The physico-chemical characterization of novel celecoxib-loaded beta-casein micelles (Cx/bCN) was recently described and its superiority in enhancing celecoxib bioavailability after intraduodenal administration to pigs was demonstrated. Here, using solution differential scanning calorimetry (DSC) combined with analysis of size distribution by DLS, zeta potential and changes in composition we demonstrate that the above superiority may be related to the thermotropic behavior of these micelles under physiological conditions. DSC of Cx/bCN reveals a characteristic irreversible exotherm upon heating, having its temperature of maximal change in heat capacity (Tm) at 40–46°C, depending on the Cx/bCN ratio. The higher the Cx/bCN ratio, the lower is Tm. While the thermodynamically stable bCN (alone) micelles lack any phase transition, the heat-induced, irreversible structural change of Cx/bCN micelles was associated with almost complete drug release, while micelle diameter and zeta potential decreased. Heating drug-free bCN micelles was effect-less. Altogether, our results suggest that Cx/bCN micelles are metastable supramolecular assemblies that transform upon heating to thermodynamically stable drug-free bCN micelles while releasing their drug-load. These findings also indicate the utility of DSC for future development of bCN micelles as nano-drugs, as well as of other supramolecular-assembly nano-drugs.
The physico-chemical characterization of novel celecoxib-loaded beta-casein micelles (Cx/bCN) was recently described (1) and its superiority in enhancing celecoxib bioavailability after intraduodenal administration to pigs was demonstrated (2). Here, using solution differential scanning calorimetry (DSC) combined with analysis of size distribution by DLS, zeta potential and changes in composition we demonstrate that the above superiority may be related to the thermotropic behavior of these micelles under physiological conditions. The thermotropic behavior of an assembly is studied best by high-sensitivity differential scanning calorimetry (DSC), as this technique enables one to examine if the assembly is thermodynamically stable or metastable. It also enables one to characterize and follow phase changes defined as phase transitions, and the energy and cooperativity involved in these phase transitions. DSC is widely used to investigate the phase behavior of pharmaceutical solids (3), as well as of various types of aqueous dispersions (4, 5). In drug development, DSC can be used for following phase changes for stability screening, purity determinations, drug-excipient compatibility studies and determining miscibility of amorphous mixtures (5, 6). As an example of celecoxib-relevant systems, Andrews (7) and Albers (8) and their co-workers used hot-melt extrusion for the production of glassy solid solutions containing celecoxib (Cx) and used DSC to confirm their formation. DSC is also a powerful tool to assess the thermotropic behavior of liposomes and proteins in aqueous solutions (4, 5, 9) and to provide thermodynamic parameters of biomolecules and nanomaterials (10). MicroCal™ VP-DSC (Malvern Instruments) is a high-sensitive, easy-to-use differential scanning calorimeter for the study of samples in solution, applied in studies of stability and unfolding of proteins, of lipid assemblies such as liposomes, or of nucleic acids, and in the study of liquid biopharmaceutical formulations (4, 5, 9, 11–15; http://www.microcal.com/products/dsc/vp-dsc.asp), for example, stability assessment studies of monoclonal antibody formulations in which the indicator for conformational stability is Tm (11, 14).
In a basic MicroCal VP-DSC experiment, energy is introduced simultaneously into a sample cell and a reference cell, and the temperatures of both cells are raised identically over time. The difference in the input energy required to match the temperature of the sample to that of the reference is the amount of excess heat absorbed or released by the macromolecules in the sample during an endothermic or exothermic process, respectively (http://www.microcal.com/products/dsc/vp-dsc.asp).
An exotherm upon heating is usually indicative of a thermodynamically metastable assembly. The direct measurement of heat changes during controlled increase or decrease in temperature provides an accurate determination of temperature of maximal change in the heat capacity, defined as Tm, as well as the enthalpy. Factors that stabilize the assembly shift the phase transition to a higher temperature range and higher Tm, while destabilizing factors lower them. The reversibility of a phase transition indicates whether the assembly is a stable equilibrium structure or a metastable “kinetically-trapped” structure. Calorimetric specific enthalpy (ΔHcal) is the total integrated heat capacity below the thermogram peak, indicating total energy change upon phase transition: energy uptake (positive values for endotherm) or release (negative values for exotherm) per mole of the assembly molecule. The thermodynamic parameters obtained from DSC experiments are sensitive to the structural state and the purity of the system under investigation. Any change in the physical state or increase of impurity due to degradation would affect the position, sharpness and shape of transition(s) of DSC scans.
In this study the thermal properties of beta-casein (bCN) micelles loaded with Cx were determined, focusing on the effect of drug loading, as part of research on the application of Cx/bCN to improve bioavailability of Cx via intraduodenal administration (2). The description of the physico-chemical features of Cx/bCN micellar assemblies are described elsewhere (1). Our studies also demonstrate that bCN micelles serve as a superior carrier for drug delivery of low-solubility drugs such as Cx (1). Additional studies, performed in pigs, showed improved bioavailability of Cx after intraduodenal administration of Cx/bCN (2). bCN is a major casein protein. The caseins are a group of unique open-structure, proline-rich, small phosphoproteins in mammalian milk. The MW of bCN is ~24,000 Da, and it is composed of 209 amino acids (bCN variant A2-5P). The N-terminal region of bCN is highly charged, while the remainder of the molecule is neutral and hydrophobic. Due to its amino acid composition and sequence, bCN is amphipathic and therefore in aqueous solution it self-assembles into nanometric micelles having a hydrophobic core and a hydrophilic corona (16). Cx, a selective COX-2 inhibitor NSAID, served as a model of a poorly water-soluble drug loaded into the bCN micelles.
This is the first report of the application of solution DSC to study the thermal properties of Cx/bCN micelles in a liquid formulation. DSC has been used in the study of a bCN micellization model, which showed that the micellization transition originated at temperatures very close to 0°C and that the process was dependent on bCN concentration and solvent composition (17). Reports on the thermodynamic analyses of drug-loaded micelles using DSC often involve drug-loaded micelles in their dry solid state after lyophilization, for example, the paclitaxel-loaded micelles prepared using a micelle-forming chitosan derivative, N-octyl-O-sulfate chitosan. The DSC analysis of the lyophilized drug-loaded micelles suggested a solid dispersion of amorphous paclitaxel in the polymer (18). Our study is unique as it focuses on the analysis of the thermotropic behavior of an aqueous dispersion of a new drug-loaded micellar system (Cx/bCN). This is a direct stability measurement of the native state of the drug-loaded micelles, thus avoiding a possible effect of lyophilization on the stability of the drug-loaded micellar formulation. It is also relevant to drug release from the nano-carrier in vivo.
Bovine β-casein (Sigma-Aldrich catalog no. C6905), Hepes free acid (MP Biomedicals), MgCl2 (Sigma-Aldrich), EGTA (Sigma-Aldrich), NaCl (Frutarom), celecoxib (Teva), acetonitrile (Bio-Lab), methanol (Bio-Lab), glacial acetic acid (Frutarom), absolute ethanol (Bio-Lab), NaOH (Merck), HCl (Gadot), Coomassie Brilliant Blue G-250 (Aldrich), ortho-phosphoric acid (Fluka), Dowex1x8-200 anion exchange resin (chloride form, 100–200 mesh, Sigma-Aldrich catalog no. 217425). The Dowex resin was pre-washed with 1 M HCl while stirring for 30 min, extensively washed with water to reach pH 6 of the wash solution, followed by overnight desiccation. Highly pure water of conductivity 18.2 MΩ was prepared using the Barnstead™ Nanopure™ ultra-pure water purification system (Thermo Fisher Scientific Inc., Waltham, MA, USA).
For the DSC experiments, bCN micelle dispersions were prepared and used for drug loading to obtain Cx/bCN formulations of increasing encapsulated Cx:bCN mole ratios. Dispersions were examined visually and analyzed for Cx and protein content and percent encapsulation. Micelle diameter and zeta potential were also measured. The bCN micellar dispersions without the drug and the drug-loaded Cx/bCN micellar formulations were scanned using DSC, and additional composition analyses were performed on the Cx/bCN samples after the DSC scans. Micelle diameter and zeta potential were measured again after heating all samples to 65°C. Additionally, 1% bCN micellar dispersions with and without drug (encapsulated Cx:bCN mole ratio of ~6) were prepared and characterized after heating experiments at 37°C.
Bovine bCN was dissolved in 20 mM Hepes buffer (pH 6.8) containing 1 mM MgCl2, 2 mM EGTA and 10 mM NaCl, and was stirred overnight at 4°C. Two dispersions of ~1% (10 mg/mL, 0.4 mM) bCN were prepared (samples 028 and 029). This concentration is above the bCN critical micellar concentration (CMC) of 0.5–2 mg/mL (0.021–0.083 mM) (http://www.microcal.com/products/dsc/vp-dsc.asp). The two protein dispersions were filtered through a 0.45-μm polyvinylidene fluoride (PVDF) membrane filter to remove any residual large protein aggregates that may exist, and each was divided into two aliquots for drug loading of four Cx/bCN formulations (A, B, C and D) of increasing Cx concentrations. Cx (MW 381) was dissolved in absolute ethanol at concentrations of 6, 12, 24 and 30 mg/mL. An appropriate volume from each Cx solution was added dropwise to each of the four protein dispersions while stirring, keeping the ethanol concentration in the final dispersions at 5% v/v, followed by additional stirring for 30 min at room temperature. Additional bCN micellar dispersions of 1% with and without Cx (in encapsulated Cx:bCN mole ratio of ~6) were prepared for heating experiments at 37°C using the same procedure with a Cx solution of 24 mg/mL.
HPLC method for Cx quantification and % encapsulation
Quantification of Cx was done by HPLC (Hewlett Packard 1050, equipped with UV/Vis detector), using LiChroCART 100 RP-18 column (250 mm×4 mm, 5-μm particle size, Merck) and a guard column LiChroCART 4-4, flow rate of 0.5 mL/min, and UV detection at 254 nm at ambient temperature. Injection volume was 50 μL. The mobile phase consisted of acetonitrile: methanol: water, 45:45:10 (v/v/v), containing 0.5% glacial acetic acid adjusted to pH 3.5 with NaOH (19). Separation between encapsulated and non-encapsulated Cx and determination of total and non-encapsulated Cx were performed as follows: (A) For total (encapsulated plus non-encapsulated) Cx determination, a protein precipitation procedure was carried out. One hundred and forty micro-liters of cold acetonitrile was added to 120 μL of Cx/bCN micelle sample in triplicate, mixed vigorously by vortex for 1 min and immersed for 20 min in crushed ice. (B) In order to determine non-encapsulated (free) and encapsulated Cx, the free Cx was separated from the encapsulated Cx using a strong anionic exchange resin (Dowex1x8-200), which binds the negatively charged bCN, with or without Cx, while the charge-less free drug does not bind to the anionic exchange resin. To 50 mg of Dowex, 100 μL of Cx/bCN micelle sample was added in triplicate, and the mixture was mixed vigorously by vortex for 1 min. Both total (A) and Dowex-treated (B) samples were centrifuged for 10 min at 3000 g at 4°C. Supernatants were collected, diluted with mobile phase, and 300 μL was transferred into the HPLC vials for analysis. The Cx calibration standards were prepared in the range of 0.3–10.0 μg/mL. The Cx calibration curve was constructed by plotting area under the peak (y) of Cx vs. Cx concentration (x). Good linearity was achieved over the range tested, as shown in Figure 1.
Encapsulated Cx concentration was calculated by subtracting the free, non-encapsulated, drug concentration obtained after the Dowex treatment from the total drug concentration. A typical HPLC chromatogram of Cx is shown in Figure 2.
Bradford protein assay for bCN quantification
Quantification of bCN was done using Coomassie Blue dye protein binding assay, a spectrophotometric protein quantitation method originally described by Bradford (20). The samples were loaded onto a microplate, and the absorbance at 595 nm was determined using Synergy 4 HT, Multi-Mode Microplate Reader (BioTek). The calibration curve for bCN in Hepes buffer was linear in the range of 0–60 μg/mL of bCN.
Dynamic light scattering (DLS) and zeta potential
The size distribution (by intensity-averaged diameter) and zeta potential of bCN micelles and drug-loaded bCN micelles were determined using a combined DLS and zeta potential analyzer (Zetasizer Nano-ZS, Malvern Instruments) at 25°C. For DLS, a 633-nm He-Ne laser beam and a detector oriented at 173° (back-scattering detection) were used. Measurements were carried out before and after heating the micellar dispersions at 65°C for 5 min. After heating, the micellar dispersions were centrifuged for 2 min at 14,000 rpm before measurements were made. Software provided by the manufacturer was used to calculate the zeta potential and DLS intensity-averaged diameter of triplicate samples (n=3).
Calorimetric measurements were carried out using MicroCal VP-DSC (Malvern Instruments, MA, USA), comparing empty (drug-free) bCN micelles and Cx-loaded bCN micelles, with the same Hepes buffer in the reference cell. In a preliminary study, the Cx/bCN micelles were scanned in the range 10–90°C, which revealed a phase transition with a Tm in the range 40–50°C. The subsequent scans were performed in the range 25–65°C. Each sample was scanned three times: heating from 25 to 65°C (scan 1), cooling from 65 to 25°C (scan 2) and reheating from 25 to 65°C (scan 3). The scanning rate for the heating and cooling modes was 1.0°C per min. Processing the calorimetric data was done using Origin 7.0 software.
Results and discussion
The four Cx/bCN preparations (A, B, C and D) were analyzed for bCN concentration, Cx concentration, percent encapsulation, particle size and zeta potential. bCN concentrations ranged from 7.9 to 10.7 mg/mL (0.33–0.45 mM, respectively); these values were used for normalization of the Cp data in the DSC. The formulations contained increasing amounts of Cx (0.3–1.2 mg/mL), with 96%–99% encapsulation, in order to study the effect of amount of drug loaded per mole bCN on the thermodynamic properties of Cx/bCN. The Cx/bCN analytical results are summarized in Table 1.
|Cx/bCN preparation||Encapsulated Cx||% encaps.||bCN||Encapsulated Cx:bCN (mole ratio)|
Thermograms of the two bCN preparations alone, without the drug (028 and 029), show no peak in any of the DSC scans (Figure 3), while for the four Cx/bCN preparations, the first heating scans showed a characteristic exothermic peak (Tm) between 40 and 46°C (Figure 4).
DSC scan results (related to the exotherm of the first heating scans) are summarized in Table 2.
|Cx/bCN preparation||Encapsulated Cx:bCN mole ratio||Range of phase transition, °C||ΔT1/2, °C||Tm, °C||Exotherm ΔH, kcal/mole|
aMain peak; ΔT1/2 is the width in °C of the phase transition at its half height; ΔH (kcal/mole) is related to mole of bCN.
Thermograms A and C (first heating scans), obtained for the preparations with higher Cx-loadings, show that the transition already starts below 40°C. The characteristic peak was a single peak for three preparations (A, B and D), while for C, a double peak was observed, with a smaller peak appearing on the shoulder of a main peak. It is assumed that the smaller peak represents a sub-population of Cx/bCN micelles within the total micelle dispersion. Therefore the data obtained for this batch were re-analyzed using the software curve-fitting module of Origin 7, which allowed integration of the main peak area following separation of the overlapping peaks, as presented in Figure 5.
The characteristic peak was absent in the second (cooling) or third (reheating) scans (Figure 6), indicating that the phase transition observed during the first heating scan is irreversible. The exotherms of all four Cx/bCN preparations at their first heating scan are shown in Figure 7.
For the four Cx/bCN preparations, the exothermic peak area ranged from –7426 to –37,964 kcal/mole, with the enthalpy magnitude increasing with higher encapsulated Cx:bCN mole ratio. As the enthalpies were collected at different Tm’s and ΔCp was not accounted for, the direct comparison of enthalpy is only to be seen as indicative and perhaps a subject for future investigation. The large enthalpy variation may be indicative of larger Cx/bCN micelles being formed with an increase of Cx loading.
The shift of the exothermic peak to lower temperatures in the Cx/bCN preparations with the higher encapsulated Cx:bCN mole ratio, as shown in Figure 8, indicates lower stability of the bCN micelles with increase in the Cx ratio in Cx/bCN.
The initially transparent Cx/bCN samples become turbid after being aged at 37°C or higher temperature (as shown in Figure 9, right) and visual observations of Cx/bCN samples after DSC scans indicate that the heat-induced, irreversible rearrangement of the bCN micelles was associated with drug release.
HPLC analysis was performed on the DSC-scanned Cx/bCN samples to determine the percent of Cx released after the DSC scans. Results (Table 3) show that after the DSC scans, 92%–99% of the drug was released from the micelles. At higher Cx concentrations (0.7 and 1.2 mg/mL), almost all (99%) of the drug was released after the DSC scans, while at lower Cx concentrations (~0.3 mg/mL), a small amount of drug (7%–8%) remained with the bCN micelles after the DSC scans.
|Cx/bCN formulation||Cx mg/mL||% Cx releaseda|
|Before DSC||After DSC|
aCalculated as (Cx conc.- Cx conc. after DSC/Cx conc.)×100.
The exothermic nature of the transition reflects energy release associated with drug release, which shows that the energetically-favored state is the Cx-free bCN micelles. It is possible that the loading of the drug into the core of the bCN micelle leads to weakening of the attractive forces between bCN molecules, thus destabilizing the bCN micelle. Perhaps the hydrophobic interactions that drive the association of bCN monomers are decreased in the presence of the drug, whose rigid structure may impair the ability of the bCN hydrophobic regions to tightly pack.
DLS and zeta potential measurements were conducted before and after drug loading and before and after drug release by heating to 65°C in order to evaluate the effect of heating on these parameters. The results, summarized in Tables 4 and 5, show that, as in our previous report (1), empty bCN micelle diameter was about 21 nm, and after Cx-loading the diameter increased to 26–32 nm in a Cx:bCN mole-ratio-dependent manner. The zeta potential slightly increased with higher drug loading. The data show that after heating to 65°C, zeta potential and size of the empty bCN micelles remained unchanged, demonstrating thermal stability of the empty micelles. However, upon heating the drug-loaded bCN micelles, their diameter decreased; this was more pronounced for the moderate and high Cx loading, where almost all the drug was released (Table 3). The heating resulted in a decrease in zeta potential in three out of four Cx/bCN preparations, being more pronounced at the higher encapsulated Cx:bCN mole ratios. These observations show that the heat-induced, irreversible rearrangement of bCN micelles associated with drug release did not have a major effect on these basic properties of the bCN micelles.
|Preparation||Diameter mean±SD, nm|
|Before heating||After heating|
|Before Cx loading||028||21±0.5||22±0.4|
|After Cx loading||D (low Cx)||26±0.5||25±0.8|
|B (low Cx)||26±0.6||24±0.7|
|A (moderate Cx)||31±1.3||26±1.8|
|C (high Cx)||32±1.0||26±0.9|
|Preparation||Zeta potential mean±SD, mV|
|Before heating||After heating|
|Before Cx loading||028||–20±3.0||–20±1.4|
|After Cx loading||D (low Cx)||–24±1.8||–21±1.0|
|B (low Cx)||–22±0.6||–23±1.8|
|A (moderate Cx)||–24±1.4||–22±1.9|
|C (high Cx)||–29±1.2||–25±0.7|
The Cx/bCN system was thermodynamically characterized by solution DSC, and shown to have an exothermic phase transition (upon heating), with Tm in the range of 40–46°C. No peak was present for the protein (alone) micelles, indicating that the exotherm is associated with the presence of drug in the assembly. The heat-induced, irreversible rearrangement of the bCN micelles was associated with drug release, as supported by HPLC and by the DLS showing decreased particle diameter after heating to 65°C. The exotherm and the irreversibility upon heating indicate that the drug-loaded bCN micelles are metastable and transform to stable bCN micelles upon heat-induced drug release. The shift of the exothermic peak to lower temperatures with increasing encapsulated Cx:bCN mole ratios indicates lower stability of the bCN micelles with increase in the Cx ratio in Cx/bCN. This study suggests that the Cx/bCN micelles are metastable assemblies in an intermediate energy state (21). These results help to understand the superiority of Cx/bCN micelles in enhancing celecoxib bioavailability over Celebra, the commercial Cx product (2) and supports the use of DSC especially in future development of bCN micelles as a drug carrier, and as a general tool in understanding the composition-structure-function relationships of supramolecular-assembly nano-drugs.
This study was supported in part by the Barenholz Fund. The authors would like to thank Dr. Simcha Even-Chen, formerly from the Laboratory of Membrane and Liposome Research, for her help in this research and Mr. Sigmund Geller for editing the manuscript.
Conflict of interest statement
Competing financial interests: Yechezkel Barenholz and Dganit Danino are holders of the following recently allowed patents:
Beta-casein assemblies for mucosal delivery of therapeutic bioactive agents
Y Barenholz, D Danino
US Patent 8,871,276
Beta-casein assemblies for mucosal delivery of therapeutic bioactive agents
Y Barenholz, D Danino
US Patent 8,865,223
Beta-casein assemblies for enrichment of food and beverages and methods of preparation thereof
D Danino, YD Livney, O Ramon, I Portnoy, U Cogan
US Patent 8,865,222
Funding: The Hebrew University received royalties from licensing of Doxil patents of Yechezkel Barenholz until patents expired in March 2010. Part of these royalties established the Barenholz Fund that was used to support the research in the Barenholz Lab including this study.
Besides the above-mentioned patents, the authors state no conflict of interest. All authors have read the journal’s Publication ethics and publication malpractice statement available at the journal’s website and hereby confirm that they comply with all parts applicable to the present scientific work.
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