Polyelectrolyte pH-Responsive Protein-Containing Nanoparticles: The

Article pubs.acs.org/Langmuir

Polyelectrolyte pH-Responsive Protein-Containing Nanoparticles: The Physicochemical Supramolecular Approach Anna Riabtseva, Leonid I. Kaberov, Jan Kučka, Anna Bogomolova, Petr Stepanek, Sergey K. Filippov,* and Martin Hruby* Institute of Macromolecular Chemistry, AS CR, Heyrovsky Sq. 2, Prague, Prague 6 162 06, Czech Republic S Supporting Information *

ABSTRACT: We report on the physicochemical properties and self-assembly behavior of novel efficient pH-sensitive nanocontainers based on the Food and Drug Administration-approved anionic polymer Eudragit L100-55 (poly(methacrylic acid-coethyl acrylate) 1:1) and nonionic surfactant Brij98. The features of the interaction between Eudragit L100-55 and Brij98 at different pH values and their optimal ratio for nanoparticle formation were studied using isothermal titration calorimetry. The influence of the polymer-to-surfactant ratio on the size and structure of particles was studied at different pH values using dynamic light scattering and small-angle X-ray scattering methods. It was shown that stable nanoparticles are formed at acidic pH at polymer-tosurfactant molar ratios from 1:43 to 1:139. Trypsin was successfully encapsulated into Eudragit−Brij98 nanoparticles as a model bioactive component. The loading efficiency was determined by labeling trypsin with radioactive iodine-125. Eudragit−Brij98 nanoparticles effectively protected trypsin against pepsin digestion. The results showed that trypsin encapsulated into novel pH-sensitive nanocontainers retained more than 50% of its activity after treatment with pepsin compared with nonencapsulated trypsin. The described concept will contribute both to understanding the principles of and designing next-generation nanocontainers.

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INTRODUCTION Oral drug administration is the most patient-friendly approach, and therefore development of novel dosage forms for the oral delivery of drugs and enzymes occupies one of the leading positions in chemistry, pharmacy, and medicine. It is easy and painless and does not violate the integrity of body tissues.1,2 Specific delivery of drugs to the selected location or segment of the gastrointestinal tract (GI) is advisable for therapies of a wide variety of different diseases. Targeted drug delivery to specific segments of the GI tract provides the ability for local treatment reducing the side effects, greatly increases the effectiveness of drugs, and makes it possible to reduce the effective dose. Moreover, targeted delivery to separate parts of the GI tract may be advantageous in the case when the absorption of the drug into the bloodstream is limited to part of the GI tract. The main obstacles to effective oral delivery of many protein drugs are their low stability at acidic pH in stomach, poor permeability across intestinal mucosa, and proteolytic degradation, which limit the activity and duration of therapeutic effects.3,4 For oral delivery of some enzymebased drugs, for example, trypsin, it is necessary to protect them in the acidic media of stomach and from the action of pepsin for the subsequent release in the small intestine. pH-dependent delivery systems allow delivery and quick release of drugs under the influence of the pH of the intestinal fluids.5−7 The most useful substances for this purpose are nano- and microcontainers based on acrylic and methacrylic acid-based pH-sensitive polymers and hydrogels.8−13 Enteric-soluble pH© 2017 American Chemical Society

sensitive polymers are primarily weak acids containing acidic functional groups which are capable of ionization at high pH values. Eudragits have been known to be nonbiodegradable, nonabsorbable, nontoxic synthetic soluble pH-sensitive polymethacrylate-based copolymers and are approved by the Food and Drug Administration.14−16 Eudragits L and S usually are used as coating polymers for the delivery of various drugs, their protection from gastric fluids, and increase in drug effectiveness. In the present work, we propose to use Eudragit L100-55 (poly(methacylic acid-co-ethyl acrylate) copolymer 1:1) for the preparation of nanoparticles for oral drug delivery. The presence of carboxylic groups in the structure of Eudragit L100-55 provides solubility at basic and neutral pH in the region of the GI tract, but the polymer precipitates at acidic pH. This is the mechanism by which such nanoparticles protect trypsin from degradation by gastric juice (Figure 1). To ensure stability of Eudragit L100-55 nanoparticles, we propose to use the surfactant Brij98. It was previously shown that addition of Brij98 prevents phase separation and promotes rearrangement of pH-responsive polymer molecules.17−19 Nanoparticles made of anionic Eudragit (L100, L100-55, and S100) and different surfactants such as anionic surfactants (bile salts, sodium cholate, sodium lauryl sulfate, and phosphatidylcholine) and cationic surfactants (dodecyl ammonium bromide and Received: October 19, 2016 Revised: December 21, 2016 Published: January 3, 2017 764

DOI: 10.1021/acs.langmuir.6b03778 Langmuir 2017, 33, 764−772

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Czech Republic). All other chemicals were purchased from SigmaAldrich Ltd. (Prague, Czech Republic). Dynamic Light Scattering. Dynamic light scattering (DLS) measurements were recorded using an ALV/CGS instrument equipped with a 22 mW He−Ne laser in the angular range from 30 to 150° and with an ALV 6010 multibit, multi-τ autocorrelator covering approximately 12 decades in the delay time τ. Normalized autocorrelation functions g2(t) were analyzed using the Gendist software, which employs the REPES algorithm23 to perform inverse Laplace transformation 2 ⎡ ⎛ t⎞ ⎤ g2(t ) − 1 = β ⎢ G(τ ) exp⎜ − ⎟dτ ⎥ ⎝ τ⎠ ⎦ ⎣

∫

where t is the delay time of the correlation function and β is an instrumental parameter known as contrast. The resultant G(τ) is a distribution of relaxation times τ that generally consists of several peaks representing individual dynamic processes. Finally, the apparent hydrodynamic radius Rh is derived from the relaxation times obtained using the well-known Stokes−Einstein relation. The DLS data are taken from intensity-weighted distribution functions.8 To account for the logarithmic scale on the Rh axis, all DLS distribution diagrams are displayed in the equal area representation, RhG(Rh).24 The value of Rh for each system was averaged over three runs. The pH dependence of the hydrodynamic radius of particles, Rh, and the scattering intensity, Is, were measured at a scattering angle of θ = 173° using a Zetasizer Nano ZS instrument, model ZEN3600 (Malvern Instruments, UK). The DTS (Nano) program was used to evaluate the data. It provides intensity-, volume-, and numberweighted Rh distribution functions G(Rh). The number-weighted value of the apparent Rh was chosen to monitor the pH-dependent changes in the system. The DTS (Nano) software also provides polydispersity index (PDI) derived from cumulant analysis (PDI = Γ2/Γ12). Here, Γ2 and Γ1 are the second and first cumulants.25 Three measurements were recorded after each pH change. Standard deviation for the Rh value was calculated using three measurements and was less than 5% for all samples. All measurements were recorded with a polymer concentration of 2.1 mg/mL in water; the concentration of surfactant Brij98 was changed depending on the desired ratio. All solutions were filtered through a 0.45 μm polyvinyl difluoride syringe filter at pH = 11 before measurement. Zeta Potential Measurements. Electrophoretic mobility was measured using the same Zetasizer Nano ZS instrument used for DLS measurements. The DTS software was used to compute the zeta potential using the Henry equation (eq 1). The electrophoretic mobility of a spherical particle is related to the zeta potential by

Figure 1. Scheme of drug release from pH-sensitive nanocontainers in the digestive system.

cetyltrymethylammonium chloride), Brij35, Brij78, Pluronic 28, and others were studied by Pereira et al.20 It was shown that the addition of nonionic surfactants leads to a significant increase in the drug incorporation efficiency, reaching a maximum of 43%. Dai and co-workers reported21 the synthesis of oral cyclosporine A delivery systems with the pH-sensitive property based on Eudragit E100, Eudragit L100, Eudragit L100-55, and Eudragit S100. The drug-loading efficiency was very high, ranging from 90.9 to 99.9%. Trypsin is a model drug, a digestive enzyme, normally present in the intestine, and its activity is easy to determine so one can easily follow the protection of cargo against gastric juice. There are only a few publications concerning the immobilization of trypsin on polymers. For example, polyanhydride microspheres based on poly(fatty acid dimer), poly(sebacic acid), and their copolymers [P(FAD−SA)] were described.22 P(FAD−SA) spherical microspheres, with diameters of 50−125 μm, containing proteins of different molecular sizeslysozyme, trypsin, heparinase, ovalbumin, albumin, and immunoglobulinwere prepared. It was shown by enzymatic activity studies that encapsulation of enzymes inside of polyanhydride microspheres can protect them from activity loss. However, to the best of our knowledge, systems based on polyelectrolytes and surfactants for enzyme delivery have not been described before. Thus, the aim of this work was to investigate pH-sensitive nanocontainers based on the polyelectrolyte−colloidal complex of Eudragit L100-55 and nonionic surfactant Brij98, by studying their structure and features of formation as well as prospects of their application for the delivery of drugs. In particular, here we present the results of trypsin loading into Eudragit/Brij98 nanocontainers.

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(1)

UE =

ξ 2εrf (ka) 3η

(2)

where εr is the dielectric constant of the sample, η is the dynamic viscosity (Pa s), and ξ is the zeta potential (V); f(ka) is the Henry’s function, which is calculated within the approximation of Smoluchowski ( f(ka) = 1.5) for aqueous solutions with moderate ionic strength. Each mobility value presented in the text is an average of 15−100 values. Isothermal Titration Calorimetry. The microcalorimetry study was performed using a MicroCal 200 isothermal titration calorimeter. The experiment was performed with consecutive injections of the concentrated surfactant solution into the calorimeter cell; the cell contained 280 μL of the polymer solution (2.1 mg/mL) or water. A surfactant solution was added to a 40 μL injection syringe, the tip of which was modified to act as a stirrer. The chosen stirring speed was 1000 rpm. The injection volume was 2 μL. The time between injections was usually 200 s. The measurements were recorded at 30 °C. The data were analyzed using Microcal Origin software. Experimental enthalpy was obtained by integrating the raw data signal, and the integrated molar enthalpy change per injection was obtained by dividing the experimentally measured enthalpy by the number of moles of the surfactant added. The final enthalpograms are

MATERIALS AND METHODS

Materials. Trypsin from bovine pancreas, Nα-p-tosyl-L-arginine methyl ester hydrochloride (TAME), pepsin from porcine gastric mucosa, Tris base, and Brij98 were purchased from Sigma-Aldrich. Eudragit L100-55 (poly(methacrylic acid-co-ethyl acrylate) 1:1) was obtained from Evonik Industries AG (Germany) and was used as free acid. Hydrochloric acid and calcium chloride were received from LachNer (Czech Republic). Na125I was received from Lacomed Ltd. (Ř ež, 765

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Figure 2. ITC curves for Brij98 titration of polymer Eudragit L100-55 at different pH values (a); and dependence of saturation molar ratio of Brij98 to Eudragit L100-55 on the pH value (b). solution was adjusted to 7, 28 μg of trypsin was added to the solution and stirred for 10 min, and then the pH was adjusted to 3.0−5.0. Radiolabeling of Trypsin with 125I. For the labeling, trypsin (1.1 mg) was dissolved in phosphate-buffered saline (PBS) (200 μL), chloramine T solution (5 mg/mL in PBS, 5 μL) and [125I]−NaI solution (5 μL, 15.6 MBq) were added, and the mixture was incubated at room temperature for 15 min. After the addition of an ascorbic acid solution (10 μL, 26 μg/mL in PBS) and an additional 10 min of incubation at room temperature, the mixture was separated onto a preparative Sephadex PD-10 SEC column using distilled water as the mobile phase. The amount of radioactivity that was bound to the trypsin was calculated as the radioactivity of the trypsin fractions divided by the initial radioactivity applied to the column. The radiochemical yield was 37%. Determination of Trypsin Loading (Labeling of Trypsin). Eudragit/Brij nanoparticles were prepared as described previously, with different amounts of trypsin mixed with a specified volume of 125Ilabeled trypsin. The obtained solution was centrifuged using Amicon ultra centrifugal filters with 100 000 NMWL at 6000 × g for 5 min, which were prewashed with 400 μL of 10 μM trypsin in 1 mM HCl. This procedure was repeated at least three times by washing the tubes with 1 mM HCl until all solvent with unbound trypsin was filtered off. The amount of loaded trypsin was calculated by measuring the radioactivity of sediments in the filtration tube. The loading efficiency (LE) was calculated using the following equation

the plots of the integrated molar enthalpy as a function of the total surfactant concentration in the calorimeter sample cell. Cryogenic Transmission Electron Microscopy. Cryogenic transmission electron microscopy (cryo-TEM) measurements were recorded using a Tecnai G2Sphera 20 electron microscope (FEI Company, Hillsboro, OR, USA) equipped with a Gatan 626 cryospecimen holder (Gatan, Pleasanton, CA, USA), and a LaB6 gun. The samples for cryo-TEM were prepared by plunge-freezing.26 Briefly, 3 μL of the sample solution was applied to a copper electron microscopy grid covered with a perforated carbon film forming wovenmesh-like openings of different sizes and shapes (lacey carbon grids #LC-200 Cu, Electron Microscopy Sciences, Hatfield, PA, USA), glowdischarged for 40 s with a current of 5 mA. Most of the sample was removed by blotting (Whatman no. 1 filter paper) for approximately 1 s, and the grid was immediately plunged into liquid ethane held at −183 °C. The grid was then transferred into the microscope without rewarming. Images were recorded at the accelerating voltage of 120 kV, with magnifications ranging from 11 500× to 50 000× using a Gatan UltraScan 1000 slow scan CCD camera in the low-dose imaging mode, with the electron dose not exceeding 1500 electrons per nm2. The magnifications resulted in final pixel sizes ranging from 1 to 0.2 nm, and the typical value of the applied underfocus ranged between 0.5 and 2.5 μm. The applied blotting conditions resulted in the specimen thicknesses varying between 100 and approximately 300 nm. All cryo-TEM images were carefully inspected for possible artifacts such as radiation damage and ice crystals. Small-Angle X-ray Scattering. Synchrotron small-angle X-ray scattering (SAXS) experiments were performed at the beamline B21 (Diamond Light Source, Didcot, UK) using a pixel detector (2M PILATUS). The X-ray scattering images were recorded for a sampleto-detector distance of 3.9 m, using a monochromatic incident X-ray beam (λ = 1 Å) covering the range of momentum transfer 0.0025 Å−1 < q < 0.4 Å−1 (q = 4π sin θ/λ, where 2θ is the scattering angle). Most of the samples had no measurable radiation damage, detected by the comparison of 20 successive time frames with 50 ms exposures. In all cases reported in this paper, the two-dimensional scattering patterns were isotropic. They were azimuthally averaged to yield the dependence of the scattered intensity Is(q) on the momentum transfer q. Before fitting analysis, the solvent scattering had been subtracted. For further modeling, the data were brought to an absolute scale. Encapsulation of Trypsin. Encapsulation of trypsin was performed as follows: 2.1 mg of Eudragit L100-55 and 0.7 mg of Brij98 were dissolved in 1 mL of purified water, the pH was adjusted to 11.0, and the solution was stirred for 1 h. The pH of the obtained

LE(%) = m(trypsin in nanoparticles)/m(trypsin loaded) × 100% (3) Determination of Trypsin Activity. To confirm the encapsulation of trypsin inside of the Eudragit/Brij nanoparticles, an excess of pepsin (1.5 mg) was added to the solution of Eudragit/Brij containing trypsin and the solution was shaken for 1 h. Then, the pH was adjusted to 11 for pepsin deactivation. Trypsin activity (a) was measured using a method adapted from the study of Hummel.27 Into quartz cuvettes were added 2.6 mL of 46 mM Tris/HCl buffer (pH = 8.1) containing 11.5 mM CaCl2 and 0.3 mL of 10 mM TAME in purified water and mixed by inversion, and the resultant mixture was incubated at 25 °C for 5 min to achieve temperature equilibration. Then, 100 μL of 1 mM HCl (for blank assay) or 100 μL of trypsin containing solution was added. The absorbance at 247 nm was measured as a function of time using an Evolution 220 spectrometer (Thermo Scientific, USA) during 10−30 min until leveling-off. The slopes ΔA247 from the linear portion of the curves were determined for blank and test assays, and the activity of trypsin (a) was calculated using the equation 766

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Figure 3. Dependence of hydrodynamic radii of nanoparticles at different molar ratios of Eudragit L100-55/Brij98 on pH (a); and dependence of the PDI of nanoparticles of Eudragit L100-55/Brij98 on pH at the Eudragit to Brij98 ratio of 1:93 (b).

Figure 4. Dependence of hydrodynamic radius and zeta potential of particles in Eudragit/Brij98 system at different pH values, with a Eudragit/Brij98 ratio of 1:93 (a); and hydrodynamic size distributions for samples of Eudragit/Brij98 ratio 1:93 at pH = 9 (black) and 3 (red) (b).

a=

(ΔA 247 test − ΔA 247 blank) × 1000 × 3 540 × X

can be described in a few stages. At the first stage, a decrease in the enthalpy change can be explained by the dissociation of aggregates of anionic polymers because of their interaction with Brij98 molecules, whereas the second process describes the complex formation between a single Eudragit chain and Brij98. The enthalpy change passes through a minimum and starts increasing with increasing Brij98 concentration in the system. Such a conclusion is based on our previous ITC studies on the interaction of thermosensitive polyoxazoline-based copolymers with surfactants.28 We can conclude that the strong interaction of Eudragit polymer and Brij98 at pH values lower than 6.5 leads to the formation of Eudragit/Brij98 complexes. It should be noted here that above a certain value of the concentration of Brij98 in solution, the enthalpy change reaches a plateau that corresponds to Brij98 to water titration. Such a phenomenon is generally observed for polymer/surfactant systems and called critical saturation concentration or c2.28−31 Above that point, no polymer−surfactant interactions exist in solution; only the effect of surfactant micelle titration with water is detectable. From the obtained ITC curves, (Figure 2a) we can calculate the c2 value of the amount of Brij98 that is needed to cover all interaction that centers on Eudragit molecules. It can be seen from Figure 2b that a decrease in the pH value leads to a significant increase in the Brij98 amount that binds with Eudragit polymer. The highest ratio between these two components is calculated for pH = 5.75 and is equal to 43:1 mol (Brij98:Eudragit L100-55). It is obvious that if a significant amount of Brij98 is present in the solution, it can protect Eudragit single chains from aggregation. Nevertheless, a reasonable compromise between unshielded hydrophobic acrylic monomers and Brij98

(4)

where a is the trypsin activity (units/mg), A247 is the slope of the initial linear portion of the Δ curve for the test (with enzymes) and blank (unit absorbance/min), 540 represents the molar extinction coefficient [L/(mol × cm)] of TAME at 247 nm, 3 represents the volume (in milliliters) of the reaction mixture, and X represents the quantity of trypsin in the final reaction mixture (mg).

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RESULTS AND DISCUSSION The presence of nonionic surfactant Brij98 in the solution prevents the sedimentation of Eudragit nanoparticles at acidic pH owing to the interaction of hydrophobic moieties of the polymer and Brij98. To characterize this interaction in details and to define the features of complex formation as well as optimal ratio between Eudragit L100-55 and Brij98, isothermal titration calorimetry (ITC) experiments were conducted. Eudragit L100-55 solution was titrated using a solution of nonionic surfactants at different pH values. We could not perform ITC experiments at pH lower than 5.6 because of the sedimentation of Eudragit. Titration of Brij98 into water was performed for comparison. One can see from Figures 2 and S1 that the interaction between the polymer and the surfactant results in the appearance of exothermic peaks. Analysis of data obtained for the titration of the Eudragit L100-55 molecules by Brij98 shows that at basic pH there is a very weak interaction between these two components. At pH from 10 to 7, enthalpy changes almost do not differ from the value of this parameter for Brij98 titration into water. On the contrary, in the case of pH = 6, one can see strong interaction; this process is nonmonotonous and 767

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Langmuir surfactant is needed to form stable nanoparticles. If there is not enough Brij98 molecules in solution, Eudragit will precipitate; if Brij98 is in a huge excess, it will cover all hydrophobic groups in Eudragit polymer, which appear at acidic pH, and nanoparticles will not form. Therefore, we performed a detailed study on the appropriate ratio of Eudragit and Brij98 using DLS. A set of DLS experiments at different molar ratios of Eudragit L100-55 and Brij98 (1:277, 1:139, 1:93, and 1:56) were carried out at different pH values (Figure 3). The obtained results showed that in the case of the lowest ratio of Eudragit L100-55/Brij98, 1:277 micelles are not formed at acidic pH. It is well-known that pH sensitivity of polyacids is provided by the presence of carboxylic groups.32 Addition of high amounts of surfactant to the polymer solution causes covering of all available carboxylic groups, thereby decreasing its pH sensitivity. At a certain molar ratio of Eudragit L100-55/ Brij98 (1:139) in the system, one can see the formation of nanoparticles at pH = 5.6. However, further reduction in the Brij98 concentration in the system leads to rapid aggregation and sedimentation of the formed nanoparticles. Figure 3b shows that for pH values lower than 5.6, polydispersity of the obtained nanoparticles is quite low and does not exceed 0.2. A more detailed DLS study of polymer−surfactant systems at different pH values was conducted using an ALV/CGS instrument. It is evident from Figure 4 that there are two modes at basic and neutral pH values. The peak at larger sizes for basic pH values is a classical manifestation of the slow mode, which is always observed for polyelectrolytes33 (Figure 4b). The presence of the second peak could be attributed to the aggregates with hydrodynamic radii from 141 to 428 nm whose annihilation was nicely demonstrated by ITC experiments with an increase in the Brij98 concentration. The fast mode with a hydrodynamic radius near 6 nm belongs to Brij98 micelles. One can see that at pH lower than 5.6 only one mode with a hydrodynamic radius of 53 nm appears, and this mode belongs to the mixed nanoparticles Eudragit/Brij98. To verify the results of DLS, the dependence of zeta potential as a function of pH was measured for the Eudragit/ Brij98 ratio of 1:93. Electrophoretic mobility measurements (Figures 4a and S2) show gradual growth up to pH 5 where the transition jump to neutral values is observed. The gradual growth is attributed to the neutralization of anionic Eudragit, whereas the jump at the pH of 5 correlates with the formation of nanoparticles observed using the DLS data at the same pH value (Figure 4a). Cryo-TEM experiments were conducted to obtain additional information on the shape and structure of Eudragit/Brij98 nanoparticles. One can see from Figure 5 that Eudragit/Brij98 nanoparticles at pH 3 are polydisperse spherical objects with a radius of 30−80 nm, which is in agreement with the results of the DLS study. To support the conclusion on the formation of nanoparticles by changing the pH and to study their structure in detail, we performed SAXS experiments as a next step. The experiments were conducted at four different pH values. The SAXS curves show significant changes with varying pH values from 3 to 10 (Figure 6). The scattering curve for pH = 3 manifests a decay at low q values, whereas at higher q values > 0.04 Å−1, a second decay becomes clearly resolved. A similar behavior was observed for pH 5. The only difference between the two pH values is that the first decay is much broader for pH 5. At higher values of pH, the scattering pattern is changed. The first decay at low

Figure 5. Cryo-TEM image of Eudragit/Brij98 nanoparticles at pH = 3.

values of q is evolving into an upturn. The scattered intensity extrapolated to zero q drops 1 order of magnitude in comparison with the one under acidic conditions. By contrast, another decay appears at the middle q range. The interpretation of the scattering curves presented in Figure 6 is rather straightforward. The first decay at low values of q obviously results from the presence of large particles in the solution at pH 3 and 5. The disappearance of this decay and its evolution into the upturn should be related to the absence of corresponding particles under basic conditions. Moreover, the depression in the scattering intensity at q → 0 and the appearance of an additional peak at q > 0.02 Å−1 for basic pH are manifestations of the small polydisperse entities that were formed at pH above 6. To fit the scattering curves, we have used a combination of several basic models: the model of a polydisperse sphere, the model of a generalized Gaussian coil, and the model of fractal aggregates. The generalized Gaussian coil model34,35 (parameters: Rg, radius of gyration of a polymer chain; v, Flory exponent) was taken for the case of pH = 7 and 10 to describe the decay at the middle q range. The hard sphere model (Rbig, radius of hard spheres; σ, polydispersity) with Schulz−Zimm distribution function of polydispersity was used to describe the decays at low and high q. At basic pH, where an upturn at low q values was observed, we used instead of the hard sphere model an additional contribution of fractal aggregates of which the distribution of sizes is described by power law with a scaling exponent value. The resulting fitting parameters are presented in Figure 6 and Table 1. The size of small spheres at acidic pH corresponds to the size of Brij98 micelles reported previously. The size of large spheres that are observed using SAXS at acidic pH is in reasonable agreement with DLS data (Figure 4). The possible reason for discrepancy between Rh values measured using the DLS method (53 nm) and R fitted from SAXS could be due to different sensitivities of the two methods and limited q ranges of the SAXS setup. Keeping in mind the presence of smaller objects with size 36 Å observed using SAXS, we can assume that the nanoparticles have a spherical shape composed of Eudragit molecules with Brij98 micelles attached to the Eudragit chain. 768

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Figure 6. SAXS curves for solutions of Eudragit/Brij98 (molar ratio Eudragit/Brij98 = 1:93) taken at T = 25 °C. The solid lines are fits.

Table 1. Fitting Parameters for Eudragit/Brij98 Systems at Different pH Valuesa big spherical particles

a

pH

Rbig, Å

σ

3 5 7 10

268 ± 1 128 ± 1 NA NA

0.29 ± 0.01 0.58 ± 0.01 NA NA

small spherical particles

generalized Gaussian coil

fractal aggregates

Rsmall, Å

Rg , Å

ν

α

± ± ± ±

NA NA 58.5 ± 0.1 58.2 ± 0.1

NA NA 0.4 ± 0.1 0.4 ± 0.1

NA NA 2.5 ± 0.1 2.6 ± 0.1

36 35 14 14

1 1 1 1

NAnot applicable.

At basic pH, a distribution function of Rh shows two peaks. This observation is further supported by SAXS data that were successfully fitted by a combination of form factors of larger aggregates, macromolecular coils, and small spheres. Because coils and small spheres have sizes in the same range, they give one unresolved peak on DLS distribution function at 4 nm. After a detailed study on the formation, structure, and size of pH-sensitive nanoparticles based on Eudragit and nonionic surfactant Brij98, we examined the possibility of using this system for the immobilization of drugs. For this purpose, we used trypsin as a model enzyme to investigate the possibility of loading into the Eudragit/Brij98 nanoparticles. The dependence of nanoparticle size on the initial amount of loaded trypsin was studied at pH = 3. Figure 7 shows that the increase in trypsin concentration in the nanoparticles causes a slight increase in their hydrodynamic radii. The most plausible explanation of such a phenomenon could be the swelling of nanoparticles with 3 orders of magnitude increase in trypsin concentration in the interior of the nanoparticles. Because the stability of nanoparticles for drug delivery is one of the critical factors that influence the successful administration, sizes of such systems should be monitored over time. It can be seen from the obtained results (Figure 8) that the hydrodynamic radii of Eudragit/Brij98 nanoparticles have changed slightly over 1 week.

Figure 7. Dependence of hydrodynamic radius of trypsin-loaded Eudragit/Brij98 nanoparticles on the amount of trypsin loaded at pH = 3 and the Eudragit/Brij98 ratio of 1:93.

To determine the LE of Eudragit/Brij98 nanoparticles, trypsin was labeled with iodine 125I. Labeling of trypsin with 125I was performed using the chloramine method36 with a yield of 37% after 16 min of incubation at room temperature. To avoid manufacturing problems (especially large volumes of liquid radioactive waste), we decided to label a small amount of trypsin and add it to a known amount of the unlabeled enzyme. Then, different amounts of trypsin (1, 4, and 10 wt %) labeled with iodine were encapsulated into Eudragit/Brij98 nanocontainers, and the resultant water-based systems were 769

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Figure 8. Dependence of Eudragit/Brij98 nanoparticles at different pH levels over time: (■), pH = 3; (●), pH = 4; and (▼), trypsin-loaded nanoparticles, pH = 3.

Figure 10. Dependence of trypsin activity on pH when loaded into Eudragit/Brij nanoparticles before and after treatment by pepsin.

Because the immobilized trypsin is subjected to the pepsin action in stomach before entering the small intestine, we also studied the activity of unbounded and immobilized trypsin after treating it with a 50-fold excess of pepsin (purple and orange bars). One can see (Figure 10) that the activity of trypsin immobilized into Eudragit/Brij98 nanoparticles remains at nearly 50% of its activity, and this value is almost independent of the pH of solution in stomach. These results are in good agreement with the trypsin-loading data. The stability of trypsin activity in Eudragit/Brij98 nanoparticles was also evaluated over time (Figure 11). It can be

centrifuged to remove the unloaded trypsin. The LE was calculated from the values of nanoparticle radioactivity after the removal of unloaded trypsin by centrifugation. We have observed the highest LE (56%) for 4 wt % of the initial trypsin. However, the increase in the initial amount of trypsin to 10 wt % leads to a decrease in the LE to 29%. We can assume that the encapsulation of trypsin into Eudragit/Brij98 nanocontainers is due to several interactions, namely, hydrophobic−hydrophobic and electrostatic. The latter occurs while reducing the pH below pK of trypsin, which is equal to 10.5. For lower values of pH, trypsin is positively charged and can interact with polyanionic chains of Eudragit that are stabilized by Brij98 surfactant (Figure 9).

Figure 11. Dependence of trypsin activity on time when loaded into Eudragit/Brij nanoparticles before and after treatment by pepsin.

concluded that despite a slight drop in the enzyme activity over time when it is encapsulated into the nanoparticles, this decrease is not as dramatic as in the case of unloaded trypsin. Moreover, trypsin loaded into nanoparticles retains 35% of its activity after the treatment by pepsin after 3 days of storage. As it was mentioned above, trypsin can be encapsulated into Eudragit/Brij98 nanocontainers owing to the hydrophobic or/ and electrostatic interactions, and this assumption is confirmed by the determination of trypsin activity. It is known37 that the conformation of enzymes significantly affects the proteolysis, which affects the activity of enzymes. Thus, the ionic interaction between positively charged trypsin and carboxylate groups of Eudragit can cause changes in trypsin conformation and in turn decrease the autolysis of enzymes, hence increasing its activity.

Figure 9. Scheme of interaction between trypsin and Eudragit/Brij98 system.

The influence of trypsin immobilization onto Eudragit/Brij98 nanoparticles at different pH on its activity was studied with the standard substrate TAME. The comparison of trypsin activities, which was stored for 1 h in the unbounded form and later on immobilized into nanoparticles, is represented in Figure 10. It is known that trypsin is capable of autolysis, which is prevented by its storage at pH = 3. Therefore, it is evident that with an increase in the pH value of trypsin storage, its activity decreases. Although trypsin immobilization in nanoparticles leads to a partial loss of its activity, the pH dependence of this process is not so dramatic. Depending on the pH of storage, the activity of trypsin remains at the level of 68.4−50.5% compared with that of the theoretical one (green and blue bars, respectively).

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CONCLUSIONS Formation of Eudragit L100-55 nanoparticles in the presence of the nonionic surfactant Brij98 has been studied. The addition of 770

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a certain amount of surfactant Brij98 to polymer Eudragit L100-55 provides the possibility to obtain stable nanoparticles and prevent a macroscopic polymer phase separation at acidic pH. A detailed analysis of this system using SAXS and DLS techniques showed that at low pH values, nanoparticles have a spherical shape composed of Eudragit−Brij98 complexes. Trypsin delivery systems based on Eudragit/Brij98 nanoparticles were obtained. It was shown that encapsulation of trypsin in Eudragit/Brij98 nanocontainers prevents its deactivation when it is treated with pepsin at different pH values.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b03778. ITC curves for the titration of Eudragit L100-55 with Brij98 at different pH values; normalized zeta potential distribution functions at different pH values; and description of the generalized Gaussian coil model (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: sfi[email protected]. Phone: +420 608 720 561 (S.K.F.). *E-mail: [email protected]. Phone: +420 296 809 130 (M.H.). ORCID

Sergey K. Filippov: 0000-0002-4253-5076 Martin Hruby: 0000-0002-5075-261X Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.H. acknowledges financial support of the Ministry of Education, Youth, and Sports of the Czech Republic, grant no. LH14292 and the Ministry of Health of the Czech Republic, grant no. 15-25781A. S.K.F. acknowledges support of the Grant Agency of the Czech Republic (15-10527J). The authors thank the Diamond Light Source Synchrotron (Oxfordshire, UK) for providing the synchrotron beam time and Evonic Industries for providing the Eudragit L100-55 polymer. The authors acknowledge Lubomir Kovacik and Sami ̈ Kereiche for Cryo-TEM measurements.

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REFERENCES

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DOI: 10.1021/acs.langmuir.6b03778 Langmuir 2017, 33, 764−772

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DOI: 10.1021/acs.langmuir.6b03778 Langmuir 2017, 33, 764−772