polyelectrolyte core–shell nanoparticles for controlled release of encapsulated ibuprofen

Colloids and Surfaces B: Biointerfaces 123 (2014) 616–622

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BSA/polyelectrolyte core–shell nanoparticles for controlled release of encapsulated ibuprofen ˝ b , I. Dékány b,c,∗ N. Varga a , M. Benko˝ b , D. Sebok a b c

Department of Physical Chemistry and Materials Science, Faculty of Science and Informatics, University of Szeged, Aradi Vt. 1, Szeged H-6720, Hungary MTA-SZTE Supramolecular and Nanostructured Materials Research Group, University of Szeged, Dóm tér. 8, Szeged H-6720, Hungary Department of Medical Chemistry, Faculty of Medicine, University of Szeged, Dóm tér. 8, Szeged H-6720, Hungary

a r t i c l e

i n f o

Article history: Received 9 June 2014 Received in revised form 20 September 2014 Accepted 1 October 2014 Available online 27 October 2014 Keywords: Bovine serum albumin Core–shell nanoparticles Ibuprofen Polyelectrolytes Controlled release Kinetic models

a b s t r a c t Bovine serum albumin (BSA) based core–shell nanoparticles were developed as carrier systems for drug transportation. At pH = 3, the oppositely charged polyelectrolytes: poly(sodium-4-styrene)sulphonate (PSS) and the chitosan (Chit) bind to the positively charged protein via electrostatic interactions. We applied ibuprofen (IBU) as model molecule which has low solubility. The changes in the BSA’s secondary structure during the steps of the synthesis were inspected by FT-IR measurements. The size and the zeta potential were determined by dynamic light scattering (DLS). The changes in the structure and in the size were investigated by small angle X-ray scattering (SAXS) too, for each composite. The release of the ibuprofen was studied by vertical diffusion cell (Franz cell) at pH 7.4 at 25 and 37.5 ◦ C. The structure of the core–shell nanoparticles have significantly changed as the pH has risen from 3.0 to 7.4. Kinetic models were used to describe the release mechanism. The experimental results demonstrated that the BSA has an ordered structure at pH = 3 which will become random coil by adding ibuprofen. The first shell restores the ordered structure of the protein. The controlled release was carried out; the IBU release decreased by 40% in the case of two-layered composites compared with the “naked” BSA. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Core–shell nanoparticles, in nano-scale range, are widely used in therapeutic applications [1,2]. Delivery to appropriate place at appropriate concentration of poorly soluble, small and hydrophilic drug molecules is challenging [3]. These systems are able to carry drug molecules to target tissues. The drug-delivery systems contain a nanosphere where the drug can be encapsulated or absorbed onto their surfaces [4]. Serum albumins are the most commonly used proteins which play an important role in transport processes [5,6]. The protein–drug interactions have been investigated in many studies [7,8]. The human serum albumin (HSA) creates reversible bonds with a very high number of therapeutic agents. Drugs usually bind to high-affinity sites with at least one, at most with a few bonds [9]. The fluorescence probe reveals two high-affinity sites where drugs mainly like to bind. They are called site I and site II. Ligands, that

∗ Corresponding author at: Department of Medical Chemistry, Faculty of Medicine, University of Szeged, Dóm tér. 8, Szeged H-6720, Hungary. Tel.: +36 62 544476; fax: +36 62 544042. E-mail address: [email protected] (I. Dékány). http://dx.doi.org/10.1016/j.colsurfb.2014.10.005 0927-7765/© 2014 Elsevier B.V. All rights reserved.

typically like to bind to site I, are warfarin, salicylate, while ligands that tend to bind to site II are IBU and the diazepam, for example [10]. The binding to site I happens through hydrophobic interaction, while the binding at site II can consist of hydrogen binding, hydrophobic and electrostatic interactions [11]. Non-toxic, biocompatible and biodegradable materials, such as the bovine serum albumin (BSA), are excellent for transporting different drug molecules. This protein is often used as a protein model due to its stability, low cost and structural homology with HSA [12]. The BSA is known to be composed of 582 amino acid residues [13]. It is mainly made of ␭-helices (66%), the rest of the structure is composed of ␤-sheet turns and side chains (34%) [14]. Infrared spectroscopy is a well establish research technique for the analysis of the secondary structure of proteins. There are nine characteristic IR absorption bands in the infrared spectra, amide A, B, I–VII [15]. The changes in the protein’s secondary structure are due to the ligand–protein interaction. The amid I band is the most sensitive to the changes in the secondary structure among the absorption bands [16]. The BSA contains tryptophan (TRP) residues which show intrinsic fluorescence in the spectra at 340 nm. While TRP212 is located within the hydrophobic pocket of IIA sub-domain of the protein, the TRP-134 is located on the surface of the BSA [17]. When we excited the BSA at 280 nm it had an emission peak at

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340 nm which can be attributed to the presence of the tyrosine and the tryptophan [18]. IBU is a non-steroidal-anti-inflammatory-drug (NSAID) which is used at analgesic, antipyretic and anti-rheumatic medical treatments [19,20]. The therapeutic concentration of the IBU in blood is about 50 mg/L, however it is toxic above 250 mg/L [21]. Herein we report biodegradable, non-toxic Chit/PSS coated BSA nanoparticles for encapsulation. They target poorly soluble drugs, like IBU molecules. Core–shell nanoparticles were developed through layer-by-layer technique at room temperature. The change in the protein’s secondary structure during the preparation process was observed by IR spectroscopy. The structural properties of the composites were investigated by SAXS measurements. The release processes of the IBU were measured by vertical diffusion cell at 25 and 37.5 ◦ C. Kinetic models are applicable to describe the release mechanism of the IBU molecules from the core–shell nanoparticles. 2. Materials and methods 2.1. Materials

2.2. Synthesis of core–shell nanoparticles We prepared all samples in buffer solutions at pH = 3 at 25 ◦ C with constant ionic strength (0.9% NaCl). At this pH the BSA is a positively charged macromolecule and is still below the IBU’s pK value (∼4.5 [22]) and the isoelectric point of the BSA (∼4.7 [23]). The IBU was added to the BSA (3.9%) solution in an excess amount (BSA:IBU molar ratio is 1:10). The solubility of the IBU is very low in water (0.021 mg/ml [24]). During the preparation process more and more the IBU bind to the protein; the system continuously gets more and more diluted thus more drug molecules becomes able to dissolve in water. The BSA/IBU composite dispersion (15 ml) was precipitated by 2 ml Na2 SO4 (2 M) after an overnight stirring. This was then centrifuged (9000 rpm, 20 min) and dispersed in PSS (0.3%, 15 ml) solution, finally was stirred for one more hour then centrifuged again. The Chit (in 4, v% acidic acid) solution has been treated the same way. Samples were taken for further investigations at each step. The concentration of the IBU in the solution after each centrifugation process was determined by UV–vis spectroscopy. The products were freeze dried (liophylized) and stored at −80 ◦ C. The schematic picture of the formation of the core–shell nanoparticles and the release processes are shown in Fig. 1. The encapsulation efficiency of the IBU molecules was calculated by using the following equation: Encapsulation efficiency % =

2.3. In vitro drug release experiments For the in vitro IBU release investigations we used a vertical diffusion cell (Franz cell). The height of the cell is 61 mm, the diameter of the orifice is 9 mm and the volume is 4 ml. The encapsulated IBU in nanoparticle dispersion was separated by cellulose membrane from the physiological solution. The cell was connected to a UV-1800 spectrophotometer forming a closed system. The absorbance of IBU was detected at 264 nm (the error of the calculated concentrations are less than ±6%). The core–shell nanocomposites containing IBU were dispersed in phosphate solution (PBS, pH = 7.4). The solution was stirred continuously with a magnetic stirrer and peristaltic pump at 25 ◦ C and 37.5 ◦ C. Samples were taken every 10 min in the first hour, then once every hour. We continued the measurement for 500 min. Each experiment was carried out twice. Kinetic models were used for describing the release mechanism; the correlation coefficient values show which model fits best for the dissolution. For calculations we have used the following equations: Zero-order rate model

The poly(sodium-4-styrene-sulphonate) (PSS) with MW of 70,000 g/mol and the low molecular weight chitosan (Chit) were purchased from Sigma–Aldrich. The bovine serum albumin (BSA) (fraction V), the IBU (C13 H18 O2 ) and the component of the Mc Ilvaine’s buffer (pH = 3) were purchased from Sigma–Aldrich. The sodium-chloride (NaCl), the sodium-sulphate (Na2 SO4) , the sodium-hydroxide (NaOH) and the hydrogen-chloride (HCl) were bought from Molar Chemicals. Highly purified water was obtained by deionization and filtration with a Millipore purification apparatus. All solvents and reagents used were of analytical grade and no further purifications were made.

M  t

Mi

× 100%,

where Mt is the total amount of the drug in the composites and Mi is the total quantity of the drug added initially during preparation [25,26]. The absorbance of the drug was measured to know the remaining IBU concentration in supernatant by a UV–vis spectrophotometer at 264 nm.

617

ct = co + kd t

(1)

(the release rate is independent from the concentration of the dissolved substance). First-order rate model

ln

c  t

co

= −kd t

(2)

(the release rate depends on the concentration). Higuchi model

ct = kH + t 0.5

(3)

(the release mechanism is diffusion-controlled process). In these models, co is the amount of the IBU in the composites at t = 0, ct is the amount of the IBU in the composites at time t. The release rates are kd and kH [27]. 2.4. Materials characterization The streaming potential of the BSA, PSS and the Chit was measured by particle charge detector (PCD-04 MÜTEK). The rate of the addition was 0.2 min/␮l. Attenuated total reflection Fourier transform infrared (ATRFTIR) spectroscopy measurements were performed by a Biorad FTS-60A FT-IR spectrometer by accumulation of 256 scans at a resolution of 4 cm−1 between 4000 and 400 cm−1 . All spectral manipulations were performed using Thermo Scientific GRAMS/AI Suite software. Small angle X-ray scattering (SAXS) measurements were used to analyze the inner structure of the materials. SAXS curves were recorded with a slit-collimated Kratky compact small-angle system (KCEC/3 Anton-Paar KG, Graz, Austria) equipped with a positionsensitive detector (PSD 50M from M. Braun AG, Munich, Germany). Cu K␣ radiation was generated by a Philips PW1830 X-ray generator operating at 40 kV and 30 mA. The fluorescence emission properties of the samples were determined by a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer at wavelength of 280 nm excitation with 2 nm slit. The size and the zeta potential were calculated by DLS measurements with a Zetasizer Nano ZS ZEN 4003 apparatus (Malvern Ins., UK). A FEI Tecnai G2 20 X-TWIN microscope with tungsten cathode was used to carry out the transmission electron microscopy (TEM) measurements at 200 kV. The absorption of the IBU was studied by a UV-1800 (Shimadzu) spectrophotometer. The concentration of the IBU was determined based on the absorption at 264 nm in the spectrum.

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Fig. 1. Development of BSA based core–shell nanoparticles for release of IBU, the process of the release and the molecular structure of ibuprofen.

3. Results and discussion The charge of the components was determined by titration method using the particle charge detector (Fig. S1, Supporting information). First, PSS solution (0.3%, pH = 3 buffer) was added to the BSA solution (10 ml, 2%, pH = 3, buffer) and the streaming potential was monitored. The BSA has a positive charge at this pH so the surface needs 20 ml of PSS to compensate the positive charge. The calculated specific charge of the neutral charge state was 1.42 mequiv./g BSA, which value shows close resemblance with previous experiments [28]. Finally, positively charged Chit (0.3%, pH = 3, in 4, v% acetic acid) was added to the BSA/PSS core–shell nanoparticles. The charge becomes positive when the two shells form on the BSA core. The BSA/PSS composite reaches neutral charge by adding small amount of Chit (93 ␮l). The Chit needs only a minimal amount of 0.0046 mequiv./g to compensate the charge of the BSA/PSS core–shell nanoparticles. We have studied the formation of the core–shell nanoparticles by infrared spectroscopy (Fig. 2). The bands found within the range of 1650–1640 cm−1 and 1540–1528 cm−1 wavenumber values correspond to amide I, caused by C O stretching and amide II, caused by C N stretching coupled with N H bending, respectively [29,30]. These bands are sensitive to the changes in the protein’s secondary structure, but the most sensitive is the amid I band. This band of the BSA at 1642 cm−1 suggests a ␤-sheet structure. The shift of this band is caused by the IBU binding to the BSA, the protein chain partially unfolds. At this wavenumber (1648 cm−1 ) the ␤-sheet content decreases and random structure becomes the determinative conformation. The amid II band is shifted too by adding IBU molecules (from 1528 cm−1 to 1536 cm−1 ). These indicate that the IBU can bind to the C O, C N or N H groups of the polypeptide chain of the BSA and hydrogen bond may be formed between the drug and the BSA. The structure of the BSA/IBU/PSS core–shell nanoparticles will become ordered, the maximum of amid I band shifts toward lower wavenumbers (1639 cm−1 ). A disordered, random structure becomes characteristic for the protein after the formation of the second shell.

The SAXS method is appropriate to determine some structural parameters [31,32], including the fractal dimension. The I(h) is plotted versus h can be obtained a straight line, this is the power law representation [I(h) = Cha ], where the C is a constant. The fractal slope, the a value represents the surface morphology of the samples. The scattering curves in log–log representation are shown in Fig. 3. The a value is 2, so the mass fractals are characteristic for the “naked” BSA and the core–shell nanoparticles (BSA/IBU/PSS, BSA/IBU/PSS/Chit). In case of the BSA/IBU composites this value is 1 which means that the system has a chain-like structure. This result supports the infrared measurements since the BSA contains ␤-sheets in largest percentage. If the IBU binds to the BSA, the protein’s compact structure changes, the random coil structure will be the determining unit in the protein’s secondary structure. Fig. 4(A) representation shows the Guinier-plot of the samples. There are individual scattering centers in the case of the BSA and the BSA/IBU. This is confirmed by the h × Rg value which is lower than 1.3. This value increases (h × Rg > 1.3) during the formation of the core–shell nanoparticles because of the aggregation and the development of the shells. The ATSAS Gnom software was used to evaluate the particle distance distribution function [33]. The p(r) plots are shown in Fig. 4(B). The maximum values of the curves are found at the same r values but they will become more and more prolonged and flat. This suggests that the system becomes ellipsoid shape. This indicates that the protein keeps partially its initial structure, while a significant fraction of the protein chain unfolds. The shapes of these curves imply the increase in size, too. The results of the SAXS measurements are collected in Table S1 (Supporting information). The fluorescent spectra of the initial materials and the core–shell nanoparticles are shown in Fig. 5. The IBU and the Chit do not have emission at this wavelength-range excitation, so these are not represented in Fig. 5. The BSA has two aromatic residues: the tryptophan (TRP) and the tyrosine (TYR). They show a strong emission peak at 335 nm when excited at 280 nm. This emission peak is attributable to the TRP. By adding IBU to the BSA solution the intensity of this peak decreases. This verifies that the IBU binds to the BSA, close to TRP residues. The conformation of the

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619

Fig. 2. Infrared spectra of the formation of the core–shell nanoparticles and the secondary structure units assigned to the peaks.

polypeptide chain changes due to the effect of the IBU. The protein can bind much more IBU in an unfolded structure compared to the chain form. This effect covers the aromatic amino acid residues, causing a decrease in the emission peak. In the case of the polymers, the PSS has two strong emission peaks in the UV-range while the Chit does not. By forming the first shell, the emission peak decreases further. This is caused by the PSS covering the protein, hiding the fluorescent emission of the TRP, the protein’s secondary structure will become ordered. The formation of the second shell shows an increasing in the emission which probably means that the system has obtained a disordered structure again (the emission peak at 336 nm). This phenomenon implies that the protein chain unfolds. The infrared results support this statement. We have followed the increase in size and the change in zeta potential during the formation of the core–shell nanocomposites (Table S1, Supporting information). The charge of the surface is determined by the external shell. At pH = 3 the core of the BSA is positively charged, the zeta potential is +2.5 mV. If the IBU bind to

Fig. 3. The scattering curves of the SAXS measurements in log–log representation.

the BSA, the protein’s chains unfold, the dissociation of the protein increases, so we observe a slight rise in zeta potential (+5.1 mV). When the PSS envelopes the core, the charge of the nanocomposite changes, the surface becomes negatively charged (−10.3 mV). The Chit, as the outermost shell, grants positive charge to the surface (+6.1). The two-layered nanoparticles are applicable for oral administration, because their positive charge allows them to stick efficiently to the gastro-intestinal mucosa, with a further possible diffusion through the epithelium, thus providing a continuous drug delivery into the blood stream [34]. The TEM images (Fig. 6(A)) of the BSA/IBU/PSS core–shell nanocomposites verify the formation of the core–shell structure. The average diameter of these composites is about 140 nm. Fig. 6(B) shows the size distribution of each samples. The diameters of the BSA and the BSA/IBU nanoparticles are below 10 nm while the diameters of the monolayer nanocomposites are between 90 and 150 nm. The size of the core–shell nanoparticles grows with another 100 nm with the development of the second shell. The average diameters are shown in Table S1 (Supporting information). A system with a size of about 250 nm is able to cross the blood–brain barrier (BBB) [35]. The BSA has two absorption bands in UV range, one is around 220 nm and the other one is around 280 nm. The first peak, which is at 220 nm, derives from the polypeptide bonds and the carboxylgroups of the side chains. Its place and intensity can be disturbed by the absorption of IBU. The second peak, which is at 280 nm, is caused by the absorption of tryptophan and tyrosine residues [36]. The concentration of the IBU was determined by the absorption at 264 nm of the spectrum. At this wavelength the absorbance of BSA does not intervene with the absorbance of IBU. We have investigated how the number of shells influences the release of the drug from the core–shell nanoparticles. The release % was calculated based on the encapsulated efficiency (Table S1, Supporting information). The highest encapsulation efficiency value of 93.7% (close to the 1:10 molar ratio) was obtained in the case of the BSA/IBU. This result suggest that the IBU binds not only the high affinity binding sites but to other binding sites of the protein molecule. The IBU content decreases during the formation of the core–shell nanoparticles, which is due to the continuous release of the IBU during the synthesis. Lower efficiency value was measured at the BSA/IBU/PSS core–shell nanocomposites, only 77% of the IBU remained in the composites. In the case of

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Fig. 4. The Guinier plot form SAXS measurements (A), the curve of BSA is shifted by −0.5 and the curve of BSA/IBU/PSS/Chit is shifted by 0.5. The p(r) vs. r plot (pair distant distribution function) (B) of each samples.

the two-layered composites this value further decreases (69.4%). The lower efficiency value in case of the one-layered core–shell nanoparticles was predicted compared to the value of the twolayered composites since it is easier for the IBU to escape from the “naked” BSA than from the mono-layered version. The release profile at 25 ◦ C is shown in Fig. 7(A). In the first hour the dissolution of the IBU is the slowest in the BSA/IBU and the fastest in the BSA/IBU/PSS case. The retarding effect of the shell appears after 1 h. 70% of the IBU dissolute from the “naked” BSA after 8 h. The release rate (40% of the IBU) significantly decreases by the formation of the first shell. With more than 8% IBU remain in

Fig. 5. Fluorescence intensity of (a) BSA, (b) BSA/IBU, (c) BSA/IBU/PSS, (d) BSA/IBU/PSS/Chit and (e) PSS at ex = 280 nm.

the BSA/IBU/PSS/Chit core–shell nanocomposites than in the case of BSA/IBU/PSS after 500 min. The release of the IBU in the first hour was minimal at 37.5 ◦ C (Fig. 7(B)), 10% was dissolved from all composites. In the cases of BSA/IBU and BSA/IBU/PSS core–shell nanoparticles, the release rates were equivalent, after 8 h 67% of the IBU dissolved. From the BSA/IBU/PSS composites were the fastest the release rate of the IBU at 37.5 ◦ C. There is a strong repulsion between the BSA and the PSS are at pH 7.4 due to those molecules having the same charge. Due to the weaker electrostatic interactions the IBU rapidly dissolves with the polyelectrolyte into the surrounding medium. There is a stronger attraction between the drug and the protein, so the release process is slower in the case of the BSA/IBU composite. The two-layered core–shell nanoparticles show a significant decrease, only 39% dissolved after 500 min. The release profile of the IBU “naked” core–shell nanoparticles is the same, only 1% is the difference at two different temperatures. So the release of the IBU was controlled only by its binding strength to the BSA. The one-layered core–shell nanoparticles show difference at 25 and 37.5 ◦ C. The release of the IBU starts faster in the initial time at room temperature, than the rate of the release slows down. On the other hand, the release of the IBU slowly increases continuously at higher temperature: 18% more IBU remains in core–shell nanoparticles after 500 min at 37.5 ◦ C than at room temperature. In the first hour the IBU releases faster from the BSA/IBU/PSS/Chit composite at 25 ◦ C, but the values of the final release are the same at both temperatures (39% – 25 ◦ C, 40% – 37.5 ◦ C). Kinetic models were applied to describe the release mechanism of the IBU from the core–shell nanoparticles. The correlation coefficient and the release rate values at the two temperatures are represented Table 1. The R2 values show how well the models explain the dissolution processes. The first-order rate model fits best for the BSA/IBU core–shell nanoparticles at both temperatures, which means that the drug release rate depends on its concentration. The release profiles are analogous; the temperature does not influence the rate of the IBU release. The utmost R2 value for the BSA/IBU/PSS core–shell nanoparticles is 0.973 based on Higuchi model at 25 ◦ C. This model suggests a diffusion-controlled release mechanism. At higher temperature the zero-order rate (0.976) is

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Fig. 6. TEM image of the BSA/IBU/PSS (A) and the size distribution (B) during the formation of the core–shell nanoparticles.

Fig. 7. Release profile of the IBU from the core–shell nanoparticles at 25 ◦ C (A) and at 37.5 ◦ C (B) (circle – BSA/IBU; square – BSA/IBU/PSS; triangle – BSA/IBU/PSS/Chit).

the most convenient model; it implies that the drug release rate is independent from the concentration of the dissolved substance. The release of the IBU from the two-layered core–shell nanocomposites fits well with the Higuchi model at room temperature. The kinetic models do not describe well the release processes at 37.5 ◦ C; the correlation coefficient values are under 0.96. At 25 ◦ C the polyelectrolyte layers slow down the IBU release by diffusion. At 37.5 ◦ C other processes influence the drug release as well; diffusion is not the most determinative process. Some methods (FT-IR, SAXS, fluorescence, CD, etc.) are perfectly applicable to study the changes in the proteins’ structure since ligands can cause ligand-dependent allosteric conformation transition(s) in the protein’s structure. The proteins undergo pH- and allosteric effector-dependent reversible conformational isomerization(s) too [37]. Between pH values of 2.7 and 4.3 the BSA shows

a fast (F) form, characterized by a significant loss in its ␣-helical content [37]. This is in good agreement with our results since the ␤-sheet structure unit is the determined in BSA’s secondary structure at pH 3. The proteins were tested mostly in their native form, above pH 7. The HSA–IBU interaction was investigated by Galantini et al. [38]. The HSA keeps the original structure by adding IBU (1:10 molar ratio). The stability of the HSA is also enhanced upon binding to the drug molecules. The SAXS measurements, the Kratky plots and the p(r) functions are most important in the study of the unfolding process of the protein. The maximum of the characteris˚ of a native globular protein decreases when tic peak (Dmax = 85 A) partially folded states appear and keeps transforming into a flat shape that is typical for denaturated structure (random coil) [38]. This phenomenon appears due to the effect of polyelectrolytes during the synthesis of the core–shell nanoparticles. We can see that

Table 1 Interpretation of the release experiments using different various models. BSA/IBU

BSA/IBU/PSS

◦

◦

25 C 2

◦

37.5 C 2

25 C 2

BSA/IBU/PSS/Chit ◦

25 ◦ C

37.5 C 2

2

37.5 ◦ C

Kinetic models

R

kd

R

kd

R

kd

R

kd

R

kd

R2

kd

Zero-order rate (s−1 ) First-order rate (s−1 ) Higuchi (s−1/2 )

0.945 0.989 0.964

6.48 × 10−2 1.50 × 10−3 6.06 × 10−1

0.971 0.970 0.935

2.19 × 10−1 2.30 × 10−3 2.37 × 100

0.945 0.935 0.973

5.21 × 10−2 7.00 × 10−4 5.43 × 10−1

0.976 0.974 0.930

1.34 × 10−1 1.50 × 10−3 1.44 × 100

0.977 0.971 0.986

1.50 × 10−1 1.60 × 10−3 1.66 × 100

0.959 0.959 0.906

5.00 × 10−4 5.00 × 10−4 5.30 × 10−3

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the proteins are in different state at different pH but they behave similarly after connecting with any kinds of ligand. 4. Conclusion Core–shell nanoparticles with protein core, covered with polyelectrolytes were developed as carrier system for poorly soluble drug molecules. The positively charged BSA molecules have determinative ␤-sheet conformation at pH = 3. The protein’s secondary structures show continuously alteration by the addition of the drug molecules, the PSS and the Chit polyelectrolytes, based on IR spectroscopy. In the case of the BSA/IBU composites the random coil structure is present with highest percentage in the protein’s structure, which was verified by the SAXS measurements, too. The emission intensity of the BSA decreases by adding IBU, so the BSA uncoils, some of the IBU can bind close to the TRP groups. The protein becomes orderly by the appearance of the first shell, hiding the strong TRP emission. The core–shell structure aggregates by the addition of Chit, the polymer chains unfold; random coil structure becomes the determinative formation. The h × Rg values (>1.3) from the SAXS measurements prove the presence of the aggregate systems as well. The IBU release profiles show that the shells slow down the release rate at both temperatures. More than 30% less IBU is released from the BSA/IBU/PSS/Chit core–shell nanocomposites than from the “naked” BSA. The small size and the results of the in vitro release studies show that these core–shell nanoparticles are applicable in future in the controlled delivery of therapeutic agents. Acknowledgement This research was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP-4.2.2.A-11/1/KONV-2012-0047. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2014. 10.005. References [1] A. Takami, K. Tatsuo, K. Toshiyuki, A. Mitsuru, Preparation and characterization of biodegradable nanoparticles based on poly(g-glutamic acid) with l-phenylalanine as a protein carrier, J. Control. Release 108 (2005) 226–236. [2] F. Danhier, et al., PLGA-based nanoparticles: an overview of biomedical applications, J. Control. Release 161 (2012) 505–522. [3] P.L. Lama, R. Gambari, Advanced progress of microencapsulation technologies: in vivo and in vitro models for studying oral and transdermal drug deliveries, J. Control. Release 178 (2014) 25–45. [4] K. Sparnacci, M. Laus, L. Tondelli, L. Magnani, C. Bernardi, Core–shell microspheres by dispersion polymerization as drug delivery systems, Macromol. Chem. Phys. 203 (2002) 1364–1369. [5] N. Wang, L. Ye, B.Q. Zhao, J.X. Yu, Spectroscopic studies on the interaction of efonidipine with bovine serum albumin, Braz. J. Med. Biol. Res. 41 (2008) 589–595. [6] A.O. Elzoghby, W.M. Samy, N.A. Elgindy, Albumin-based nanoparticles as potential controlled release drug delivery systems, J. Control. Release 157 (2012) 168–182. [7] P.B. Kandagal, et al., Study of the interaction of an anticancer drug with human and bovine serum albumin: spectroscopic approach, J. Pharm. Biomed. Anal. 41 (2006) 393–399.

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