A protein-polysaccharide-based potential nanocarrier system have been developed via a simple, one-step preparation protocol without the use of long-term heating and the utilization of hardly removable crosslinking agents, surfactants, and toxic organic solvents.
Carbohydrate Polymers 251 (2021) 117047 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Serum protein-hyaluronic acid complex nanocarriers: Structural characterisation and encapsulation possibilities ´ ´ ´cs a, Norbert Varga a, Ad ´sz a, b, Edit Csapo ´ a, b, * Alexandra N Kova am Juha a b Department of Physical Chemistry and Materials Science, University of Szeged, H-6720, Rerrich B Square 1, Szeged, Hungary MTA-SZTE Biomimetic Systems Research Group, Department of Medical Chemistry, Faculty of Medicine, University of Szeged, H-6720 D´ om Square 8, Szeged, Hungary A R T I C L E I N F O A B S T R A C T Keywords: Hyaluronic acid Bovine serum albumin Protein-polymer nanoconjugates Charge neutralisation Encapsulation capacity Drug release A protein-polysaccharide-based potential nanocarrier system have been developed via a simple, one-step prep aration protocol without the use of long-term heating and the utilization of hardly removable crosslinking agents, surfactants, and toxic organic solvents To the best of our knowledge, this article is the first which summarizes in detail the pH-dependent quantitative relationship between the bovine serum albumin (BSA) and hyaluronic acid (HyA) confirmed by several physico-chemical techniques The formation of colloidal complex nanoconjugates with average diameter of ca 210–240 nm is strongly depend on the pH and the applied BSA:HyA mass ratio Particle charge titrations studies strongly support the core-shell type structure, where the BSA core is covered by a thick HyA shell Besides the optimization of these conditions, the drug encapsulation capacity and the disso lution profiles have been also studied for ibuprofen (IBU) and 2-picolinic acid (2-PA) as model drugs Introduction Due to the outstanding biocompatible and biodegradable nature, the utilization of natural polysaccharides as sustained-release carriers is beneficial for pharmaceutical fields (Mohamed, El-Sakhawy, & ´, 2020) The targeted drug El-Sakhawy, 2020; Turcs´ anyi, Varga, & Csapo delivery is possible by using HyA, which is a negatively charged glycosaminoglycan, but the chemical modification of its hydroxyland/or carboxyl-group is required (Yamanlar, Sant, Boudou, Picart, & Khademhosseini, 2011) The HyA has a prominent role at biomedical ´ applications as well (Huerta-Angeles et al., 2020) The HyA-based nanohydrogels/nanoparticles (NPs) and films are implied as a prom ising area of the cancer treatment, tissue engineering, gene delivery etc (Graỗa, Miguel, Cabral, & Correia, 2020) BSA is a water soluble glob ular protein consist of 583 amino acid residues (Ghosh & Dey, 2015); wide ranges of active compounds are able to bind at the appropriate ă mo ăto ăr et al., 2018) Depending on the pH, binding sites of the protein (Do the charge of the BSA is shifted from the positive to the negative value reaching the isoelectric point (pI⁓5.1), that regulates the interaction between the BSA-polymers and BSA-drugs via electrostatic interactions (Varga, Hornok, Sebok, & D´ek´ any, 2016) Polysaccharide-protein conjugates may represent a new dimension in the design of drug delivery systems The utilization of these complex conjugates enhances the colloid stability, targeted efficiency, biocom patibility or the reduced drug toxicity (Gaber et al., 2018), which is published previously for e.g BSA/Chitosan (Karimi, Avci, Mobasseri, Hamblin, & Naderi-Manesh, 2013), Ovalbumin/Chitosan (Yu, Hu, Pan, Yao, & Jiang, 2006), Protamine/HyA (Mok, Ji, & Tae, 2007) or Lyso zyme/Alginate (Fuenzalida et al., 2016) nanocarriers The polysaccharide-protein nanoconjugates are generally prepared by elec trostatic complexation (Antonov et al., 2019), chemical conjugation ´n, & Blanco, 2011) and electrospinning (Martínez, Iglesias, Lozano, Teijo techniques (Torres-Giner, Ocio, & Lagaron, 2009) In some cases, the Maillard reaction is also implied, but the reaction requires long term heating (60 ◦ C; for at least h) (Edelman, Assaraf, Levitzky, Shahar, & Livney, 2017) which is specifically unfavourable for heat-sensitive drugs The fabrication of carrier NPs is usually carried out by chemical coupling in the presence of crosslinking agents, like glutaraldehyde ´nyi et al., 2020), which (Chen et al., 2013), or tripolyphosphate (Turcsa components can be difficult to remove during the purification process Based on these facts, the demand for the development of an effective polysaccharide-protein nanoconjugates with tuneable-size is strongly required, where the long-term heating and the utilization of hardly removable crosslinkers and surfactants is excluded In this work we first demonstrate a preparation possibility of BSA/ HyA conjugates by a simple, controllable charge neutralization * Corresponding author at: Department of Physical Chemistry and Materials Science, University of Szeged, H-6720, Rerrich B Square 1, Szeged, Hungary E-mail address: juhaszne.csapo.edit@med.u-szeged.hu (E Csap´ o) https://doi.org/10.1016/j.carbpol.2020.117047 Received 15 July 2020; Received in revised form September 2020; Accepted September 2020 Available online September 2020 0144-8617/© 2020 The Authors Published by Elsevier Ltd This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) A.N Kov´ acs et al Carbohydrate Polymers 251 (2021) 117047 technique without protein degradation and in the absence of cross linking agents and organic solvents Moreover, the pH-dependent quantitative characterisation of the interaction between BSA and HyA was also investigated by multiple techniques, which is not published previously in detail All the previous studies present only the charac terization of the drug-containing composites by using only dynamic light scattering (DLS) and transmission electron microscopy (TEM) images, however the pharmacokinetics of these complex nanocarriers are demonstrated (Huang, Chen, & Rupenthal, 2017; Martins et al., 2014; ˘u et al., 2020; Pigman, Gramling, & Holley, 1961; Shen & Li, Pas¸cala 2018) Only one article was found (Chen et al., 2013) which contains some information on the interaction between BSA and the encapsulated drug in the presence of HyA by differential scanning calorimetry (DSC) and infrared spectroscopy studies, but the interaction between serum protein and HyA was not investigated Two articles focus on the inter pretation of the HyA- serum protein interaction by NMR (Filippov, Artamonova, Rudakova, Gimatdinov, & Skirda, 2012) and integrated computer modeling (Grymonpr´ e, Staggemeier, Dubin, & Mattison, 2001), but only the presence and strength of the electrostatic interaction was mentioned Moreover, we also considered to demonstrate the suc cessful encapsulation of two drugs with slightly different hydrophilicity (2-PA: logP = -0.1; IBU: logP = 1.74) at the optimized weight ratio Both molecules have one aromatic ring and one carboxylic group, but the charge of the molecules at pH = 4.5 is different The IBU was selected for our experiments as a model drug because this molecule is often studied non-steroidal anti-inflammatory compound and its binding mechanism to serum albumin binding sites is well-known 2-PA is a neuroprotective, immunological and antiproliferative compound, it has not been encap sulated as active component in any drug delivery system Drug loading (DL%) and the release mechanism of the encapsulated molecules are examined, and the results are compared with other nanocapsule-based carrier systems containing same drugs Alpha 1–2 LD plus) and the solid samples were stored at -70 ◦ C 2.2.2 Synthesis of the 2-PA- and IBU-loaded BSA/HyA conjugates For the drug-loaded derivatives, similar preparation and purification protocol was used than for fabrication of unloaded NPs, but 1− mg of drugs was also dissolved in the BSA solution (2 mL, cBSA = mg mL− 1) The synthesis was carried out at mBSA/mHyA = 2.00 mass ratio based on the experimental results presented in Chapter 2.2.3 Surface plasmon resonance (SPR) The SPR studies were performed in a two-channel device improved at the Institute of Photonics and Electronics (Prague, Czech Republic) The light source was an Ocean Optics HL-2000 type tungsten halogen light source with 6.8 mW output power, while the reflected light intensity is monitored in the 574–1000 nm spectrum range using an IPE AS CR S2010 spectrometer The sensorgrams were registered by SPR UP 1.1.11.3 (2014 IPE AS CR) control software Firstly, the protein solution (cBSA = 10 μM) was flowed under a constant flow rate (50 μL min− 1) above the gold-coated SPR chip in order to immobilize the protein to the ´ et al., 2016) On the surface of the sensor via Au-S covalent bond (Csapo next step, the HyA solution was flowed across the protein-functionalized surface with 50 μL min-1 flow rate During studies, the following con ditions were used: T = 15− 30 ◦ C, cHyA = 2.5–10.0 μg mL− and the pH range of 3.6− 5.5 2.2.4 Particle charge detector (PCD) The PCD measurements were performed by a PCD-04 Particle Charge Detector (Mütek Analytic GmbH, Germany) with manual titration ac ´nyi et al., 2020) cording to our previously detailed technique (Turcsa Firstly, the HyA was dissolved in acetate buffer at four different pH values (pH = 3.6; 4.0; 4.5; 5.0), while the concentration kept constant (20 mL of cHyA=0.36 mg mL− 1) The BSA solution (10 mg mL− 1) was added dropwise in 100− 100 μL portions to the HyA solution at 25 ◦ C and the streaming potential values (mV) were registered The acquired re sults were analyzed and fitted with the modified version of the sigmoidal Boltzman equation Experimental 2.1 Materials BSA (~66,000 Da), HyA sodium salt (1.5–1.8⋅106 Da), IBU sodium salt (≥98 %), and 2-PA (≥99 %) were purchased from Sigma-Aldrich The disodium hydrogen phosphate (Na2HPO4; ≥99 %), the sodium dihydrogen phosphate monohydrate (NaH2PO4⋅H2O; ≥99 %), the so dium acetate 3-hydrate (CH3COONa⋅3H2O; ≥99 %), and sodium hy droxide (NaOH; ≥96 %) pastilles and the hydrochloric acid (HCl, ≥99 %) were bought from Molar Chemicals Acetic acid (AcOH, ≥99 %) was ˝k´ purchased from Erdo emia Ltd Company Highly purified water was obtained by deionisation and filtration with a Millipore purification apparatus (18.2 MΩ cm at 25 ◦ C) All reagents and solvents used were of analytical grade without further purification 2.2.5 Rheology Anton Paar Physica MCR 301 Rheometer (Anton Paar, GmbH, Ger many) equipped with cylinder geometry (CC27-SN12793) was used; the changing of the viscosity was followed at 25 ± 0.1 ◦ C and at 37 ± 0.1 ◦ C at different pH values using acetate buffer (pH = 3.6; 4.0; 4.5) 10 mg mL− BSA solution was added drop by drop in 19 mL of 0.1 mg mL− HyA solution at 40 μL/3 dosing speed The effect of solvent dilution ´ et al., was also considered as described in our previous article (Csapo 2018) 2.2.6 Thermal behaviour The thermal behaviour of the BSA, HyA, and the lyophilized powders of BSA/HyA conjugates at different mass ratios were studied with thermogravimetric (TG) and DSC The TG studies were performed with the use of a Mettler-Toledo TGA/SDTA851e instrument with ◦ C min− between the range of 25− 1000 ◦ C, under constant air flow (50 mL min− 1) The DSC studies were performed with the use of a MettlerToledo DSC822e calorimeter with ◦ C min− in the range of 25− 500 ◦ C under nitrogen stream (50 mL min− 1) 2.2 Methods 2.2.1 Synthesis of the unloaded BSA/HyA conjugates The BSA/HyA conjugates were prepared by charge neutralization method The 1.6 mg mL− HyA and the mg mL− BSA stock solutions were prepared in acetate buffer (0.010 M acetic acid/0.0057 M sodiumacetate; pH = 4.5) and in MilliQ water, respectively In the first step, the HyA stock solution was stirred at 350 rpm for h and stored overnight at ◦ C During the synthesis, several BSA/HyA conjugates were prepared within the range of mBSA/mHyA = 0.25–5.00 mass ratios Namely, mL of mg mL− BSA was added dropwise into the 10 mL of 0.08–1.6 mg mL− HyA solutions under 1000 rpm magnetic stirring at 25 ◦ C After mixing the appropriate amounts of BSA to HyA, the samples were further stirred for h under 500 rpm Finally, the samples were cen trifugated at 5000 rpm for min; the supernatant was removed, and the samples were redispersed in acetate buffer The cleaning method was repeated three times The cleaned products were freeze-dried (Christ 2.2.7 Fourier transformed infrared spectroscopy The FT-IR spectra were registered with a Jasco FT/IR-4700 spec trometer with the use of an ATR PRO ONE Single-reflection accessory (ABL&E-JASCO, Hungary) The spectra were recorded at a resolution of cm− between 4000 and 500 cm− by accumulating 128 interfero gram The samples prepared in the same method discussed in the pre vious section A.N Kov´ acs et al Carbohydrate Polymers 251 (2021) 117047 Fig (A) Representative SPR reflectance curves before (black) and after (blue) addition of HyA solution at pH = 3.6 and (B) the registered sensorgrams at different pH values (cHyA = 2.5 μg mL− 1, 50 μL min− flow rate, t = 25 ± 0.1 ◦ C) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 2.2.8 Circular dichroism spectroscopy CD spectra were recorded by using Jasco J-1100 CD spectrometer (ABL&E-JASCO, Hungary) at 25 ± ◦ C using cm optical pathlength quartz cuvette All spectra were recorded at 100 nm min− scanning speed in the middle UV-region (200− 300 nm) under N2 flow (3 L min− 1) and represents the average of three scans The light source was a watercooled, high-energy xenon lamp (450 W) The raw data was converted into mean residue ellipticity (MRE) using the Eq (1), and the ratio of the α-helix content was calculated from the Eq (2) MRE208 = observed CD(mdeg) 10Cp nl α − helix (%) = − MRE208 − 4000 × 100 33000 − 4000 spectrophotometer in a cm quartz cuvette The measurements were carried out at room temperature in 200− 500 nm wavelength range The exact concentration of the non-encapsulated free IBU and 2-PA was calculated from the calibration curves, where the characteristic absor bance band of the IBU and 2-PA were appeared at λ = 222 nm and λ = 264 nm in acetate buffer (pH = 4.5) medium, respectively (Fig S1) The DL% and EE% values were defined by Eqs (3) and (4) (1) (2) DL% = encapsulatedmassofdrug × 100 totalmassofthenanoparticles (3) EE% = encapsulatedmassofdrug × 100 totalmassofdruginsynthesis (4) 2.2.10 In vitro release study The in vitro dissolution profiles of the IBU- and 2-PA-containing BSA/ HyA conjugates were measured by UV–vis The release measurements were performed in phosphate buffer solution (pH = 7.4 ± 0.1) at 37 ± 0.5 ◦ C and a semipermeable cellulose membrane (avg flat width = 25 mm; Mw cut-off = 14,000; Sigma-Aldrich) was used The data points were registered for 250 The concentrations of the IBU and 2-PA in the release medium were determined by calibration curves (Fig S2) The possible release kinetics and the proposed mechanism can be defined ´nyi, Hor from the fitting of the Weibull kinetic models (Varga, Turcsa nok, & Csap´ o, 2019; Veres et al., 2017) where the Cp is the molar concentration of the protein, n is the number of amino acid residues, and l is the pathlength of the cuvette 2.2.9 Characterisation of the BSA/HyA NPs The average size, size distribution, morphology, polydispersity index and the Zeta-potential values were measured by DLS using a HORIBA SZ100 NanoParticle Analyzer (Retsch Technology GmbH, Germany) The light source was a semiconductor laser (λ = 532 nm, 10 mW) and photomultiplier tubes (PMT) were used as detector at 90◦ scattering angle For registration of TEM images a Jeol JEM-1400plus equipment (Japan) at 120 keV accelerating voltage was applied To determine the encapsulation efficiency (EE%) and DL%, the absorbance spectra of the supernatants of the centrifuged drug-loaded BSA/HyA conjugates were registered by Shimadzu UV-1800 UV–vis double beam Fig (A) Change of the streaming potential of pure BSA and HyA as a function of pH (B) Change of the streaming potential of HyA titrated with BSA at different pH values (starting concentrations: cHyA =0.36 mg mL− 1, cBSA =10.0 mg mL-1, VBSA = 100-100 μL) A.N Kov´ acs et al Carbohydrate Polymers 251 (2021) 117047 Fig Apparent viscosity values of BSA/HyA conjugates (marked with (⸰)) and the calculated streaming potential curves (grey continuous lines) as a function of mBSA/mHyA at pH = 3.6 (A); pH = 4.0 (B) and pH = 4.5 (C) at 25 ◦ C (starting concentrations: cHyA =0.10 mg mL− 1, cBSA =10.0 mg mL-1, VBSA = 40-40 μL) Results opposite charges Fig 2B shows that the initial negative charge of the HyA is shifted to higher values by the addition of BSA At pH = 3.6 and 4.0 the course of the curves is steeper, which is much longer at pH = 4.5 In accordance with SPR results, we found that there is no measurable change in the streaming potential values at pH = 5.0 The following neutralization points (where the streaming potential is mV) are ob tained by fitting the measured points by the modified Boltzmann equation: mBSA/mHyA = 2.04 ± 0.01 (pH = 3.6), 2.69 ± 0.01 (pH = 4.0) and 5.05 ± 0.01 (pH = 4.5) It is also observed that the inflection points of the titration curves (1.97, 2.51 and 4.46, respectively) appear before the neutralization points, which suggests structural changes between the macromolecules and the possible formation of BSA/HyA colloidal NPs before charge neutralization To confirm this observation rheological studies were also performed 3.1 Surface plasmon resonance spectroscopy The pH-dependence SPR studies have been performed at five different pH using acetate buffers (pH = 3.6; 4.0; 4.5; 5.0; 5.5) at 25 ± 0.1 ◦ C The concentration of HyA solution was fixed at 2.5 μg mL− in every cases The registered sensorgrams are presented in Fig The decrease in the pH of the HyA solutions causes a greater shift of the signal of the sensor response which suggests a pH-dependent inter action between the HyA and BSA If the pH of the HyA solution exceeds the pH = 5.0, no significant interaction can be observed (pH = 5.5) The concentration- and the temperature-dependence of the interaction of the two studied macromolecules was also investigated, while the pH of the HyA solution was remain the same (pH = 4.5; acetate buffer) At this pH = 4.5, the HyA are in fully deprotonated form and it is well-known that no measurable structural change occurs in the secondary structure of ´ et al., 2016) For both series BSA at this slightly acidic conditions (Csapo of measurements, merely a slight shift can be observed in the signal of the SPR gold-coated biosensor (Fig S3) Based on these results, it can be concluded that the interaction between the polysaccharide and the protein is strongly depends on pH, and the effect of the temperature and the concentration is negligible under the studied conditions 3.3 Rheological studies By the addition of the BSA stock solution to HyA the viscosity values are continuously decreased to a given point and then a slightly constant values are measured The intersection point of the fitted lines gives a breaking point This trend is observed at all the studied pH values (Fig 3) The breaking points can be given at the following mBSA/mHyA ratios at 25 ◦ C: 1.43 (pH = 3.6), 2.26 (pH = 4.0) and 4.14 (pH = 4.5) The determined breaking points can be obtained at nearly similar mBSA/ mHyA ratios than the inflection points of the PCD curves (1.97 (pH = 3.6), 2.51 (pH = 4.0) and 4.46 (pH = 4.5)) The rheological studies have been carried out at 37 ◦ C and similar trend was observed that at 25 ◦ C, but the breaking points shifted towards the smaller values because of the different solvatated states of the macromolecules (breaking points at 37 ◦ C: 0.91 (pH = 3.6), 1.79 (pH = 4.0) and 3.63 (pH = 4.5) It can be concluded that, before neutralization, a structural change is occurred, which strongly indicates the possible formation of colloidal NPs via electrostatic interaction of BSA and HyA 3.2 PCD measurements The interaction between HyA and BSA was also confirmed in detail by PCD titrations, where the neutralization points were determined at different pH values However, the acid-base property of HyA is well´nyi et al., 2020)), but for quantitative known (pKa = 2.83 (Turcsa interpretation of the results the isoelelectric points of BSA and HyA were determined by PCD (Fig 2A) The titration clearly proved that the BSA has positive charge below pH = 5.0; the neutralization point is obtained at pH = 5.10 which value is in good correspondence with the pI of BSA (Varga et al., 2016) For HyA, the negative surface charge is dominant in wide pH range (pH = 2–11) In case of BSA/HyA system the titrations were carried out in the pH range of pH = 3.5–5.0, where the macromolecules have well-defined A.N Kov´ acs et al Carbohydrate Polymers 251 (2021) 117047 Fig (A) The hydrodynamic diameter (●, left y-axis) and the turbidity values ( , right y-axis) of the BSA/HyA system as a function of increasing mBSA/mHyA (cBSA = mg mL− 1) with the representative photos of the samples at mBSA/mHyA = and mBSA/mHyA = (B) DLS curve of BSA/HyA NPs using mBSA/mHyA = with the TEM images of the sample Fig CD curves of BSA (continuous red line) and BSA/HyA conjugates (dotted grey lines) in MilliQ water (A) and in acetate buffer (pH = 4.5) (B) t = 25 ◦ C, cBSA = 2.78 μg mL− (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 3.4 Characterisation of the drug-free BSA/HyA nanocarrier systems negatively charged HyA To confirm these theory, further PCD studies have been performed The pure HyA solution and the probably HyAcoated BSA-based particles-containing dispersion were titrated with the same CTABr solution under similar conditions We hypothesized that, if HyA functions as a thick shell, nearly the same amount of CTAB will compensate the negative surface charge as would be expected on the free macromolecule as well For pure HyA solution and for composites the charge compensation points are obtained at nCTABr /nHyA(monomer) = 0.88:1.0 and at 0.93:1.0, respectively (Fig S5) This means that ca one CTABr compensates one HyA monomer unit This observation is in good ´ et al., 2018) The presence of agreement with our previous result (Csapo core-shell-type NPs instead of “alloy-like” structure is more preferred The adsorption of the BSA on the surface of HyA can be ruled out 3.4.1 DLS and TEM investigations To prove the formation of BSA/HyA colloidal NPs, DLS and turbidity studies have been also performed The measured hydrodynamic di ameters (Z-average) and the turbidity values of the BSA/HyA conjugates-containing aqueous dispersion at pH = 4.5 are presented in Fig The parallel registered Zeta-potential values are seen in Fig S4 Fig 4A clearly indicates the formation of colloidal NPs according to the increasing turbidity values within the range of mBSA/mHyA = 0.25–3.75 mass ratios If the mass ratio exceeds the mBSA/mHyA = 4.0 value, the aggregation of NPs can be observed, as the inserted photo also represents This observation is confirmed by DLS At the above mentioned mBSA/mHyA = 0.75–3.50 mass ratios the average diameter of 240 – 210 nm is obtained depending on the mass ratios For small BSA content (mBSA/mHyA 3.75) both the adhesion and the aggregation of NPs is feasible The results of DLS and turbidity studies are in good agreement with the main conclusions of both PCD and rheological measurements The change of the Zeta-potential values as a function of mBSA/mHyA also shows similar trend The values continually decrease with increasing mBSA/mHyA (ζ = − 50.2 ± 1.2 mV (mBSA/mHyA = 2); ζ = − 37.4 ± 1.5 mV (mBSA/mHyA = 4)) Fig 4B represents the DLS curve of BSA/HyA NPs at mBSA/mHyA = 2.0, where the average diameter is the smallest The representative TEM image of this system also supports the formation of NPs with nearly 200 nm average diameter and a welldefined core-shell structure The negative surface charge may indicate the formation of core-shell structure, where the BSA is covered by 3.4.2 CD, FT-IR and thermal behaviour Detailed structural studies of the BSA/HyA conjugates at several mBSA/mHyA ratios have been also carried out by CD, FT-IR as well as TG/ DSC The main text contains the results of BSA/HyA NPs prepared at mBSA/mHyA = 2.0 ratio; other data are summarized in (Figs S6–S7) Fig 5A, B represents the CD curves registered for the pure BSA and BSA/ HyA conjugates In both cases the characteristic negative bands of the pure BSA are occurred at 208 and 220 nm (Zhou, Wu, Zhang, & Wang, 2017) In MilliQ water (Fig 5A), by the presence of HyA, only a slight shift is observed in the intensity of the negative band at 208 nm, which confirms that, there is no significant change in the secondary structure of BSA According to Eqs (1) and (2), the calculated α-helix content: 54.49 % for pure BSA and 57.76 % for BSA/HyA nanoconjugates This is in good correspondence with previous values (Zhou et al., 2017) At nearly A.N Kov´ acs et al Carbohydrate Polymers 251 (2021) 117047 Fig FT-IR spectra (A), DSC (B) and TG (C) curves of BSA, HyA and the lyophilized powder of BSA/HyA nanoconjugates neutral pH, both macromolecules have negative charge and thus the potential for electrostatic interactions is low In contrast, the α-helix content of the BSA in the BSA/HyA conjugates is drastically decreased in the presence of HyA at acidic conditions (Fig 5B) The proportion of the α-helix content in acidic conditions is: 41.52 % for pure BSA and 13.56 % for BSA/HyA nanoconjugates Parallel with the decreasing α-helix content the ratio of the β-sheets is increased to ca 75 % supported by the fitting of the measured CD curve by Reed model (Reed & Reed, 1997) At pH = 4.5 the macromolecules have well-defined opposite charge and most probably the serum protein chains are charge compensated with the approx 20-fold larger HyA and the protein chains are partially unfolded and arranged to form a core(BSA)-shell(HyA) structure sup ported by TEM, Zeta-potential and PCD studies (Fig S5) The FT-IR measurements clearly indicate that the presence of both HyA and the protein in the composite; the determinative bands of amide I and II as well as the vibrations of COO− and C-O(H) are presented in Fig 6A The data are in good agreement with previously published values for same macromolecules (Zhou et al., 2017) The FT-IR results did not confirm obviously the observations of CD studies, which can be explained by the fact that the FT-IR spectra were recorded in solid powder form, while the CD studies were measured in aqueous solution The degradation temperature (Tg) and the composition of the BSA/HyA conjugates were determined by TG and DSC (Fig 6B, C) Based on Fig 6B, it can be seen that the intensive exotherm peak of the HyA (230 ◦ C) and the endotherm peak of the BSA (222 ◦ C) does not appear in the conjugates which indicates the effective washing procedure and the composite does not contain macromolecules in free form By fitting of the DSC curves, the Tg values of the BSA and HyA are 202 ◦ C and 222 ◦ C, while for conjugates is 181 ◦ C The decrease is presumably due to the formation of electrostatic interaction between the macromolecules The Tg value was also determined by TG, where similar data can be obtained: 204 ◦ C (BSA), 224 ◦ C (HyA) and 185 ◦ C (BSA/HyA conjugates) Considering the weight changes and the shape of the curves, the com posite contains both BSA and HyA, but the BSA content is more domi nant and the presence of only physical mixture can be excluded (Figs S8, S9) 3.5 Characterisation of the drug-loaded BSA/HyA nanocarrier systems After the comprehensive study of the drug-free BSA/HyA NPs, the encapsulation of two model drugs is performed using mBSA/mHyA = 2.0 mass ratio The average size, the size distributions and the morphology Fig Size distribution curves of IBU- and 2-PA-loaded BSA/HyA colloidal particles by DLS with the representative TEM images of these particles A.N Kov´ acs et al Carbohydrate Polymers 251 (2021) 117047 Fig Dissolution profiles of the 2-PA (A) and IBU (B) molecules before (⸰) and after loading () at pH = 7.4 ± 0.1 (in phosphate buffer solution) at 37 ◦ C (the dotted lines represents the fitting of the primer data via Second-order (free molecules) and Weibull kinetic models (drug-loaded particles) • of the drug-loaded BSA/HyA conjugates are presented in Fig It can be stated that the capsulation was successful; the size of the drug-loaded NPs is greater than the size of the drug-free NPs (dDLS = 210 ± 56 nm) and spherical morphology is observed The results of DLS (dDLS, IBU-loaded = 250 ± 80 nm, ζ = -38.9 ± 1.4 mV; dDLS, 2-PA-loaded = 276 ± 74 nm; ζ = -42.0 ± 1.1 mV) and TEM (dTEM, IBU-loaded = 247 ± 92 nm; dTEM, 2-PA-loaded = 264 ± 80 nm) are in good agreement However, the TEM images not present core-shell structure but based on strongly supported structure of unloaded NPs and the preparation conditions, most probably the BSA-drug conjugates form the inner core and the outer shell contains dominantly HyA The measured Zetapotential values (presented above) also indicate this supposition The EE% and the drug loading also calculated by the Eqs (3)− (4) For IBUloaded BSA/HyA NPs the EE % is 40 % and the DL % is % In case of the 2-PA-loaded BSA/HyA NPs the EE % is 14 % and the DL% is % Although no previously published data on the encapsulation of 2-PA were found, but it can be clearly stated for IBU that the presence of ´ HyA slightly increases the drug content from DL% = 4–4.5 % (Csapo et al., 2016) to % compared the DL% of our previously published pure BSA-based systems After encapsulating the active substance, the dissolution profiles of the drugs are also investigated at pH = 7.4 (in phosphate buffer solution) at 37 ◦ C The registered curves are presented in Fig For the 2-PA-loaded BSA/HyA conjugates almost the 28 % of the encapsulated drug is released in the examined period (t = 240 min), while for IBU-loaded BSA/HyA conjugates ca 52 % of the encapsulated IBU is liberated The primer data points are fitted by different kinetic models (First-Order, Second-Order, Weibull, Korsmeyer–Peppas, Higu chi), but for IBU- and 2-PA-loaded particles the measured data fit well to the Weibull expression To compare the rate of dissolutions, the disso lution data of several IBU-containing nanocomposites synthesized in our lab were considered and the corresponding half-time (t1/2) data were compared We can compare the t1/2 values because similar technique was used for the registration of the dissolution profiles and same kinetic models were applied for fitting In case of mesoporous SiO2 (Varga et al., ´ et al., 2016), the change of t1/2 of the IBU is 2015) and pure BSA (Csapo not determinative in the presence of these carriers (t1/2(IBU) =0.11 h, t1/2 (SiO2/IBU) =0.08 h, t1/2(BSA/IBU) = 0.13 h) In case of our BSA/HyA complex NPs the following t1/2 values are calculated under the applied conditions: t1/2(IBU) =0.6 h, t1/2(BSA/HyA/IBU) = 2.2 h These data clearly confirm that the combination of HyA with BSA strongly facilitates the prolonged release of IBU at pH = 7.4 (in phosphate buffer solution), where nearly fourfold drug retention is achieved rheology, turbidity, DLS, TEM and CD proved that the optimized fabri cation as well purification protocols resulted in the formation of colloidal drug carriers with average diameter of ca 200− 210 nm via the partially charge compensation of these macromolecules The commonly used long-term heating as well as the application of crosslinking agents, surfactants and toxic organic solvents were eliminated It was confirmed that the pH as well as the applied BSA/HyA mass ratios strongly influ ence the size, the size distribution, and the proposed core-shell structure of the potential drug carrier particles This work clearly highlights the importance of detailed physico-chemical characterization of the pure carriers and drug-containing carriers to design effective nanosystem for encapsulation Our results may successfully contribute to the develop ment of promising drug delivery and controlled drug release colloidal systems in the future CRediT authorship contribution statement ´cs: Methodology, Investigation, Writing - orig Alexandra N Kova inal draft Norbert Varga: Methodology, Investigation, Visualization ´ a ´m Juha ´sz: Validation, Formal analysis, Visualization Edit Csapo ´: Ad Conceptualization, Resources, Writing - review & editing, Supervision Acknowledgements This research was supported by the National Research, Development and Innovation Office -NKDIH through GINOP-2.3.2-15-2016-0034, GINOP-2.3.2-15-2016-0060 and FK131446 The research is supported ´nos Bolyai Research Fellowship of the Hungarian Academy of by the Ja Sciences (E Csap´ o) The authors thank the registration of TEM images ´ria Hornok (University of 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Thermodynamic and kinetic characterization of pH-dependent interactions between bovine serum albumin and ibuprofen in 2D and 3D systems Colloids and Surfaces A: Physicochemical and Engineering... between BSA and the encapsulated drug in the presence of HyA by differential scanning calorimetry (DSC) and infrared spectroscopy studies, but the interaction between serum protein and HyA was... 3-hydrate (CH3COONa⋅3H2O; ≥99 %), and sodium hy droxide (NaOH; ≥96 %) pastilles and the hydrochloric acid (HCl, ≥99 %) were bought from Molar Chemicals Acetic acid (AcOH, ≥99 %) was ˝k´ purchased