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Figure 8 shows the SEM diameters of PEI-silica particles produced with a PEI concentration of 2.5 g/L and 29 mM sodium nitrate, sodium sulfate, sodium citrate buffer, sodium phosphate buffer,
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or piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES). The charge of the nitrate anion and sulfonic acid groups of PIPES is 1 at neutral pH, whereas the sulfate, citrate and hydrogen phosphate anions
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are all multivalent at pH 7, as all their pKa values are substantially lower than pH 7 [43]. The data show a clear trend in terms of particle size related to charge since the multivalent anions produced
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particles almost twice the size of the particles produced with sodium nitrate or PIPES (Figure 8). At
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pH 7, the dibasic PIPES is expected to have only half of its sulfonic acid groups charged given its
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pKa of 6.8 [37]. Therefore, the ionic strength of the PIPES and sodium nitrate should be the same (Table S2 supplementary data). Conversely, the ionic strength of the sodium sulfate, sodium citrate buffer and sodium phosphate buffer should be significantly greater as shown in Table S2 (supplementary data).
The particle sizing data also suggest that there is a linear relationship of PEI-silica particle size and ionic strength for particles made with monovalent ions (Figure 9) as the particle size increases with ionic strength for the particles made with sodium nitrate and PIPES. However, particle growth does not seem to be greatly affected by ionic strength for particles made with multivalent anions, since the particles are of a similar size range independent of the ionic strength in the range 87-1687 mM (Figure 9). This may be due to the principle that multivalent anions are involved in the assembly of
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polyamines where silica condensation occurs since they contain multiple points for electrostatic interactions. Instead, monovalent anions are perhaps only involved in charge balance of the template and the growing surface of the particle [31] due to reduced charge density. Furthermore, it is proposed that the recruitment of PEI by multivalent anions to make aggregate nucleation points
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decreases the time required for the nucleation and allows growth to proceed for longer, therefore
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producing larger PEI-silica particles than when only monovalent anions are present.
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In nature silaffin peptides involved in natural silica formation (biomineralisation) are
phosphorylated, but the data show that PEI-silica formation occurs in the presence of a number of
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anions and so silica formation is not phosphate-specific when using PEI and TMOMS. The study using different anions and salt concentrations shows a clear change in particle size depending on
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whether the anion used is mono- or multivalent (Figures S2 and S3, supplementary data) suggesting that the anion charge is important in producing larger particles. This novel trend found with PEI is
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consistent with work on poly-L-arginine [30] which found that divalent anions were effective at
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converting the poly-L-arginine from a random coil to a helical structure, whereas most of the monovalent anions examined were not efficient in producing this transition. This suggests that the
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higher valence of the anion is an important factor in driving conversion and strengthening the secondary structure of the polyamine. However, it was found that this effect was specific for polyL-arginine and was not observed for poly-L-lysine or poly-L-ornithine [30].
The data suggest that the mechanism of PEI-silica particle formation using TMOMS as the silane and PEI as the polyamine is different depending on the anions used in the synthesis. The data presented here are in agreement with the polyamine-salt aggregate mechanism proposed by a number of authors [13, 20, 23-24, 26-29] who have used polyamines other than PEI, such as polyallylamine hydrochloride and poly-L-arginine to form silica particles and capsules. The polyamine-salt aggregate mechanism involves the combination of the cationic polyamine with a multivalent anionic salt and results in electrostatically cross-linked polyamine-salt aggregates [27Page 15 of 37
29]. Figure 10 shows a representation of the difference in PEI-silica particle formation when using monovalent and multivalent anions, based on the experimental results we have presented. The pH is also important in the formation of polyamine-silica as it must be below the pKa of the polyamine but high enough that the effective charge of the anion is controlled by the pH of the solution [27].
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This information sheds light on the mechanism of PEI-silica formation using TMOMS as the silane and PEI as the polyamine, since the pH of the syntheses carried out was optimal for particle
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formation to occur. Therefore, we propose that larger PEI-silica particles formed with multivalent
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anions due to polymer-anion aggregation which acts as a starting point for silica growth. This
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process did not occur with monovalent salts, in which case producing much smaller particles.
In order to test the hypothesis that multivalent anions aggregate with PEI, experiments were
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performed to determine the hydrodynamic diameter of PEI when combined with nitrate anions (monovalent) and phosphate anions (multivalent). Our hypothesis is supported by results which
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show the aggregation of single PEI molecules with multivalent anions, but not monovalent anions
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(Figure 11). The data (Figure 11) show the particle size distribution of PEI alone and the changes
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which occurred with the addition of sodium nitrate and sodium phosphate buffer. The mean PEI hydrodynamic diameter was determined to be 9.6 ± 1.3 nm which is similar to a previously published value [49]. This value only increased to 11.4 ± 1.3 nm after sodium nitrate was added to the PEI. However, when sodium phosphate buffer was added to the PEI the hydrodynamic diameter increased by nearly 65 % to 15.8 ± 2.4 nm, indicating a different behaviour of the PEI towards mono- and multivalent anions. This data support our hypothesis that the multivalent anions aggregate with PEI to later form larger PEI-silica hybrid particles than the PEI-silica particles made with monovalent anions which are smaller, most likely due to the absence of PEI-anion aggregation.
4. Conclusions
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A study of bioinspired silication was presented in which polyethyleneimine (PEI) was used to induce the condensation polymerisation of hydrolysed trimethoxymethylsilane (TMOMS) to form PEI-silica hybrid particles. The effects of PEI concentration, sodium phosphate buffer conditioning prior to silication, ionic strength and the presence of different anions during the silication reaction
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were examined in order to gain insight into the role of different anions in silica particle formation. Scanning electron microscopy, dynamic light scattering and zeta potential, ATR-FTIR and EDS
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measurements were used to characterise particle size, morphology, surface charge, elemental
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composition and bonding.
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The net positive charge of the PEI-silica particles in the pH range of 2-11 was confirmed with zeta potential measurements for the first time, suggesting that some of the PEI involved in particle
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formation is also present on the silica surface. The use of different salts has lead us to conclude that electrostatic interactions between multivalent anions and PEI polyamine groups are the major force
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in the production of polyamine aggregates which act as condensation sites for the formation of
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larger PEI-silica particles than particles produced with monovalent anions. In addition, particle size
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is affected by ionic strength, only when the anions present are monovalent, since very little change in particle diameter was observed over a range of ionic strengths of multivalent anions. The increased knowledge of this tuneable silica synthesis which uses more environmentally friendly reagents than previous methods is of significant benefit in producing silica particles which have a wide range of applications and the global demand of which increases annually.
In conclusion, it is clear that a superior understanding of silica particle growth is of great use in order to control the parameters needed to refine and improve the production of silica particles made using less toxic reagents, since the use of silica particles has applications in many fields of science, engineering and medicine.
Page 17 of 37
Acknowledgements The authors wish to acknowledge the University of Newcastle Electron Microscope X-ray Unit. FN is the recipient of a University of Newcastle fellowship. TM acknowledges the Faculty of Science
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and IT, University of Newcastle for summer scholarship funding.
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[41] L. Avadiar, Y.K. Leong, Interactions of PEI (polyethylenimine)–silica particles with citric acid in dispersions, Colloid Polym. Sci., 289 (2011) 237-245. [42] B.C. Ong, Y.K. Leong, S.B. Chen, Interparticle forces in spherical monodispersed silica dispersions: Effects of branched polyethylenimine and molecular weight, J. Coll. Interf. Sci. 337
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[43] "Middle-Range Infrared Absorption Correlation Charts," in CRC Handbook of Chemistry
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[44] V.V. Ganbavle, U.K.H. Bangi, S.S. Latthe, S.A. Mahadik, A.Venkateswara Rao, Self-cleaning
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silica coatings on glass by single step sol-gel route, Surf. Coat. Tech. 205 (2011) 5338-5344. [45]R. Al-Oweini, H. El-Rassy, Synthesis and characterization by FTIR spectroscopy of silica
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aerogels prepared using several Si(OR)4 and R´´Si(OR´)3 precursors, J. Mol. Struct. 919 (2009) 140145.
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[46] S. Amoriello, A. Bianco, L. Eusebio, P. Gronchi, Evolution of two acid steps sol-gel phases by
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FTIR, J. Sol-Gel Sci. Technol. 58 (2011) 209-217.
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[47] M. Badertscher, P. Bühlman, E. Pretsch, Structure determination of organic compounds. Tables of spectral data, 4th edition, Springer, 2009. [48] P.J. Launer, P.J., Infrared Analysis of Organosilicon Compounds: Spectra-Structure Correlations. Silicone Compounds Register and Review, 100-103, Bristol USA, Petrarch Systems, 1987.
[49] M.M. Andersson, R. Hatti-Kaul, Dynamic and static light scattering and fluorescence studies of the interactions between lactate dehydrogenase and poly(ethyleneimine), J. Phys. Chem. 104 (2000) 3660-3667.
Page 22 of 37
Figure Legends
Fig. 1. Effect of [Pi]/[r.u.] ratio on PEI-silica particle diameter for 25 and 750 kDa PEI and sodium
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phosphate buffer (Pi) concentrations of 29 mM and 250 mM.
Fig. 2. SEM images showing the effect of [Pi]/[r.u.] on PEI-silica particle growth. The [Pi]/[r.u.]
us
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ratios are as follows: A) 0.025; B) 0.2; C) 0.5; D) 0.72 and E) 1.25. The scale bar is 1 µm.
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Fig. 3. Zeta potential as a function of pH of PEI-silica particles made with TMOMS and PEI (25 kDa ). The trend line follows a third order polynomial (y = -0.047x3 + 0.202x2 - 0.185x + 46.00, R²
M
= 0.994).
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Fig. 4. EDS pattern for PEI-silica particles made with sodium phosphate showing the presence of
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nitrogen from the PEI as well as silicon, oxygen and carbon.
Fig.5. ATR-FTIR of PEI (black line), washed PEI-silica hybrid particles (dotted), TMOMS hydrolysed in acidic conditions (grey line) and sodium phosphate buffer (dashed line). Data are offset vertically for clarity.
Fig. 6. SEM images showing the effect of different sodium phosphate buffer conditioning times on PEI-silica particle formation. (A) 25 kDa PEI, 0 min; (B) 750 kDa PEI, 0 min; (C) 25 kDa PEI, 120 min; (D) 750 kDa PEI 120 min. The arrows indicated uncondensed silica. Scale bar is 1 µm.
Fig. 7. Effect of different sodium phosphate buffer conditioning times on PEI-silica particle diameter determined using dynamic light scattering.
Page 23 of 37
Fig. 8. Effect of anion identity on particle size. PEI-silica particles were made with 29 mM salt concentration.
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Fig.9. Hydrodynamic diameter of PEI-silica particles produced with multivalent and monovalent anions as a function of ionic strength. The arrows indicate that data for PEI-silica particles made
cr
with sodium citrate buffer at 1600 and 1687 mM ionic strength, for both molecular weights of PEI
us
is included in the linear fit of the data. The values are given in Table S3, supplementary data.
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Fig. 10. Proposed mechanism of formation of PEI-silica particles using mono- or multivalent
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anions.
Fig.11. Particle size distribution of PEI (black) with addition of sodium nitrate (grey) and sodium
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induced polymer aggregation.
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phosphate buffer (grey hatched line), indicating that there is no substantial monovalent anion
Page 24 of 37
Highlights Hybrid particles were made with polyethylenimine (PEI) and trimethoxymethylsilane
(TMOMS). The effects of PEI concentration and different anions were studied.
Multivalent anions produced larger particles than monovalent anions.
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te
d
M
an
us
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PEI aggregates with multivalent anions followed by silica growth.
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PEI is present at the surface of the silica hybrid particles made with TMOMS.
Page 25 of 37
*Graphical Abstract (for review)
700
multivalent anions: ionic strength does not affect diameter
600 550
450 400 1 μm
multivalent anions: ionic strength does not affect diameter 700
300
650600
150
550 ionic strength is proportional to diameter monovalent anions: 500
0
231 38 nm
600
Sodium phosphate buffer
500 500 1000 450 ionic strength (mM) 400 400
1500
350
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200
25 kDa PEI + multivalent anions multivalent anions: ionic strength does not affect diameter 750 kDa PEI + multivalent anions 25 kDa PEI + monovalent anions 750 kDa PEI + monovalent anions
cr
250
Hydrodynamic diameter (nm)
350
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500
hydrodynamic diameter (nm)
Sodium nitrate
hydrodynamic diameter (nm)
650
25 kDa PEI + multivalent anions 750 kDa PEI + multivalent anions 25 kDa PEI + monovalent anions 750 kDa PEI + monovalent anions
300300 250 200200
an
monovalent anions: to 400 diameter 500 0 100 ionic strength 200 is proportional 300 (mM) 1500 0 500 Ionic strength 1000 monovalent anions: diameter is proportional to ionic strength ionic strength (mM)
150
Ac ce
pt
ed
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476 42 nm
Page 26 of 37
ep t
600 Figure 1
400
Ac c
SEM particle diameter (nm)
500
300
200
100
0
0
0.2
0.4
25 kDa PEI + 29 mM Pi 750 kDa PEI + 29 mM Pi 25 kDa PEI + 250 mM Pi Page of mM 37 Pi 750 kDa PEI27 + 250
0.6 0.8 [Pi]/[r.u.]
1
1.2
1 μm
A
Ac
Figure 2
B
C
D
E Page 28 of 37
pt
Figure 3 60
ce
40 30 20 10
Ac
zeta potential (mV)
50
0
-10 -20
Page 29 of 37
-30 0
2
4
6
8 pH
10
12
14
3000 2500 2000
counts per second/eV
ce
O
Ac
counts per second/eV
Figure 3500 4
1.0
C
0.5 N 0.0 0.2
0.3 keV
0.4
Si
1500
C
1000
500
NN
Page 30 of 37
0 0
1
keV
2
3
Figure 5
M
950
1040
1113
C-N 1O, 2O, 3O
P
750
1039
1120
ep 1016
1113
Si-O-Si
Ac c
1272
Si-CH3 1410
te
950
d
Si-O-Si
1272
1410
Si-CH3
1600
1400
941
1077
1153
P=O
1200 1000 Page 800 31 of 37600 wavenumber (cm-1)
pt
1 μm
B
ce
Figure 6 A
C
Ac
330 37 nm
312 24 nm
D
Page 32 of 37 459 30 nm
410 24 nm
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600
25 kDa PEI
750 kDa PEI
500 400
Ac
Hydrodynamic diameter (nm)
700 Figure 7
300 200 100
Page 33 of 37 0 0
30 60 Conditioning time (min)
90
120
500
25 kDa PEI 750 kDa PEI
Ac c
400
SEM diameter (nm)
ep
Figure 8
300
200
100
Page 34 of 37
0 sodium nitrate
PIPES
sodium sulfate
sodium citrate buffer
sodium phosphate buffer
ep t
650 9 Figure
550 500
Ac c
Hydrodynamic diameter (nm)
600
450
400 350 300
25 kDa PEI + multivalent anions 750 kDa PEI + multivalent anions 25 kDa PEI + monovalent anions 750 kDa PEI + monovalent anions
250
Page 35 of 37
200 0
100
200 300 400 Ionic strength (mM)
500
600
-
+ + +
+ +
+
+
+
+ +
+ +
+ +
+
+
+
+
+
+
+
+
+
+
+
+ +
+
- +
+
+
+
+ +
+ TMOMS
+
+ +
PEI
+ TMOMS
Smaller PEI-silica hybrid particles
+ Multivalent anions
+ +
-
- -
+
Ac
+
ce
+
+
+
+ Monovalent anions
+
+
+
-
+
+
pt
Figure 10
+ +
PEI-multivalent anion aggregate
Page 36 of 37 Larger PEI-silica hybrid particles
45
35
Ac c
Mean intensity %
40
ep
Figure 11 50
30 25 20 15 10
5
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0 5
10
15
20
Hydrodynamic diameter (nm)
25
S0927-7757(13)00306-3 http://dx.doi.org/doi:10.1016/j.colsurfa.2013.04.022 COLSUA 18351
To appear in:
Colloids and Surfaces A: Physicochem. Eng. Aspects
Received date: Revised date: Accepted date:
21-1-2013 11-4-2013 13-4-2013
Please cite this article as: F. Neville, T. Murphy, E.J. Wanless, The formation of polyethyleneimine-trimethoxymethylsilane organic-inorganic hybrid particles, Colloids and Surfaces A: Physicochemical and Engineering Aspects (2013), http://dx.doi.org/10.1016/j.colsurfa.2013.04.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The formation of polyethyleneimine-trimethoxymethylsilane organic-inorganic hybrid particles
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Frances Neville a,*, Thomas Murphy a, Erica J. Wanless a
a
us
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School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia.
an
* Corresponding author. Dr Frances Neville, Chemistry Building, University Drive,
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NSW 2308,
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Callaghan,
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Postal address:
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Australia
Telephone: +61 249216458; Fax: +61 249215472; Email: [email protected]
Page 1 of 37
Abstract Polyethyleneimine-silica (PEI-silica) organic-inorganic hybrid particles were formed in the presence of multivalent anions, using trimethoxymethylsilane as the silica source. Here we present new understanding on the difference in particle formation when multi- and monovalent anions were
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used as well as providing clear evidence that anions other than phosphate may be used to produce PEI-silica particles, unlike the phosphorylated peptides involved in silication in nature. We also
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show that PEI-silica particle size increases with ionic strength when monovalent anions were used,
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but that the diameter is not ionic strength dependent with the use of multivalent anions. Furthermore, our results suggest that PEI-silica particle production involves the formation of
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polyethyleneimine aggregates with multivalent anions due to electrostatic interactions, producing sites from which silica particles can grow. In addition, for the first time zeta potential measurement
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data show the surface charge of the PEI-silica particles, giving evidence of the presence of the polyethyleneimine at the surface of the particles, without the need of an additional surface
d
modification step. The superior understanding of silica particle growth in order to control the
te
parameters needed to refine and improve the production of silica particles made using less toxic
Ac ce p
reagents is of great significance in silica particle fabrication and processing. In addition, the potential application of the polyethyleneimine on the silica particle surface is of great benefit since hybrid organic-inorganic silica particles have several applications from carbon dioxide capture to drug delivery in many fields of science, engineering and medicine.
Keywords: polyethyleneimine; trimethoxymethylsilane; silica particles; hybrid particles.
Abbreviations: PEI, polyethyleneimine; TMOMS, trimethoxymethylsilane, TMOS, tetramethoxysilane; DLS, dynamic light scattering; SEM, scanning electron microscopy; ATRFTIR, attenuated total reflectance Fourier transform infrared spectroscopy; EDS, Energy-dispersive X-ray spectroscopy.
Page 2 of 37
1. Introduction
Spherical silica particles are used in a wide range of applications including coatings, cosmetics, catalysis, separation materials, enzyme immobilization and sensors, [1-4] and the current global
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demand for them is forecast to grow by more than 6 % per year [5]. For optimum use it is important to characterise the particles, in particular paying attention to particle size and morphology since
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physical, electrical, and optical properties of the silica particles vary with size. In addition, it is
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advantageous if the particles can be fabricated using neutral conditions and more environmentally
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friendly reagents than commonly used methods which often involve harsh reaction conditions [6-8].
Polyethyleneimine (PEI) is a polyamine which is very much in use at present for a number of
M
important applications involving particles. For instance, PEI functionalized silica is used for carbon dioxide capture from the air [9] as well as for separation applications, [10] and PEI functionalized
d
particles are also used for gene delivery [11]. Furthermore, there is great potential to produce
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improved functionalised silica particles for these applications as well as biomedical materials,
Ac ce p
sensors and composite materials [10, 12]. However, it is vital to know the parameters which have to be tuned to be able to control the formation of the silica with the desired properties, such as size, shape and surface charge.
Recent work [4, 6, 13-18] has demonstrated that polybasic peptide mimics such as polyethyleneimines (PEIs) and polyallylamine hydrochloride are efficient at directing silica deposition into nanoparticles in a similar manner to silaffin peptides involved in biomineralisation [2, 6, 19]. This is due to the multiple amine functional groups of PEI, as is found in nature where the silaffin peptides are highly decorated with polyaminated hydrocarbon chains [19]. The method of silica formation involves hydrolysis and condensation polymerisation of silane precursors to form silica nanostructures with different morphologies including particles, fibres and star-like
Page 3 of 37
architectures amongst others [1, 3, 14, 17]. However, there is very little work [6] on the use of PEI for controlled silica particle synthesis, or indeed on the effect of different anions on particle growth.
Phosphate is thought to act as a bridge between amine groups to form polyamine aggregates [13, 21,
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23-24, 25]. It is believed that the polyamines in solution associate with multivalent anions forming aggregates and that silicic acid derivatives are adsorbed on to the aggregate or are incorporated into
cr
the aggregate which then solidifies into silica [13]. It has been proposed that the silica particle
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formation occurs after the aggregation of polyamines associated with multivalent anions via electrostatic interactions [13, 20, 23-24, 26-29]. However, hydrogen bonding interactions between
an
the hydrogen phosphate anion and PEI are also thought to be important [3, 13, 21, 23, 25] since hydrogen bonding may occur between the silanol groups of silicic acid and proton donors such as
M
amines in polyethyleneimine and free hydroxyl groups in other additives [30] . However, there is no previous work published on the hypothesis that trimethoxymethylsilane (TMOMS) polymerises
d
around PEI-multivalent anion aggregates to form PEI-silica particles. In addition, most studies to
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date have used tetraethoxysilane or tetramethoxysilane for the silicic acid precursor [3, 9, 13, 23,
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31]. Other studies have used sodium silicate [6, 32], silicon catecholate which dissociates to silicic acid [33-34] and glycol modified silanes [35], but in general these silica sources do not produce uniform sized spherical particles, which was our aim. The results presented here are for trimethoxymethylsilane which is substantially less toxic and harmful than TMOS [8].
This work presents the study of bioinspired silication of TMOMS using the polyamine PEI, to form PEI-silica hybrid particles. Zeta potential measurements were made to determine the surface charge of the PEI-silica particles. Furthermore, the effects of PEI concentration, sodium phosphate buffer conditioning prior to incubation and the presence of different anions during particle synthesis were examined in order to gain insight into the mechanism of PEI-silica particle formation. ATR-FTIR
Page 4 of 37
and EDS measurements were also used to characterise the PEI-silica hybrid particles formed in this one-pot method.
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2. Experimental Section
2.1. Materials
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Hydrochloric acid, polyethyleneimine (PEI) (molecular weight 25 kDa & 750 kDa), sodium
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dihydrogen phosphate, disodium hydrogen phosphate, sodium nitrate, sodium sulfate, trisodium citrate, sodium chloride and PIPES (Piperazine-N,N'-bis(2-ethanesulfonic acid) were purchased
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from Sigma-Aldrich. The alkoxysilane precursor was trimethoxymethylsilane (TMOMS) and was also supplied by Sigma-Aldrich. Unless otherwise noted, all reagent-grade chemicals were used as
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received and deionised water (18.2 MΩ) was used in the preparation of all aqueous solutions. In general the PEI-silica particles were produced in the presence of a mixture of NaH2PO4 and
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Na2HPO4, which from now on is referred to as sodium phosphate buffer, [36] for easy comparison
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of anions. In addition, trisodium citrate was combined with sodium chloride at ten times the
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trisodium citrate concentration and this will be referred to as sodium citrate buffer [36]. All salt solutions were adjusted to pH 7.0 before use in particle synthesis using 10 mM sodium hydroxide or hydrochloric acid.
2.2. Preparation of particles.
A summary of all the experimental conditions is given in Table S1 (supplementary data). In general, polyethyleneimine (2.5 g/L) of the desired molecular weight was added to the sodium phosphate buffer followed by TMOMS. Prior to use in the reaction 1000 mM TMOMS was hydrolysed for 15 min with 1 mM hydrochloric acid. It was then added to the reaction to give a final concentration of 100 mM TMOMS in all reactions. The molar ratio of sodium phosphate buffer concentration to the concentration of the ethyleneimine moiety ([Pi]/[r.u.]), where the molar sodium phosphate buffer
Page 5 of 37
concentration is represented as [Pi] and the ethyleneimine moiety, or repeat unit, molar concentration as [r.u.] [6, 18, 23, 31] was maintained, independent of the PEI molecular weight. For example, a sodium phosphate buffer concentration of 29 mM with a 25 kDa polyethyleneimine concentration of 2.5 g/L would give a [Pi]/[r.u] ratio of 0.5, where one ethyleneimine repeat unit is
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equal to C2H5N (molar mass = 43 g/mol) [6].
cr
The reaction proceeded as a condensation polymerization reaction with a cloudy precipitate of
us
particles formed. In general, the PEI was added to the aqueous sodium salt and the hydrolysed TMOMS was added last at which point the solution was mixed by vortex mixing. The whole
an
mixture was then left to incubate for 30 min. After this time, the precipitate was washed twice by resuspension in water followed by sonication and then centrifugation at 16493 g for 2 min to
M
sediment the particles, before finally resuspending the particles in water.
d
2.2. Effect of PEI concentration and molecular weight
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Previous studies used a fixed molar concentration of PEI for comparison of particle formation [6,
Ac ce p
17]. However, this approach leads to significant variation in the concentration of ethyleneimine repeat units, especially when using a range of polymer molecular weights (25 kDa to 750 kDa). Therefore, in this study the g/L concentration was fixed in order to maintain a constant ethyleneimine repeat unit concentration, independent of the PEI molecular weight. The final polymer concentrations ranged from 1.0 to 50 g/L. The range of polymer concentrations was used with final sodium phosphate buffer concentrations of 29 mM and 250 mM.
2.3. Conditioning of PEI with phosphate prior to silication To obtain further insight into the role of phosphate ions in particle growth, the PEI was conditioned with sodium phosphate buffer for different periods of time up to 120 min prior to the addition of hydrolysed TMOMS, instead of immediately combining all the reagents together (time 0 min).
Page 6 of 37
Apart from this factor, the reaction was carried out as described in the preparation of particles section above, where the final PEI concentration was 2.5 g/L and the sodium phosphate buffer concentration was 29 mM.
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2.4. Effect of different salts
Particles were prepared using the preparation of particles procedure described above (section 2.2),
cr
but using different salts (sodium nitrate, sodium sulfate, sodium citrate buffer). Particles were also
us
made with 1, 4-piperazinediethanesulfonic acid (PIPES) [37] as it has been successfully used in a similar particle fabrication method [6, 17]. The non-phosphate salts were initially used at 29 mM in
an
the absence of phosphate. In addition, they were used at a higher concentration (100 mM) both with
M
and without 29 mM sodium phosphate buffer (see Table S2, supplementary data).
2.5. Particle Characterization
d
The PEI-silica hybrid particles made in the presence of different salts and molecular weights of
te
polyethyleneimine were characterised to determine particle diameter and morphology together with
Ac ce p
elemental composition and binding by using dynamic light scattering, scanning electron microscopy EDS and ATR-FTIR.
2.5.1. Scanning electron microscopy
The PEI-silica particles were analysed directly from the final suspension using scanning electron microscopy (SEM). A drop of a particle suspension was dried onto an aluminium stub before sputter-coating with gold. A Philips XL30 SEM was used for the characterisation. The SEM images were analysed for particle size using the freeware UTHSCSA Image Tool [38]. In general, 50 different particles were analysed. The standard deviation within each image was obtained.
Page 7 of 37
2.5.2. Dynamic light scattering Particle sizes were determined using a Malvern NanoZS ZetaSizer instrument. The intensity average (hydrodynamic diameter) size distributions were obtained. The approximate particle
runs were produced with the standard error of the mean value.
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2.5.3. Zeta potential measurements
ip t
concentration was 0.5 g/L. The mean hydrodynamic values of triplicate measurements each of 15
us
Zeta potential measurements were made on PEI-silica particle suspensions against a constant background ionic strength of 10 mM sodium nitrate. The pH was adjusted with 10 mM sodium
an
hydroxide or hydrochloric acid. Measurements were performed using a Malvern NanoZS ZetaSizer instrument. The approximate particle concentration was 0.5 g/L. The mean zeta potential values
M
from triplicate measurements each of 15 runs were produced with the standard deviation of the
te
2.5.4. ATR-FTIR
d
mean value.
Ac ce p
A Spectrum Two (Perkin Elmer) ATR-FTIR instrument was used for making measurements. The PEI-silica particle suspension, acid hydrolysed TMOMS, PEI and sodium phosphate buffer solutions were analysed directly using the viscous liquid mode. A 20 µL aliquot was used for each measurement and the data produced was the average of four runs for each sample. The background signal of water was subtracted from the data to facilitate comparison.
2.5.5. EDS The PEI-silica particles were analysed using the EDS detector of the Sigma Zeiss FEGSEM. This instrument is a high resolution scanning electron microscope which meant that the elemental composition of individual TMOMS-PEI particles could be ascertained. The particles were mounted onto clean silicon wafers, assuring that there was no contaminant signal from the underlying
Page 8 of 37
aluminium sample stub. In addition, sputter coating of the samples was not required with this instrument. The EDS data were gathered for a one minute period when the beam was focussed on a single particle.
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3. Results and Discussion
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The biomimetic silication of trimethoxymethylsilane (TMOMS) using polyethylenimine (PEI) was
us
studied in order to gain insights into the mechanism of PEI-silica hybrid particle formation and to
an
fully characterise the PEI-silica hybrid particles.
3.1. Effect of polymer molecular weight and sodium phosphate buffer concentration
M
Figure 1 shows the particle diameters obtained for two different molecular weight PEIs and two different concentrations of sodium phosphate buffer. The most important feature in Figure 1 is that
d
the particle diameters produced with TMOMS using 25 and 750 kDa PEI gave size results which
te
were similar independent of the molecular weight of the polyethyleneimine. This result indicated
Ac ce p
that the concentration of ethyleneimine repeat units is more important in the formation of PEI-silica particle than the molecular weight of the PEI.
According to the results shown in Figure 1, the PEI-silica particle diameter increased with [Pi]/[r.u.] until reaching a plateau at a value of around 0.8. In terms of [Pi]/[r.u.] ratio, the maximum particle diameter was seen at the highest [Pi]/[r.u.] value and the smallest particle diameters were seen at the lowest [Pi]/[r.u.] values (Figure 1). The trend line of all the data in Figure 1 follows the dose response equation in the form y=a+(b-a)/1+ c•10-dx, where a is the mean minimum particle size (100 nm), b is mean maximum particle size (400 nm) and c and d are constants ( c= 14, d = 3.3). This is a similar trend to that observed by Neville et al., [6] even though the previous study used tetramethoxysilane (TMOS) rather than TMOMS which contains a methyl group rather than the
Page 9 of 37
fourth methoxy moiety. It is also important to note that the same trend was observed whether the fixed sodium phosphate buffer concentration was low (29 mM) or relatively high (250 mM). Therefore, it is very interesting to note that this trend of increasing particle size with [Pi]/[r.u.] ratio is valid for different alkoxysilanes and for a range of absolute PEI and sodium phosphate buffer
ip t
concentrations. However, the results are different to polyallylamine hydrochloride-silica particles previously studied in this way [39] which required a minimum [Pi]/[r.u.] ratio of 0.3 for any
cr
significant particle formation. This difference may be due to the structure of the polyallylamine
us
hydrochloride which only contains primary amines, rather than the primary, secondary and tertiary amines found in PEI, or that Brunner et al. used sodium dihydrogen phosphate as the phosphate
an
source [39], rather than the predominantly higher anion valency disodium hydrogen phosphate used
M
in this study.
Figure 2 shows selected SEM images of the PEI-silica hybrid particles formed at selected points
d
throughout the range of [Pi]/[r.u] ratio values shown in Figure 1. The SEM images (Figure 2)
te
clearly show the increase in size with [Pi]/[r.u.] ratio and also show that the surfaces of the particles produced are fairly smooth independent of the [Pi]/[r.u.] ratio. In addition, the PEI-silica particles
Ac ce p
are all spherical which was not the case when TMOS was used [17] rather than TMOMS.
3.2. Presence of PEI on the silica surface It has long been known that silica has a negative charge above an isoelectric point of around pH 2, which may be observed by zeta potential measurements [40-41]. It has also been demonstrated that PEI may be adsorbed onto the surface of silica particles resulting in a shift in the isoelectric point to pH 10 or above [40-41]. The zeta potential measurements given in Figure 3 indicate that the isoelectric point of the PEI-silica particles synthesised is around pH 11.5. This suggests that at least some of the PEI involved in particle formation is on the silica surface since the results are very similar to those of silica particles whose surfaces have had PEI adsorbed to them in a separate surface modification step [41-42]. We propose that our particles contain PEI around which the silica Page 10 of 37
forms as well as it being present at the surface although the exact mechanism of particle formation is not fully clear. However, it is possible that sections of the PEI chains are present at the surface of the particles whilst being permanently cemented [15] into the silica. This is proposed since zeta potential measurements on PEI-silica hybrid particles that had been sonicated in ethanol showed
ip t
similar results to those in Figure 3 (Figure S1, supplementary data), suggesting that the PEI could not be easily removed. Our hypothesis is supported by work [31] which proposes that positively
cr
charged PAMAM and polypropyleneimine dendrimers attract negatively charged silanolate groups
us
from silicic acid to the growing silica particle surface, accounting for the entrapment of the
an
dendrimers within the silica as is the case for PEI.
M
EDS analysis was carried out on the PEI-silica particles to see if any elemental information could be obtained on the PEI-silica particles. Figure 4 shows the EDS pattern for a single PEI-silica particle
d
from sample made using PEI, TMOMS and sodium phosphate buffer. It is clear to see that the only
te
elements visible are silicon, oxygen, carbon and nitrogen. Since the only source of nitrogen was from the PEI it is suggested that the PEI is on the outside of the particle due to the visible signal in
Ac ce p
the data (Figure 4). Furthermore, even though the beam may be powerful enough to penetrate the whole particle sample, no other elements were detected, such as the cations and anions from the buffer, suggesting that they are more internal in the particles, rather than at the surface or are present at levels below the detection limit.
To further support the hypothesis of the presence of PEI on the surface of the particles, ATR-FTIR analysis was carried out. The individual components of the particle synthesis (PEI, phosphate buffer, acid hydrolysed TMOMS) were analysed as well as the washed hybrid particles. Figure 5 shows the FTIR data from 1600-600 cm-1 which show some of the defining components of the different samples analysed. The PEI FTIR data show three peaks at approximately 1113, 1040 and 950 cm-1 which correspond to the primary, secondary, tertiary C-N stretch of the PEI molecule Page 11 of 37
respectively [43]. The hydrolysed TMOMS has major peaks at 1016 cm-1 and 1113 cm-1 which correspond to Si-O-Si bonds found during the acid catalysed hydrolysis step before the reagents are combined [44]. Weaker peaks at 1272 and 1410 cm-1 correspond to the Si-CH3 moiety of the TMOMS molecule [45-46]. The main peaks from the sodium phosphate buffer analysis are found at
ip t
1153, 1077 and 941 cm-1 and correspond to the P=O stretch of the phosphate group [47].
cr
When the PEI-silica particles were analysed some similar peaks from the individual reagents were
us
observed such as the 1272 Si-CH3 peak from the TMOMS. In addition, a moderately strong peak at around 780 cm-1 was visible which also corresponds to the Si-CH3 group of the TMOMS molecule
an
[44, 46]. As well as this strong peaks at around 1039 and 1120 cm-1 and a weaker peak at ~950 cm-1 are present in the data. These three peaks correspond to those found in the PEI sample and
M
correspond to the C-N bonds of the primary, secondary and tertiary amines in the PEI sample. However, this region also overlaps with the vibration signals for Si-O-Si, Si-O and [RSiO1.5]n and
d
Si-NH-Si bonds [48] which mean it is difficult to definitely assign the peak to contributions from C-
te
N or Si-O-Si bonds. Nevertheless, the broadening of the peaks around 1200-1000 cm-1 indicates the
Ac ce p
siloxane polymer is becoming longer and branched during particle growth as suggested in the literature [48].
3.3. Effect of sodium phosphate buffer incubation The role of phosphate anions in silica formation has been studied for a number of different bioinspired polyamine sources [7, 13, 23-24, 26]. However, this is the first study of PEI-silica formation using TMOMS and PEI with different anions other than phosphate. We hypothesise that the multivalent anions, such as phosphate, induce ionic cross-linked PEI-anion aggregates to form, which then act as the nucleation sites for silica particle formation.
Page 12 of 37
Firstly the effect of conditioning the PEI with sodium phosphate buffer for different time periods before the addition of hydrolysed alkoxysilane was studied. A comparison of the SEM images of the PEI-silica particles prepared by the standard procedure (Figure 6A-B, conditioning time 0 min) with the particles made by conditioning the PEI with sodium phosphate buffer for 120 min (Figure
ip t
6C-D) was carried out. This conditioning step results in far greater efficiency of incorporation of material into the PEI-silica hybrid samples (Figure 6C-D) compared to the unconditioned samples
cr
(Figure 6A-B, shown by arrows). This is viewed as evidence of the importance of PEI-phosphate
us
interactions in the subsequent condensation polymerisation process, as here associations between PEI amine groups and phosphate ions can be established before silica condensation polymerisation
M
an
is initiated. This results in more efficient conversion of silane into silica particles.
In addition, the particle sizing data obtained using dynamic light scattering for the sodium
d
phosphate buffer conditioned PEI-silica particles for different time periods between 0 and 120 min
te
(Figure 7) show that in general the particles increase in size with conditioning time. Since we propose that silica formation occurs around the polyamine-phosphate aggregate, it seems logical
Ac ce p
that if this aggregation is allowed to proceed for longer, larger aggregates will be produced, explaining the trend observed in Figure 7. It should be noted however, that there was no benefit in phosphate conditioning in excess of 90 minutes.
Furthermore, the difference in PEI-silica particle size between the particles formed with 25 or 750 kDa PEI may be due to the molecular volume of the polyamine templates. The PEI molecules would occupy distinct volumes in solution depending on their molecular weight and therefore when associated with the negatively charged anions, form different sized aggregates around which the silica would condense into particles.
Page 13 of 37
3.4. Effect of different anions and salt concentration In order to gain more insight into the growth mechanism of the silica particles made with TMOMS, some variations were made to the standard procedure. Three possible factors affecting the growth of PEI-silica particles were examined: (1) the role of phosphate ions in particle growth, (2) the
ip t
resulting particles formed when sodium phosphate buffer is either replaced or supplemented by
cr
another salt and (3) the effect of ionic strength.
us
Figure 8 shows the SEM diameters of PEI-silica particles produced with a PEI concentration of 2.5 g/L and 29 mM sodium nitrate, sodium sulfate, sodium citrate buffer, sodium phosphate buffer,
an
or piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES). The charge of the nitrate anion and sulfonic acid groups of PIPES is 1 at neutral pH, whereas the sulfate, citrate and hydrogen phosphate anions
M
are all multivalent at pH 7, as all their pKa values are substantially lower than pH 7 [43]. The data show a clear trend in terms of particle size related to charge since the multivalent anions produced
d
particles almost twice the size of the particles produced with sodium nitrate or PIPES (Figure 8). At
te
pH 7, the dibasic PIPES is expected to have only half of its sulfonic acid groups charged given its
Ac ce p
pKa of 6.8 [37]. Therefore, the ionic strength of the PIPES and sodium nitrate should be the same (Table S2 supplementary data). Conversely, the ionic strength of the sodium sulfate, sodium citrate buffer and sodium phosphate buffer should be significantly greater as shown in Table S2 (supplementary data).
The particle sizing data also suggest that there is a linear relationship of PEI-silica particle size and ionic strength for particles made with monovalent ions (Figure 9) as the particle size increases with ionic strength for the particles made with sodium nitrate and PIPES. However, particle growth does not seem to be greatly affected by ionic strength for particles made with multivalent anions, since the particles are of a similar size range independent of the ionic strength in the range 87-1687 mM (Figure 9). This may be due to the principle that multivalent anions are involved in the assembly of
Page 14 of 37
polyamines where silica condensation occurs since they contain multiple points for electrostatic interactions. Instead, monovalent anions are perhaps only involved in charge balance of the template and the growing surface of the particle [31] due to reduced charge density. Furthermore, it is proposed that the recruitment of PEI by multivalent anions to make aggregate nucleation points
ip t
decreases the time required for the nucleation and allows growth to proceed for longer, therefore
cr
producing larger PEI-silica particles than when only monovalent anions are present.
us
In nature silaffin peptides involved in natural silica formation (biomineralisation) are
phosphorylated, but the data show that PEI-silica formation occurs in the presence of a number of
an
anions and so silica formation is not phosphate-specific when using PEI and TMOMS. The study using different anions and salt concentrations shows a clear change in particle size depending on
M
whether the anion used is mono- or multivalent (Figures S2 and S3, supplementary data) suggesting that the anion charge is important in producing larger particles. This novel trend found with PEI is
d
consistent with work on poly-L-arginine [30] which found that divalent anions were effective at
te
converting the poly-L-arginine from a random coil to a helical structure, whereas most of the monovalent anions examined were not efficient in producing this transition. This suggests that the
Ac ce p
higher valence of the anion is an important factor in driving conversion and strengthening the secondary structure of the polyamine. However, it was found that this effect was specific for polyL-arginine and was not observed for poly-L-lysine or poly-L-ornithine [30].
The data suggest that the mechanism of PEI-silica particle formation using TMOMS as the silane and PEI as the polyamine is different depending on the anions used in the synthesis. The data presented here are in agreement with the polyamine-salt aggregate mechanism proposed by a number of authors [13, 20, 23-24, 26-29] who have used polyamines other than PEI, such as polyallylamine hydrochloride and poly-L-arginine to form silica particles and capsules. The polyamine-salt aggregate mechanism involves the combination of the cationic polyamine with a multivalent anionic salt and results in electrostatically cross-linked polyamine-salt aggregates [27Page 15 of 37
29]. Figure 10 shows a representation of the difference in PEI-silica particle formation when using monovalent and multivalent anions, based on the experimental results we have presented. The pH is also important in the formation of polyamine-silica as it must be below the pKa of the polyamine but high enough that the effective charge of the anion is controlled by the pH of the solution [27].
ip t
This information sheds light on the mechanism of PEI-silica formation using TMOMS as the silane and PEI as the polyamine, since the pH of the syntheses carried out was optimal for particle
cr
formation to occur. Therefore, we propose that larger PEI-silica particles formed with multivalent
us
anions due to polymer-anion aggregation which acts as a starting point for silica growth. This
an
process did not occur with monovalent salts, in which case producing much smaller particles.
In order to test the hypothesis that multivalent anions aggregate with PEI, experiments were
M
performed to determine the hydrodynamic diameter of PEI when combined with nitrate anions (monovalent) and phosphate anions (multivalent). Our hypothesis is supported by results which
d
show the aggregation of single PEI molecules with multivalent anions, but not monovalent anions
te
(Figure 11). The data (Figure 11) show the particle size distribution of PEI alone and the changes
Ac ce p
which occurred with the addition of sodium nitrate and sodium phosphate buffer. The mean PEI hydrodynamic diameter was determined to be 9.6 ± 1.3 nm which is similar to a previously published value [49]. This value only increased to 11.4 ± 1.3 nm after sodium nitrate was added to the PEI. However, when sodium phosphate buffer was added to the PEI the hydrodynamic diameter increased by nearly 65 % to 15.8 ± 2.4 nm, indicating a different behaviour of the PEI towards mono- and multivalent anions. This data support our hypothesis that the multivalent anions aggregate with PEI to later form larger PEI-silica hybrid particles than the PEI-silica particles made with monovalent anions which are smaller, most likely due to the absence of PEI-anion aggregation.
4. Conclusions
Page 16 of 37
A study of bioinspired silication was presented in which polyethyleneimine (PEI) was used to induce the condensation polymerisation of hydrolysed trimethoxymethylsilane (TMOMS) to form PEI-silica hybrid particles. The effects of PEI concentration, sodium phosphate buffer conditioning prior to silication, ionic strength and the presence of different anions during the silication reaction
ip t
were examined in order to gain insight into the role of different anions in silica particle formation. Scanning electron microscopy, dynamic light scattering and zeta potential, ATR-FTIR and EDS
cr
measurements were used to characterise particle size, morphology, surface charge, elemental
us
composition and bonding.
an
The net positive charge of the PEI-silica particles in the pH range of 2-11 was confirmed with zeta potential measurements for the first time, suggesting that some of the PEI involved in particle
M
formation is also present on the silica surface. The use of different salts has lead us to conclude that electrostatic interactions between multivalent anions and PEI polyamine groups are the major force
d
in the production of polyamine aggregates which act as condensation sites for the formation of
te
larger PEI-silica particles than particles produced with monovalent anions. In addition, particle size
Ac ce p
is affected by ionic strength, only when the anions present are monovalent, since very little change in particle diameter was observed over a range of ionic strengths of multivalent anions. The increased knowledge of this tuneable silica synthesis which uses more environmentally friendly reagents than previous methods is of significant benefit in producing silica particles which have a wide range of applications and the global demand of which increases annually.
In conclusion, it is clear that a superior understanding of silica particle growth is of great use in order to control the parameters needed to refine and improve the production of silica particles made using less toxic reagents, since the use of silica particles has applications in many fields of science, engineering and medicine.
Page 17 of 37
Acknowledgements The authors wish to acknowledge the University of Newcastle Electron Microscope X-ray Unit. FN is the recipient of a University of Newcastle fellowship. TM acknowledges the Faculty of Science
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and IT, University of Newcastle for summer scholarship funding.
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synthesis of a family of propylamines derived from diatom silaffins and their activity in silicification, Chem. Commun. (2006) 1521-1523.
d
[35] A. Hardy Dessources, S. Hartmann, M. Baba, N. Huesing, J.M. Nedelec, Multiscale
Ac ce p
2713-2720.
te
characterization of hierarchically organized porous hybrid materials, J. Mater. Chem. 22 (2012)
[36] J. Sambrook, D.W. Russell, The condensed protocols from Molecular Cloning: A Laboratory Manual, Cold Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2006. [37] N.E. Good, G.D.Winget, W. Winter, T.N. Connolly, S. Izawa, R.M.M. Singh, Hydrogen Ion Buffers for Biological Research, Biochemistry 5 (1966) 467-477. [38] http://compdent.uthscsa.edu/dig/itdesc.html, accessed 7th January 2013. [39] E. Brunner, K. Lutz, M. Sumper, Biomimetic synthesis of silica nanospheres depends on the aggregation and phase separation of polyamines in aqueous solution, Phys. Chem. Chem. Phys. 6 (2004) 854-857. [40] J. Kim, D.F. Lawler, Characteristics of Zeta Potential Distribution in Silica Particles, Bull. Korean Chem. Soc. 26 (2005) 1083-1089.
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[41] L. Avadiar, Y.K. Leong, Interactions of PEI (polyethylenimine)–silica particles with citric acid in dispersions, Colloid Polym. Sci., 289 (2011) 237-245. [42] B.C. Ong, Y.K. Leong, S.B. Chen, Interparticle forces in spherical monodispersed silica dispersions: Effects of branched polyethylenimine and molecular weight, J. Coll. Interf. Sci. 337
ip t
(2009) 24-31.
[43] "Middle-Range Infrared Absorption Correlation Charts," in CRC Handbook of Chemistry
cr
and Physics, 93rd Edition (Internet Version 2013), W. M. Haynes, ed., CRC Press/Taylor and
us
Francis,Boca Raton, FL http://www.hbcpnetbase.com, accessed 7th January 2013.
[44] V.V. Ganbavle, U.K.H. Bangi, S.S. Latthe, S.A. Mahadik, A.Venkateswara Rao, Self-cleaning
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silica coatings on glass by single step sol-gel route, Surf. Coat. Tech. 205 (2011) 5338-5344. [45]R. Al-Oweini, H. El-Rassy, Synthesis and characterization by FTIR spectroscopy of silica
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aerogels prepared using several Si(OR)4 and R´´Si(OR´)3 precursors, J. Mol. Struct. 919 (2009) 140145.
d
[46] S. Amoriello, A. Bianco, L. Eusebio, P. Gronchi, Evolution of two acid steps sol-gel phases by
te
FTIR, J. Sol-Gel Sci. Technol. 58 (2011) 209-217.
Ac ce p
[47] M. Badertscher, P. Bühlman, E. Pretsch, Structure determination of organic compounds. Tables of spectral data, 4th edition, Springer, 2009. [48] P.J. Launer, P.J., Infrared Analysis of Organosilicon Compounds: Spectra-Structure Correlations. Silicone Compounds Register and Review, 100-103, Bristol USA, Petrarch Systems, 1987.
[49] M.M. Andersson, R. Hatti-Kaul, Dynamic and static light scattering and fluorescence studies of the interactions between lactate dehydrogenase and poly(ethyleneimine), J. Phys. Chem. 104 (2000) 3660-3667.
Page 22 of 37
Figure Legends
Fig. 1. Effect of [Pi]/[r.u.] ratio on PEI-silica particle diameter for 25 and 750 kDa PEI and sodium
ip t
phosphate buffer (Pi) concentrations of 29 mM and 250 mM.
Fig. 2. SEM images showing the effect of [Pi]/[r.u.] on PEI-silica particle growth. The [Pi]/[r.u.]
us
cr
ratios are as follows: A) 0.025; B) 0.2; C) 0.5; D) 0.72 and E) 1.25. The scale bar is 1 µm.
an
Fig. 3. Zeta potential as a function of pH of PEI-silica particles made with TMOMS and PEI (25 kDa ). The trend line follows a third order polynomial (y = -0.047x3 + 0.202x2 - 0.185x + 46.00, R²
M
= 0.994).
d
Fig. 4. EDS pattern for PEI-silica particles made with sodium phosphate showing the presence of
Ac ce p
te
nitrogen from the PEI as well as silicon, oxygen and carbon.
Fig.5. ATR-FTIR of PEI (black line), washed PEI-silica hybrid particles (dotted), TMOMS hydrolysed in acidic conditions (grey line) and sodium phosphate buffer (dashed line). Data are offset vertically for clarity.
Fig. 6. SEM images showing the effect of different sodium phosphate buffer conditioning times on PEI-silica particle formation. (A) 25 kDa PEI, 0 min; (B) 750 kDa PEI, 0 min; (C) 25 kDa PEI, 120 min; (D) 750 kDa PEI 120 min. The arrows indicated uncondensed silica. Scale bar is 1 µm.
Fig. 7. Effect of different sodium phosphate buffer conditioning times on PEI-silica particle diameter determined using dynamic light scattering.
Page 23 of 37
Fig. 8. Effect of anion identity on particle size. PEI-silica particles were made with 29 mM salt concentration.
ip t
Fig.9. Hydrodynamic diameter of PEI-silica particles produced with multivalent and monovalent anions as a function of ionic strength. The arrows indicate that data for PEI-silica particles made
cr
with sodium citrate buffer at 1600 and 1687 mM ionic strength, for both molecular weights of PEI
us
is included in the linear fit of the data. The values are given in Table S3, supplementary data.
an
Fig. 10. Proposed mechanism of formation of PEI-silica particles using mono- or multivalent
M
anions.
Fig.11. Particle size distribution of PEI (black) with addition of sodium nitrate (grey) and sodium
te
Ac ce p
induced polymer aggregation.
d
phosphate buffer (grey hatched line), indicating that there is no substantial monovalent anion
Page 24 of 37
Highlights Hybrid particles were made with polyethylenimine (PEI) and trimethoxymethylsilane
(TMOMS). The effects of PEI concentration and different anions were studied.
Multivalent anions produced larger particles than monovalent anions.
Ac ce p
te
d
M
an
us
cr
PEI aggregates with multivalent anions followed by silica growth.
ip t
PEI is present at the surface of the silica hybrid particles made with TMOMS.
Page 25 of 37
*Graphical Abstract (for review)
700
multivalent anions: ionic strength does not affect diameter
600 550
450 400 1 μm
multivalent anions: ionic strength does not affect diameter 700
300
650600
150
550 ionic strength is proportional to diameter monovalent anions: 500
0
231 38 nm
600
Sodium phosphate buffer
500 500 1000 450 ionic strength (mM) 400 400
1500
350
us
200
25 kDa PEI + multivalent anions multivalent anions: ionic strength does not affect diameter 750 kDa PEI + multivalent anions 25 kDa PEI + monovalent anions 750 kDa PEI + monovalent anions
cr
250
Hydrodynamic diameter (nm)
350
ip t
500
hydrodynamic diameter (nm)
Sodium nitrate
hydrodynamic diameter (nm)
650
25 kDa PEI + multivalent anions 750 kDa PEI + multivalent anions 25 kDa PEI + monovalent anions 750 kDa PEI + monovalent anions
300300 250 200200
an
monovalent anions: to 400 diameter 500 0 100 ionic strength 200 is proportional 300 (mM) 1500 0 500 Ionic strength 1000 monovalent anions: diameter is proportional to ionic strength ionic strength (mM)
150
Ac ce
pt
ed
M
476 42 nm
Page 26 of 37
ep t
600 Figure 1
400
Ac c
SEM particle diameter (nm)
500
300
200
100
0
0
0.2
0.4
25 kDa PEI + 29 mM Pi 750 kDa PEI + 29 mM Pi 25 kDa PEI + 250 mM Pi Page of mM 37 Pi 750 kDa PEI27 + 250
0.6 0.8 [Pi]/[r.u.]
1
1.2
1 μm
A
Ac
Figure 2
B
C
D
E Page 28 of 37
pt
Figure 3 60
ce
40 30 20 10
Ac
zeta potential (mV)
50
0
-10 -20
Page 29 of 37
-30 0
2
4
6
8 pH
10
12
14
3000 2500 2000
counts per second/eV
ce
O
Ac
counts per second/eV
Figure 3500 4
1.0
C
0.5 N 0.0 0.2
0.3 keV
0.4
Si
1500
C
1000
500
NN
Page 30 of 37
0 0
1
keV
2
3
Figure 5
M
950
1040
1113
C-N 1O, 2O, 3O
P
750
1039
1120
ep 1016
1113
Si-O-Si
Ac c
1272
Si-CH3 1410
te
950
d
Si-O-Si
1272
1410
Si-CH3
1600
1400
941
1077
1153
P=O
1200 1000 Page 800 31 of 37600 wavenumber (cm-1)
pt
1 μm
B
ce
Figure 6 A
C
Ac
330 37 nm
312 24 nm
D
Page 32 of 37 459 30 nm
410 24 nm
ce p
600
25 kDa PEI
750 kDa PEI
500 400
Ac
Hydrodynamic diameter (nm)
700 Figure 7
300 200 100
Page 33 of 37 0 0
30 60 Conditioning time (min)
90
120
500
25 kDa PEI 750 kDa PEI
Ac c
400
SEM diameter (nm)
ep
Figure 8
300
200
100
Page 34 of 37
0 sodium nitrate
PIPES
sodium sulfate
sodium citrate buffer
sodium phosphate buffer
ep t
650 9 Figure
550 500
Ac c
Hydrodynamic diameter (nm)
600
450
400 350 300
25 kDa PEI + multivalent anions 750 kDa PEI + multivalent anions 25 kDa PEI + monovalent anions 750 kDa PEI + monovalent anions
250
Page 35 of 37
200 0
100
200 300 400 Ionic strength (mM)
500
600
-
+ + +
+ +
+
+
+
+ +
+ +
+ +
+
+
+
+
+
+
+
+
+
+
+
+ +
+
- +
+
+
+
+ +
+ TMOMS
+
+ +
PEI
+ TMOMS
Smaller PEI-silica hybrid particles
+ Multivalent anions
+ +
-
- -
+
Ac
+
ce
+
+
+
+ Monovalent anions
+
+
+
-
+
+
pt
Figure 10
+ +
PEI-multivalent anion aggregate
Page 36 of 37 Larger PEI-silica hybrid particles
45
35
Ac c
Mean intensity %
40
ep
Figure 11 50
30 25 20 15 10
5
Page 37 of 37
0 5
10
15
20
Hydrodynamic diameter (nm)
25