Colloids and Surfaces A: Physicochem. Eng. Aspects 441 (2014) 880–884
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Albumin–furcellaran complexes as cores for nanoencapsulation b ´ E. Jamróz a , G. Para b,∗ , B. Jachimska b , K. Szczepanowicz b , P. Warszynski , A. Para a a b
Department of Chemistry and Physics University of Agriculture, ul. Balicka 122, 30-149 Kraków, Poland The Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, ul. Niezapominajek 8, 30-239 Kraków, Poland
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
Natural polysaccharide furcellaran (FUR) was used as component of polyelectrolyte/protein complexes together with bovine serum albumin (BSA). Complexes were formed at varying protein/polysaccharide concentration ratio and pH. Then, they were used as cores for encapsulation by sequential adsorption of oppositely charged polyelectrolytes. The optimal conditions for BSA/FUR complex formation for further L-b-L encapsulation were determined. Our results indicate that encapsulated furcellaran/protein complexes are very convenient, biocompatible carriers for biologically active molecules.
a r t i c l e
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Article history: Received 8 November 2012 Received in revised form 31 December 2012 Accepted 2 January 2013 Available online 29 January 2013 Keywords: Protein–furcellaran complexes Albumin (BSA) Zeta potenctial Nanocapsules
a b s t r a c t The possibility of using protein–polysaccharide complexes as cores for nanocapsules formation by sequential adsorption of polyelectrolytes were investigated. We selected the globular protein bovine serum albumin (BSA) and the natural anionic polysaccharide–furcellaran (FUR) and studied their complexation in the broad range of their concentration ratio and pH of the solutions. Analysing the zeta potential dependencies on these parameters, we selected optimal conditions for the core formation. The capsule shells were formed by the layer-by-layer adsorption of polyelectrolytes; polycation PDADMAC (polydiallyl dimethyl ammonium chloride) and polyanion PSS poly(sodium-4-styrene sulfonate). Depending on the shell thickness the size of the capsules was in the range of 60–80 nm. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Protein–polysaccharide electrostatic interactions and formation of complexes have been the subject of many studies for the past
∗ Corresponding author. Tel.: +48 126395128; fax: +48 124251923. E-mail address:
[email protected] (G. Para). 0927-7757/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfa.2013.01.002
few years [1–5]. Natural polysaccharides as: alginate, furcellaran, carrageenan, celulose or potato starch were used in many experiments as components of complexes together with various proteins as: gelatin, casein, bovine albumin, soy protein, -lactoglobulin [6–9]. The results of these experiments indicated that complexes of biocompatible polyelectrolytes with proteins, possessing unique functional properties, appear to be good candidates for delivery systems of bioactive compounds [10]. Therefore, developing a better
E. Jamróz et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 441 (2014) 880–884
understanding of these systems is a matter of importance, because of their use in a variety of formulations including pharmaceuticals, cosmetics and food industry. Recently much attention has been paid to the formation of biopolimeric, biodegradable nanocapsules with permeable or semi-permeable membrane and the investigations concerning the appropriate substrates and determination of formation parameters are still continuing. One of the most promising methods of microencapsulation is the membrane formation by sequential adsorption of polyelectrolytes having opposite charges [11]. This electrostatically and entropically driven process (called also as layer-by-layer adsorption or LbL process) was successfully applied for encapsulation of solid [12,13] or liquid (emulsion) [14] cores as well as nanoparticles/polyelectrolyte complexes [15]. That opens perspectives for application in many fields such as the cosmetic, medicine, pharmacy and food industries. For example, capsules with liquid cores can be used in drug delivery systems [16–18]. It can have great potential application in the agriculture, as substances (such as pesticides), contained in the microcapsules, can be released in the controlled amounts and at the time when it is desirable. Limiting the dosage of chemicals without hampering their efficacy will contribute to soil conservation, beneficial for the environment. The production of polymeric carriers without using synthetic chemical reagents and organic solvents is desirable for many biomedical applications [19–21]. Furcellaran is the anionic, sulphated polysacharide extracted from red algae Furcellaria lumbricalis. It is composed of D-galactose, 3,6-anhydro-D-galactose and D-galactose-4-sulphate. Its structural and functional properties are similar to -carrageenan [22]. The essential difference is that the latter has one sulphate ester residue per two sugars, while furcellaran has one sulphate ester residue per three or four sugar residues. It is used mainly as a gelling or thickening agent in food, agricultural, cosmetics and pharmaceutical industries. In the literature there are very few investigated informations about the behavior furcellaran, although the related carrageenan group were studied in detail [6,23–25]. Type of protein, in addition to the concentrations of components, ionic strength and pH, affects the process of complexation of proteins and polysacharides. It has been demonstrated that the protein complexed with the same polysacharides in various conditions, produced systems with different compatibility [26]. It was shown that polysaccharides bind more strongly to the less structured proteins as casein or gelatin, than to globular proteins as the bovine albumin (BSA). However, their thermal denaturation improves their ability to bind to polysaccharides [27]. The aim of our work was to examine the possibility and to determine the optimal conditions for protein–polysaccharide complex formation of globular protein – bovine serum albumin (BSA) and furcellaran. Then, we demonstrated the possibility to use the obtained complexes as cores for nanocapsules formation by sequential adsorption of polyelectrolytes. To our best knowledge the properties of the BSA/furcellaran complexes have been never analyzed before in respect for their use as a delivery vehicles.
2. Materials In our studies, fatty acid and globulin free bovine serum albumin (BSA) (Mw = 67.4 kDa, fraction V, lyophilized powder, 99%) which was purchased from Sigma. Fullcellaran, manufactured by neutral aqueous extraction from Baltic Sea seaweed F. lumbricalis, was purchased from Est-Agar AS, Saaremaa, Estonia. The mean molecular weight was Mw = 255 kDa. Polyelectrolytes, polyanion: poly(sodium-4-styrene sulfonate) – PSS (MW ∼70000) and polycation: polydiallyl dimethyl ammonium chloride – PDADMAC (MW ∼100000–200000) were purchased from Sigma–Aldrich. Sodium chloride (NaCl), hydrochloric acid (HCl) and sodium
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hydroxide (NaOH) were also purchased from Sigma–Aldrich. All materials were used without further purification. The distilled water used in all experiments was obtained with the three-stage Millipore Direct-Q 3UV purification system. The experiments were performed at room temperature 295 K. 3. Methods 3.1. Particle size analysis and zeta potential measurements Size of protein and protein/furcellaran complexes was determined by dynamic light scattering (DLS), using the Zetasizer Nano ZS Malvern instrument with the detection angle of 173◦ in optically homogeneous square polystyrene cells (measurement range of 0.6 nm–6 m). The zeta potential was measured by the microelectrophoretic method using Malvern Zetasizer Nano ZS apparatus. Its value was calculated using Henry’s equation =
3 e 2εF(a)
(1)
where is the dynamic viscosity, ε is the dielectric constant of water, F (a) the function of the dimensionless parameter a, −1 = (εkT/2e2 I)1/2 is the electrostatic double layer thickness, e the elementary charge, k the Boltzmann constant, T the absolute temperature, I = 12 (˙i ci zi2 ) is the ionic strength, ci represents the ionic concentrations, a is the characteristic dimension of the protein/complex. All zeta potential measurements were performed in 0.005 M NaCl, if not stated otherwise. Each value was obtained as an average from three runs with at least 10 measurements. All measurements were performed at 295 K. 3.2. Core preparation Albumin–furcellaran complexes were formed by mixing both components in 0.005 M NaCl, during permanent stirring with a magnetic stirrer at 300 rpm. The concentrations of BSA and FUR were expressed in ppm. The initial BSA and FUR concentrations were 500 and 1000 ppm respectively. The optimal ratio of albumin to furcellaran concentrations was determined by measuring zeta potential of the suspension of obtained complexes. 3.3. Layer-by-layer adsorption of polyelectrolytes on polyelectrolyte/albumine complexes After formation of cores (albumin–furcellaran complexes), the polyelectrolytes shell around such complexes were formed by the layer-by-layer technique using the saturation method. Therefore, the multilayer shells were constructed by subsequent adsorption of polyelectrolytes from their solutions, without the intermediate rinsing step. Suspension of complexes was added to the polyelectrolyte solution while mixing with a magnetic stirrer at 300 rpm. Volumes of polyelectrolyte solution used to form each layer were chosen empirically by analyzing the results of simultaneous zeta potential measurements. Addition of polyelectrolyte was ceased when the zeta potential reached a constant value. It occurred that a stable layer was obtained when zeta potential of capsules with adsorbed polyelectrolyte was close to zeta potential of the same polyelectrolyte in solution [14]. The method of nanocapsules formation is schematically presented in Fig. 1. To assess the stability of complexes/nanocapsules freshly prepared suspension were stored at room temperature for up to 10 days. Size distribution (hydrodynamic diameter) and zeta potential of nanocapsules were measured after preparation and 10 days storage.
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Fig. 1. Scheme of nanocapsule formation with protein–polyelectrolyte complexes as cores. The furcellaran albumine mixture provides the complexes, which play a role of cores that are further covered with a shell by sequential adsorption of polyelectrolytes.
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Dry nanocapsules were visualized by scanning electron microscopy (SEM). Observations were performed using a JEOL JSM-7500F field emission scanning electron microscope, at an operating voltage 15 keV. Samples were prepared by placing a drop of nanocapsules suspension on the copper cylinders and dried overnight.
40
kT RH = 6D
(2)
The average RH value for pH in the range 4–9, calculated from Eq. (2) using the measured diffusion coefficients of BSA, was 3.88 nm, independent of pH [28]. The hydrodynamic diameter of furcellaran at the ionic strength of 0.005 M NaCl was independent on pH (in the range 3–10) was equal to 56 nm. The pH dependence of the zeta potential of BSA is shown in Fig. 2. As can be seen, zeta potential of BSA decreased from 50 mV at pH 3 to −45 mV at pH 10 for I = 5 × 10−3 M. For higher ionic strength, I = 0.15, zeta potential obtained value of 20 mV at pH 3 and –20 mV at pH 10. It can be noticed that the isoelectric point (i.e.p.), which was independent on the ionic strength, is attained at pH 4.8, which is in agreement with earlier data [28–30]. Furcellaran is the polyelectrolyte with ionic (sulfate) groups. In polar solvents, such as water, these groups dissociate, leaving charges on polymer chains and releasing counterions in solution. Electrostatic interactions between charges lead to the rich behavior of polyelectrolyte solutions, qualitatively different from those of uncharged polymers [31–33]. We determined the dependence of zeta potential of furcellaran on pH at three different ionic strength: 0.005 M; 0.01 M; 0.15 M NaCl. We noticed, that the values of zeta potential are independent on pH (within experimental error) at a given ionic strength, which could be expected as the charge originates from strongly acidic sulfate groups. The values of zeta potential of furcellaran change from −48 mV, at ionic strength of 0.005 mol/dm3 , −43 mV, at ionic strength of
0.005 M NaCl 0,01 M NaCl 0,15 M NaCl
20 0 -20 -40 -60 2
4
pH
6
8
10
Fig. 2. Zeta potential of BSA as a function of pH. The points denote experimental values determined for: I = 5 × 10−3 M, I = 1 × 10−2 M, 䊉 I = 0.15 M. Lines are to guide the eye. Experimental error ±5 mV.
0.01 mol/dm3 to about −27 mV at ionic strength 0.15 mol/dm3 (experimental error ± 3 mV). Attractive electrostatic interactions between positively charged proteins and anionic polysaccharides lead to formation of complexes. Change of zeta potential upon formation of complexes can be a convenient way to study it in situ. Fig. 3. illustrates the changes of the apparent mean zeta potential of BSA/FUR mixtures at
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BSA FUR FUR/BSA FUR/BSA FUR/BSA FUR/BSA FUR/BSA
40
[mV]
For the selection of optimal conditions for formation of BSA/furcellaran complexes we characterized their components, i.e. protein and polyelectrolyte in bulk solutions by measuring the dependence of size and zeta potential on pH. The dependence of the diffusion coefficient on the concentration of BSA was previously reported in Ref. [28]. It was found that this diffusion coefficient was practically independent on BSA bulk concentration, reaching the average value of 6 × 10−7 cm2 s−1 within an ionic strength range of 0.01–0.15 M at pH 6.3. From the diffusion coefficient measurements, the Stokes hydrodynamic radius of proteins (RH ) can be determined.
zeta potential
4. Results and discussion
zeta potential [mV]
3.4. SEM
20
1/1 1/2 1/4 1,3/1 2/1
0 -20 -40 -60 0
2
4
6
8
10
pH Fig. 3. The zeta potential of albumin/furcellaran mixtures, with varying BSA/FUR ratio, as a function of pH. The points denote experimental values determined for different BSA/FUR ratio: 4/1, 2/1, 1/1, 1/1.3 and 1/2. Measurements in 0.005 M NaCl.
60
10
40
[mV]
20
0
zeta potential
zeta potential
[mV]
E. Jamróz et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 441 (2014) 880–884
-10 -20 -30
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20 0 -20
0
2
4
6
8
number of layer
-40 -60
-40 0
1
2
3
4
5
6
Fig. 5. Layer-to-layer variations of zeta potential of polyelectrolyte coated complexes with the number of PDADMAC/PSS layers for: albumin/furcellaran system.
BSA/FUR [ppm/ppm] Fig. 4. Dependence of the zeta potential of complexes on the albumin/furcellaran ratio, pH 4.
varying protein/polysaccharide concentration ratio as a function of pH. The total biopolymer concentration was 1000 ppm. As it was shown before pure BSA had the isoelectric point (i.e.p.) at pH 4.8. Decreasing the protein/polysaccharide ratio results in shift of zeta potential profile to lower pHs and therefore lower i.e.p. for BSA/FUR mixture. Clearly, there is a sharp decrease of i.e.p. around ratio 1:1. As the ratio further decreases, we don’t observe i.e.p. of BSA/FUR mixture. It is worthy to note that the DLS method used to determine the electrophoretic mobility is much more sensitive for larger particles (scattering intensity scales as a6 , where a is the particle size), therefore the signal comes from the complexes and free FUR polymer (size >50 nm) but not from free BSA (size 4 nm) in the solution even for the large excess of protein. The dependencies of the zeta potential vs. pH for BSA/FUR mixtures, which are located in between zeta potential curves for pure albumin and furcellaran, give evidence for the formation of albumin/furcellaran complexes. For the high ratio of protein to furcellaran, the polyelectrolyte chains are decorated with proteins and the zeta potential of such objects is slightly lower than BSA in the bulk. Decreasing that BSA/FUR concentration ratio the number of BSA per FUR molecule decreases and the charge compensation, i.e., the i.e.p. of the complex is observed. If BSA is strongly positively charged (pH
