The amphiphilic molecule dextrin-VA-SC16 (dexC16) was produced and studied in this work. DexC16 has a hydrophilic dextrin backbone with grafted acrylate groups (VA), substituted with hydrophobic 1- exadecanethiol (C16). The dextrin degree of substitution with the hydrophobic chains (DSC16, number of alkyl chains per 100 dextrin glucopyranoside residues) may be controlled. Materials with different DSC16 were prepared and characterized using ¹H NMR. DexC16 self assembles in water through association of the hydrophobic alkyl chains, originating hydrogel nanoparticles. The properties of the hydrogel nanoparticle were studied by dynamic light scattering (DLS), fluorescence spectroscopy and atomic force microscopy (AFM)

Gama, F. M.

Gonçalves, Catarina

Martins, J. A.

Universidade do Minho: RepositoriUM

   SELF-ASSEMBLED NANOPARTICLES MADE OF DEXTRIN   Catarina Gonçalves1, José Alberto Martins2 and Miguel Gama11Centro de Engenharia Biológica, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal fmgama@deb.uminho.pt 2Departamento de Química, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal  Abstract. The amphiphilic molecule dextrin-VA-SC16 (dexC16) was produced and studied in this work. DexC16 has a hydrophilic dextrin backbone with grafted acrylate groups (VA), substituted with hydrophobic 1-hexadecanethiol (C16). The dextrin degree of substitution with the hydrophobic chains (DSC16, number of alkyl chains per 100 dextrin glucopyranoside residues) may be controlled. Materials with different DSC16 were prepared and characterized using 1H NMR. DexC16 self assembles in water through association of the hydrophobic alkyl chains, originating hydrogel nanoparticles. The properties of the hydrogel nanoparticle were studied by dynamic light scattering (DLS), fluorescence spectroscopy and atomic force microscopy (AFM).   Keywords: Nanoparticles, Dextrin, Self-assembling  1. INTRODUCTION  Amphiphilic molecules, such as surfactants or lipids, spontaneously self-assemble in water, forming self-aggregates, such as micelles, bilayer membranes, tubes and vesicles. Amphiphilicity of biopolymers is one of the important factors for their self-organization in water (Akiyoshi and Sunamoto, 1996). Among the different types of amphiphilic polymers, water-soluble polymers with hydrophobic molecules grafted on side chains have received special attention. By self-assembling, the hydrophobic segments are segregated from the aqueous exterior, to form an inner core surrounded by hydrophilic chains. Polymer micelles or nanoparticles with hydrophobic core and hydrophilic shell are thus prepared. This kind of structure is suitable for trapping hydrophobic substances, such as fluorescent probes (Akiyoshi et al., 1993, Nichifor et al., 2004), proteins (Akiyoshi et al., 1998), and hydrophobic pharmaceuticals (Liu et al., 2005). Size, density and colloidal stability of nanoparticles can be controlled, by changing the degree of substitution of hydrophobes and its hydrophobicity (Akiyoshi and Sunamoto, 1996). The association mechanism is mainly governed by the alkyl chain concentration and length and is little influenced by the molecular weight of the polymer backbone (Petit-Agnely et al. 2000). However, with low-molecular weight polymers, the hydrophobic aggregates are not connected via the polymer backbone (Petit-Agnely and Iliopoulos, 1999).  The study of nanogels (hydrogel nanoparticles) has intensified during the last decade due to enormous potential applications in the development and implementation of new environmentally responsive materials, biomimetics, biosensors, artificial muscles and drug delivery systems (Akiyoshi and Lee, 2004). Solid nanoparticles made from biodegradable polymers have been widely investigated for long-term delivery of drugs (Hans et al., 2005). They can potentially provide benefits such as increased therapeutic effect, prolonged bioactivity, controlled release rate, and finally decreased administration frequency, thereby increasing patient compliance. Nanostructures spontaneously form when the concentration of the polymer is higher than a concentration called critical micelle concentration (cmc). The self-assembly of hydrophobized dextrin in water (dexC16) was investigated in this work. The structural change upon the dilution of the dexC16 self-aggregates, in water, was investigated by fluorometry, in the presence of pyrene as the fluorescent probe (Magdassi and Nizri, 2005). Other relevant properties of the nanoparticles, such as the size, stability, and shape were also evaluated in this work.  2. EXPERIMENTAL SECTION  2.1. Materials The dextrin-VA was synthesized as described in a previous paper (Carvalho et al., submitted). In this work, dextrin-VA with 20 acrylate groups per 100 dextrin glucopyranoside residues (DSVA 20%) was used. Dimethylsulfoxide (DMSO), triethylamine (TEA) and deuterium oxide (D2O) were from Aldrich. Regenerated cellulose tubular membranes, with 3500 MWCO, were obtained from Membrane Filtration Products. Pyrene (Py) was from Aldrich and was used after appropriate recrystallization. Distilled water was used for the preparation of aqueous solutions.  2.2. Synthesis of dexC16Dextrin-VA and 1-hexadecanethiol were dissolved in dimethylsulfoxide (conc. VA = 0.058 M). Different molar percentages (10, 20, 40, 60, 100% relatively to VA) of 1-hexadecanethiol were added to the reaction mixture such that different levels of grafting were obtained. Triethylamine (1 equivalent to VA) was added to the reaction. The medium was stirred for 24h, at 50ºC. The mixture was dialysed for 48h against water, with frequent water change. After freezing, the mixture was lyophilized and stored.   2.3. Sample Preparation The lyophilized dexC16 was dissolved in water under stirring at 50ºC, and then further sonicated for 20min. The degree of solubility depends on degree of substitution. Increasing the degree of substitution reduces the solubility.   2.4. Dynamic Light Scattering (DLS) The size distribution was determined with a Malvern Zetasizer, MODEL NANO ZS (Malvern Instruments Limited, UK). A water dispersion (1 mL) of nanoparticles in ultra-pure water were analysed in a polystyrene cell, using a He-Ne laser - wavelength of 633 nm and a detector angle of 173º. The temperature used was 25ºC.  2.5. Fluorescence spectroscopy Fluorescence measurements were performed on a JASCO FP-6200 spectrofluorometer in a quartz cell. The Pyrene spectra were obtained by exciting the solutions at 377 nm and recording the emission over the range 350-600 nm at the scan rate 250 nm/min. The slit width was set at 5 nm for the excitation and 5 nm for the emission. To increase the precision of the determination of the values of intensities of different vibronic peaks, the fluorescence intensities were measured at the maximum of each peak.  2.6. Atomic Force Microscopy (AFM)  Tapping mode imaging was carried out on a Multimode (Digital Instruments) scanning probe microscope. A silicon tip doped with phosphorus, with a radius curvature of less than 10 nm (RTESP, VEECO), was used. This tip has a typical resonance frequency of 288-328 kHz and a typical force constant of 20-80 N/m. A scan rate of 1 Hz was sufficient to maintain a good signal-to-noise ratio.  3. RESULTS AND DISCUSSION  3.1. Synthesis of the dextrin-VA- SC16The reaction between the thiol moiety and the acrylate group of dextrin-VA is a conjugate addition, with thiol acting as a nucleophile (Figure 1). Although the reaction was detected in the absence of TEA, the presence of the base substantially increases the reaction rate, as described ahead.                     Figure 1. Synthesis  3.2. Formation of nanoparticles The dissolution of dexC16 in water is expected to amphiphilic nature of the molecules. The formatiusing both 1H NMR and AFM.  Due to the limited mobility of the alkyl/hydrophshape of the proton peaks generated by the alkyl con whether the 1H NMR spectra is obtained inmethyl (0.8 ppm) and methylene (1.1 ppm) groFigure 2. They are sharp for DMSO solvent, andbase, for deuterated water solvent. As the concentincreases, the larger is the broadening of the methpeaks, in deuterated water, is characteristic of collection of chemically identical species, yet poevidence suggests that alkyl chains have different eDMSO/TEA50ºCof the dexC16. five rise to micelle formation, owing to the on of hydrogel nanoparticles was accessed obic chains inside of the nanoparticle, the hain (2.0 - 0.6 ppm) is different depending  deuterated water or DMSO. Peaks from ups are visible in all spectra presented in  sharp yet progressively broadening at the ration of water in the D2O/DMSO mixtures ylene protons. The shape of the alkyl chain a superposition of peaks representing a ssessing various degrees of mobility. This nvironments, when dispersed in water.   B B B B B B B B B B B B B B B A    B  A          (a)             Figure 2. 1H NMR spectra of dexC16 (10 mg/mL): (a) DMSO-d6, (b) 10% D2O in DMSO-d6 and (c) D2O. A B A B (c) (b)  In addition, the self-assembled particles were observed using AFM. The AFM images collected (Figure 3) allow direct visualization of the spherical nanoparticles, formed by self-assembling. The AFM results indicate that, for dexC16 with DSC16 7%, the mean diameter of nanoparticles is roughly 20 nm. In general, the nanoparticles appear to have a narrow size distribution.             (a (bFigure 3. AFM images (a) and (b) three-dimensional of dexC16 (DSC16 7%) at the concentration of 0.01g/dL.   3.3. Degree of substitution  The synthesis of dexC16 with different degrees of substitution with hydrophobic chains was successfully attempted, using different amounts of 1-hexadecanethiol in the reaction mixture. The 1H NMR spectra of dexC16, in deuterated water, was used to estimate the degree of substitution (DSC16, amount of alkyl chains per 100 dextrin glucopyranoside residues). DSC16 was calculated as a peak area ratio in the NMR spectra, according to equation 1.     10037716 ×××=yxDS C                          (1)    Where x is the average integral of the protons from alkyl moieties (2.0 - 0.6 ppm), and y is the integral of all dextrin protons (3.0 - 5.8 ppm). Table 1 presents the DS values obtained for dexC16 and the respective efficiency.  Table 1. Degree of substitution obtained relatively to acrylate groups and efficiency for dexC16.  (a) Calculated as molar ratio of 1-hexadecanethiol to acrylate groups ( 100× ). Theoretical DS a (%) Obtained DS b (%) Efficiency c (%) 10 2 20 20 4 20 40 14 35 60 23 38 100 59 59 (b) Determined by 1H NMR. The value shown is the calculated using equation 3, and multiplied by 5 to obtain the DS relatively to acrylate groups. (c) Calculated as the ratio of the obtained to the theoretical DS ( 100× ).  The results show that is possible to produce material with varying amounts of grafted alkyl chain, hence allowing the fine tunning of the properties of the obtained materials. Furthermore, different hydrophobic materials may be grafted, for instance thiocholesterol (data not shown).  3.4. Size and Size Distribution  The dynamic light scattering (DLS) studies were done using a Nano-ZS (Malvern) instrument. Samples of dexC16 with different degree of substitution and concentrations were analysed. The results of the DLS studies are given in Table 2. A representative size distribution by volume (Figure 4) is shown.            Figure 4. Size distribution by volume obtained by DLS for dexC16 with DSC16 7%.   Table 2. Results of DLS studies with dexC16.   Size /nm (fraction of material/%) DSC16 Conc. Peak 1 Peak 2 Peak 3 7% 0.001 15.4 (98.7%) 187 (1.3%)  7% 0.01 13.6 (98.5%) 57.3 (0.7%) 349 (0.8%) 7% 0.1 9 (97%) 61.6 (0.5%) 737 (2.5%)  Data in table 2 shows that the dilution process has no significant effect on the particle size, in the range of concentration used. The hydrodynamic diameter of the nanoparticles slightly increases with the decreasing of concentration. The size distribution generally presents three peaks, but the majority volume of the particles has around 10-20 nm in size.  The DLS results are in good agreement with AFM presented above. It was possible to observe the small nanoparticles, with a diameter around 20 nm, as the predominant population.  3.5. Critical Micelle Concentration of Hydrophobized Dextrin-VA As reported by Akiyoshi [1996], pyrene may be used as a fluorescent probe to evaluate aggregate formation. The fluorescent properties of pyrene change when it is transferred from the aqueous environment to the hydrophobic microenvironment of the nanoparticles. To determine the cmc, the ratio of intensities (I3/I1) in the emission spectra was used.  The pyrene concentration is in all experiments very low (1×10-6M), to prevent excimer formation (detectable at ≈ 470 nm). The I3/I1 values corresponding to samples with different polymer concentrations were calculated. The intensity ratio was plotted against polymer concentration, for dexC16 samples with different degrees of substitution. Figure 5a shows the variation of  I3/I1 with concentration for dexC16 with DSC16 7%.              0,901,001,101,201,300,00001 0,00010 0,00100 0,01000 0,10000 1,00000cp (g/dL)I0,00000,00020,00040,00060,00080,00100,00120 5 10 15DSC16 (% mol)cmc (g/dL)(b) 1/I 3(a)  Figure 5. (a) Fluorescence intensity ratio I3/I1 as a function of the dexC16 concentration with DSC16 7% and (b) variation of critical micelle concentration (cmc) of dexC16 with the degree of substitution.  At low polymer concentrations, I3/I1 is almost constant, i.e. pyrene experiences an aqueous environment. For higher polymer concentrations I3/I1 starts to increase, due to the solubilization of pyrene in the hydrophobic domains formed by self-assembly (for cp > 0.0004 g/dL), where it senses a less polar environment. The curve is sigmoidal, as observed for other amphiphilic polymers. The ratio I3/I1 reach a constant value, as there are enough nanoparticles for solubilising all pyrene molecules. The variation in pyrene fluorescence properties is due the partition of the probe between water (where it is solubilized) and the hydrophobic microdomains, until equilibrium between the two regions (or probe saturation) occurs.   Figure 6. Size and the respective % volume for dexC16 with DSC16 7% at (a) 0.00001 g/dL, (b) 0.0001 g/dL, (c) 0.0004 g/dL, 0.001 g/dL, (d) 0.01 g/dL and 0.1 g/dL.   The determination of cmc values from the fluorescence data is inaccurate, due to the large concentration over which the fluorophore response stretches. The onset and offset of the fluorophore response to polymer concentration, or the midpoint of this variation, were proposed to correspond to the cmc value (referências). In this study, the polymer concentration corresponding to the initial variation of pyrene fluorescence spectra which indicates that a significant event takes place at that particular concentration, is here considered the cmc value. This event can be an incipient association of side-chain hydrophobes (at least the very first association of two hydrophobes). As a consequence, the onset of the fluorophore response, as a function of polymer concentration, is considered the first critical micelle 0204060801001201401601802000 4 8 12 16measurementsize (nm)0102030405060708090100%volume(a) (b) 0204060801001201401601802000 4 8 12 16measurementsize (nm)0102030405060708090100%volume0204060801001201401601802000 4 8 12 16measurementsize (nm)0102030405060708090100%volume0204060801001201401601802000 4 8 12 16measurementsize (nm)0102030405060708090100%volume0204060801001201401601802000 4 8 12 16measurementsize (nm)0102030405060708090100%volume0204060801001201401601802000 2 4 6 8measurementsize (nm)0102030405060708090100%volume(c) (d) (e) (f) concentration, corresponding to the very beginning of the hydrophobe association (by intra- or inter-molecular association). Materials with different DSC16 were prepared, and the variation of the ratio I3/I1 with polymer concentration (for dexC16 with different degree of substitution 1, 5, 7 e 12%) was analysed. The influence of the degree of substitution on cmc is shown in figure 5b. The cmc value decreases with increasing degree of substitution. For a higher DSC16, the probability of finding two alkyl chains next to one another is higher, which in turn increases the association tendency.  The cmc can also be evaluated using the size distribution of the nanoparticles measured by DLS. Figure 6 shows the size and the respective % volume for dexC16 with DSC16 7% at different polymer concentrations, ranging between 0.00001 g/dL and 0.1 g/dL.   The results show that the cmc value for dexC16 with DSC16 7%, obtained through the fluorimetry experiments, was also confirmed by DLS. While at low and high polymer concentration, the measurement of particle size fine consistent results in consecutive analysis (± 120 nm and ± 18 nm for low and high concentration of polymer, respectively), for the cp values close to cmc the sample is apparently unstable, quite different values being determined in independent assays (figures 6c, d).  Indeed the nanoparticles formation was detected at around 0.0004 g/dL  The values determined are lower than the reported for the most common low molecular weight surfactants. The low cmc may be due to the low solubility of dextrin.     REFERENCES  Akiyoshi, K., Deguchi, S., Moriguchi, N., Yamaguchi, S., Sunamoto, J. (1993). Self-Aggregatesnof Hydrophobized Polysaccharides in Water. Formation and Characteristics of Nanoparticles. Macromolecules; 26: 3062-3068. Akiyoshi, K., Sunamoto, J. (1996). Supramolecular assembly of hydrophobized polysaccharides. Supramolecular Science; 3: 157-163. 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Aggregation Mechanism of Amphiphilic associating Polymers Studied by 19F and 13C Nuclear Magnetic Resonance. J. Phys. Chem. B; 103: 4803-4808. Petit-Agnely, F., Iliopoulos, I., Zana, R. (2000). Hydrophobically Modified Sodium Polyacrylates in Aqueous Solutions : Association Mechanism and Characterization of the Aggregates by Fluorescence Probing. Langmuir; 16: 9921-9927. Turro, N. J., Yekta A. (1978). Luminescent Probes for Detergent Solutions. A Simple Procedure for Determination of the Mean Aggregation Number of Micelles. J. Am. Chem. Soc.; 100: 5921-5952. Nichifor, M., Lopes, S., Bastos, M., Lopes, A. (2004). Self-Aggregation of Amphiphilic Cationic Polyelectrolytes Based on Polysaccharides. J. Phys. Chem. B; 108: 16463-16472. Nizri, G., Magdassi, S. (2005). Solubilization of hydrophobic molecules in nanoparticles formed by polymer-surfactant interactions. Journal of Colloid and Interface Science; 291: 169-174.  

Self-assembled nanoparticles made of dextrin

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Self-assembled nanoparticles made of dextrin

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