Triblock polymeric micelles as carriers for anti-inflammatory drug delivery.

http://informahealthcare.com/mnc ISSN: 0265-2048 (print), 1464-5246 (electronic) J Microencapsul, Early Online: 1–7 ! 2014 Informa UK Ltd. DOI: 10.3109/02652048.2014.995729

RESEARCH ARTICLE

Triblock polymeric micelles as carriers for anti-inflammatory drug delivery Krassimira Yoncheva1, Petar Petrov2, Ivanka Pencheva3, and Spiro Konstantinov4

Journal of Microencapsulation Downloaded from informahealthcare.com by Kainan University on 04/06/15 For personal use only.

1

Department of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy, Medical University of Sofia, Sofia, Bulgaria, 2Institute of Polymers, Bulgarian Academy of Sciences, Sofia, Bulgaria, 3Department of Pharmaceutical Chemistry, and 4Department of Pharmacology, Faculty of Pharmacy, Medical University of Sofia, Sofia, Bulgaria Abstract

Keywords

This study evaluated the properties of poly(ethylene oxide)-b-poly(n-butyl acrylate)-b-poly (acrylic acid) (PEO-PnBA-PAA) polymeric micelles as carriers for anti-inflammatory drugs (prednisolone and budesonide). The micelles comprising a hydrophobic PnBA core and a PEO/ PAA corona showed average diameter less than 40 nm. The size of the drug-loaded micelles did not change during eight hours into media that mimic physiological fluids indicating high colloidal stability. The calculation of Flory–Huggins parameter showed greater compatibility between budesonide and micellar core suggesting its location in the micellar core, whereas prednisolone was located also into the interface layer. This observation correlated further with slower release of budesonide, especially in acid medium (pH ¼ 1.2). The inclusion of budesonide into micelles showed significant protective effect against the cytotoxic damage induced by the co-cultivation of differentiated human EOL-1 and HT-29 cells. This study revealed the capacity of PEO-PnBA-PAA terpolymer as carrier of nanosized micelles suitable for oral delivery of anti-inflammatory drugs.

Cytoprotective effect, micellar stability, poly(acrylic acid), poly(ethylene oxide), poly(n-butyl acrylate), polymeric micelles

Introduction Amphiphilic polymers are widely investigated as carriers for micellar nanosized drug delivery formulations (Kabanov and Alakhow, 1997; Kataoka et al., 2001; Riess, 2003). Polymeric micelles are small nanoparticles with a diameter usually several tens of nanometers and a unique core-shell structure (Nishiyama et al., 2005; Nishiyama and Kataoka, 2006a, 2006b). This structure is associated with high capacity for loading of poorly soluble drugs into hydrophobic core of the micelles (Kwon, 2003; Torchilin, 2004; Lu and Park, 2013). On the other hand, the small size of the micelles and appropriate modification of the micellar shell are related with the achievement of longer circulation, EPR effect and improved capacity for membrane transport (Bae et al., 2003; Torchilin, 2004; Talelli et al., 2011). Regarding oral administration, the polymeric micelles could prevent enzymatic drug degradation, improve oral bioavailability of poorly absorbed drugs and facilitate the membrane transport (Blomberg, 2008; Gaucher et al., 2010; Yu et al., 2013). Mathot et al. (2007) have found that monomethyl ether poly(ethylene glycol)-poly(caprolactone-co-trimethylene carbonate) micelles can cross lipid bilayers via passive diffusion and demonstrate an oral bioavailability of 40% in rats. More recently, an improvement of oral bioavailability of paclitaxel due to its incorporation into stabilized

Address for correspondence: Krassimira Yoncheva, PhD, Department of Pharmaceutical Technology, Faculty of Pharmacy, Medical University of Sofia, 2 Dunav Str., 1000 Sofia, Bulgaria. Tel: +35929236527. Fax: +35929879874. E-mail: [email protected]

History Received 4 May 2014 Revised 10 November 2014 Accepted 14 November 2014 Published online 24 December 2014

Pluronic micelles has been reported (Yoncheva et al., 2012). Oral bioavailability of the docetaxel-loaded mixed polymeric micelles was increased 2.52 times compared with that of free drug (Dou et al., 2014). Budesonide and prednisolone are hydrophobic drugs used for the treatment of inflammatory bowel diseases like ulcerative colitis and Crohn’s disease (Campieri et al., 1997). Ulcerative colitis affects mainly the colon mucosa, whereas Crohn’s disease affects mainly the distal segments of the ileum (Cosnes et al., 2011). However, the short-term and long-term administration of these drugs is associated with side effects like headache, respiratory infection, nausea and symptoms of hypercorticism (e.g. acne, moon face, hirsutism etc.). The corticosteroid resistance has been considered another obstacle for the achievement of efficient treatment of inflamed intestinal mucosa. Recently, the poor response to prednisolone and budesonide has been related with the fact that they were identified as substrates of the intestinal drug efflux P-glycoprotein pump (Dilger et al., 2004). In addition, the short residence of drug into the distal intestinal segments hinders the interactions with mucosal cells and reduces the capacity for anti-inflammatory local therapy. Incorporation of drugs into nanoparticulate systems, such as polymeric micelles, could dissolve the problems with their side effects, cell uptake and efflux. The size has been considered one of the most important characteristics regarding nanoparticle transport. Lamprecht et al. (2001) have reported that after oral administration of polystyrene nanoparticles in experimental rat colitis model, the 100-nm nanoparticles showed higher adhesion to the inflamed colon than 10-mm particles and a 6.5-fold higher accumulation compared with healthy control. A following study


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with tacrolimus-loaded nanoparticles in rat colitis model showed that nanoparticles predominantly adhered to the inflamed tissue providing an enhanced and selective drug penetration into the inflammation site, in particular 3-fold higher penetration compared with healthy tissue when using nanoparticles as drug carriers (Lamprecht et al., 2005). The high protection of the encapsulated drug against influences from efflux systems and mucosal metabolism was considered as a reason for the more efficient drug penetration. In order to achieve long residence at desired segments of gastrointestinal tract, the physiologically governed adhesion on inflamed tissue could be combined with additional surface modification of nanoparticles. The modification with bioadhesive polymers (e.g. chitosan, sodium alginate, poly(acrylic acid), etc.) has been widely investigated because of their biocompatibility and high capacity for interactions with epithelial cells. Recent study showed that nanoparticles loaded with anti-inflammatory tripeptide Lys-ProVal (KPV) overcome physiologic barriers and target antiinflammatory agent providing similar therapeutic efficacy at concentration 12 000-fold lower than that of KPV in free solution (Laroui et al., 2010). The above considerations suggest that efficient oral therapy with prednisolone and budesonide could be achieved by development of polymeric micelles able to provide drug delivery in the distal parts of gastrointestinal tract (e.g. ileum and colon). The adhesion could be enabled by the inflammation of these segments, but the size and additional surface modifications are also required. The aim of this study was to develop polymeric micelles possessing small size and bioadhesive polymer block in the micellar corona. Our previous studies showed the possibility to prepare micelles from poly(ethylene oxide)-b-poly(n-butyl acrylate)-b-poly(acrylic acid) (PEO-PnBA-PAA) triblock terpolymers with a diameter less than 50 nm and high loading of hydrophobic drug as paclitaxel in the PnBA micellar core (Petrov et al., 2013). In this study, we aimed to evaluate the utility of PEO-PnBA-PAA micelles as a platform for oral delivery of anti-inflammatory drugs. The small size and the presence of bioadhesive poly(acrilic acid) block in the micellar corona were considered prerequisite for adhesion of micelles to inflamed intestinal segments and improved local drug delivery.

Materials and methods Materials Methoxy poly(ethylene glycol) (PEO113OH, MW: 5000, Fluka, Buchs, Switzerland) was precipitated in cold methanol (40 C), filtered and dried under a vacuum at 40 C overnight. n-Butyl acrylate and tert-butyl acrylate (kindly supplied by BASF, Ludwigshafen, Germany) were stirred overnight on calcium hydride (Merck, Darmstadt, Germany, 95%) with Irganox 1010 inhibitor (Ciba-Geigy, Basel, Switzerland) and distilled under a vacuum. CuBr (Aldrich, St Louis, MO, 98%) was stirred overnight in glacial acetic acid, filtered and rinsed successively by acetic acid, ethanol and ether to remove traces of CuBr2. 2Bromoisobutyryl bromide (Aldrich, 98%), triethylamine (Fluka, 99.5%), 1,1,4,7,10,10-hexamethyl triethylenetetramine (Aldrich, 98%), N,N,N0 ,N0 ,N00 -pentamethyldiethylenetriamine (PMDETA, Aldrich, 99%), ethyl methyl ketone (Merck, 99.8%), acetone (Aldrich, 99.8%), tetrahydrofuran (THF, Merck, 99.8%), methanol (Merck, 99.8%), 1,4-dioxane (Merck, 99.5%), SiO2 (63–200 mm, Merck) and trifluoroacetic acid (Aldrich, 99%) were used as received. Dichloromethane (Aldrich, 99.8%) was stirred overnight on calcium hydride (Merck, 95%) and distilled. Budesonide and prednisolone were provided by Sigma Aldrich. HT-29 cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ GmbH, Braunschweig, Germany).

J Microencapsul, Early Online: 1–7

Triblock terpolymer synthesis and characterization PEO113PnBA163PAA12 triblock terpolymer was synthesized by consecutive atom transfer radical polymerization (ATRP) of n-butyl acrylate and tert-butyl acrylate, initiated by a PEO113-Br macroinitiator (Petrov et al., 2013). After that, PEO113PnBA163Br macroinitiator (1.015 g, 0.039 mmol) was dissolved in acetone (1.8 mL). This solution was degassed by bubbling nitrogen under stirring for 45 min and then added with the PMDETA ligand (0.008 mL; 0.039 mmol), the CuBr catalyst (0.0056 g; 0.039 mmol) and the freshly distilled and degassed tBA monomer (0.0915 mL, 0.624 mmol). The polymerization was carried out at 50 C for 73 h (70% conversion). Purification was achieved by precipitation of the reaction mixture in cold methanol (40 C) and filtration. The terpolymer was redissolved in THF and passed through a silica column to remove the Cu(II) catalyst. Finally, THF was evaporated under a vacuum; the terpolymer was dissolved in dioxane and freeze-dried. Nuclear magnetic resonance spectra (1H NMR) were recorded in CDCl3 using a 250 MHz Bruker AC-spectrometer. DPn of PnBA was calculated by comparing the peak integral assigned to the PEO protons (4H, OCH2CH2) at 3.64 ppm to the PnBA protons at ¼ 4.03 (2H, OCH2) and at ¼ 0.91 (3H, OCH2CH2CH2CH3). DPn of PtBA was calculated on the basis of peak integrals assigned to the PEO protons (4H, OCH2CH2) at 3.64 ppm, the PnBA protons at ¼ 4.03 ¼ 1.2–2 ppm (4H, OCH2CH2 (2H, OCH2), CH2CH3) and (2H, CH2C(C¼O)H) and the tBA protons at ¼ 1.53 (2H, CH2C(C¼O)H) and ¼ 1.44 (9H, OC(CH3)3). Gel permeation chromatography (GPC) measurements were performed with PSS SDV-gel columns (5 mm, 60 cm, 1 linear ˚ ), 1 100 A ˚ ) with THF as the eluent (flow (102–105 A rate ¼ 1.0 mLmin1), at room temperature, and using refractometry for detection. The molar mass and polydispersity index (PDI) of PEO113PnBA163PtBA12 were determined using PtBA universal calibration. Preparation of drug-loaded micelles based on the triblock terpolymer The prepared PEO113PnBA163PAA12 triblock terpolymer was dissolved in 5 mL of methanol (concentration 2 mgmL1) and prednisolone or budesonide was added to this solution under stirring (700 rpm). After incubation for 30 min, approximately 2 mL purified water was added dropwise. Then, the micellar dispersion was transferred into dialysis membrane (MWCO 6000– 8000 g/mol, Spectrum Labs, Rancho Dominguez, CA) and dialyzed against water during two hours with a refreshment of the outer water every one hour. Characterization of the drug-loaded micelles The shape of the micelles was observed by cryogenic transmission electron microscopy (TEM). A drop of the micellar dispersion was deposited on an untreated bare copper TEM grid (600 mesh, Science Services, Munchen, Germany). The specimens were instantly shock vitrified by rapid immersion into liquid ethane cooled at ca. 90 K by liquid nitrogen in a temperature-controlled freezing unit (Zeiss Cryobox, Zeiss NTS GmbH, Jena, Germany). After vitrification, the specimen, the remaining ethane, was removed using blotting paper. The specimen was inserted into a cryo-transfer holder (CT3500, Gatan, Pleasanton, CA) and transferred to a Zeiss EM922 EF-TEM instrument (Zeiss NTS GmbH). The observations were carried out at ca. 90 K. The TEM was operated at an acceleration voltage of 200 kV. Zero-loss filtered images (DE ¼ 0 eV) were taken under reduced dose


DOI: 10.3109/02652048.2014.995729

Polymeric micelles as carriers for anti-inflammatory drugs

3

conditions (approximately 100–1000 e/nm2). All images were registered digitally by a bottom-mounted CCD camera system (Ultrascan 1000, Gatan) and combined and processed with a digital imaging processing system (Digital Micrograph 3.10 for GMS 1.5, Gatan). Size and zeta-potential were determined in purified water by photon correlation spectroscopy and electrophoretic laser Doppler anemometry using a Zeta master analyzer (Malvern Instruments, Worcestershire, UK). Samples were dispersed in distilled water and measured at 25 C with a scattering angle of 90 . For the stability studies, the size and PDI of micelles were determined in simulated gastric fluid (SGF) containing pepsin (Eur. Ph. 7.0, 2010), simulated intestinal fluid (Eur. Ph. 7.0, 2010) and simulated colon fluid (SCF) (Chen et al., 2007). The drugloaded PEO-PnBA-PAA micellar dispersions were mixed with the selected simulated fluids and incubated at 37 C under stirring (100 rpm). Samples were taken to determine the particle size and polydispersity at 1, 4 and 8 h of this study. The calculation of Flory–Huggins parameter sp was made applying the equation:

29 cells were placed in the microplates at a density of 3 105 cells per mL and 1.5 105 per mL, respectively. After a 24-h incubation period at 37 C, EOL-1 cells were treated with 100 mg mL1 phorbol 12-myristate 13-acetate (PMA) for 24 h and later were admixed to a HT-29 confluent monolayer. PMA treatment leads to differentiation of leukemic EOL-1 cells into functionally active eosinophilic cells, which are able to attack and damage HT-29 epithelial monolayers. HT-29 cells were untreated (control) or pretreated with pure prednisolone/budesonide (5 or 10 mM) or prednisolone/budesonide loaded micelles. After 48-h incubation, the cells were rinsed with PBS, and 3 mL of new medium (RPMI 1640) and 30 mL MTT solution were added to each well. The microplates were further incubated for 3 h at 37 C, and the MTT-formazan crystals formed were dissolved through the addition of 3 mL per well of 5% formicacidin 2-propanol. The MTT formazan absorption was determined using a microprocessor controlled microplate reader (Labexim LMR-1 Labexim, Lengau, Austria) at 550 nm.

sp ¼ Vs ðs p Þ2 =RT

The experiments were replicated three independent times, and the data are presented as mean ± SEM. Statistical analysis was carried out using Student’s t-test. Differences were considered statistically significant when p value was less than 0.01.

where Vs is the molar volume of the drug, s and p are the solubility parameters of the drug and polymer block forming the core, respectively, R is the gas constant and T is the Kelvin temperature (Flory, 1953). The solubility parameters of drug molecules were calculated applying the Fedor’s method that is based on the contribution of the chemical groups in the molecules to their cohesive energy (Fedors, 1974). The amount of prednisolone/budesonide incorporated into the micelles was calculated as a difference between its initial concentration and the concentration of free drug found in the fractions collected during the dialysis. Budesonide was determined by HPLC method. The system (Shimadzu LC-10 Advp, Shimadzu, Kyoto, Japan) was equipped with a Waters RP C18 column (Waters, Milford, MA), 250 4.6 mm (particle size 5 mm). The mobile phase, pumped at 1 mL min1, was 70:30 v/v acetonitrile–water, and effluent was monitored with UV detection at 250 nm. For the calculations, standard curve of budesonide was prepared in the concentration range of 0.499–4.15 mg mL1 (r40.9993). Prednisolone was determined by UV-Vis spectrophotometry at a wavelength of 248 nm (Hewlett Packard 8452A, Hewlett Packard, Palo Alto, CA). The standard curve of prednisolone was prepared in the concentration range of 2– 20 mg mL1 (r40.9987). Drug encapsulation efficiency (EE) was calculated using the following equation: EE ¼ (total amount of drug – free drug)/total amount of drug. In vitro release study

Statistical analysis

Results and discussion Physicochemical properties The micelles used in this study were prepared from PEO113PnBA163PAA12 triblock terpolymer (Figure 1). The triblock terpolymer was synthesized by consecutive ATRP of n-butyl acrylate and tert-butyl acrylate, initiated by a PEO113-Br macroinitiator. Finally, PtBA blocks were derivatized into PAA blocks by hydrolysis with 15 equiv of trifluoroacetic acid with respect to the tBA units according to a procedure known to preserve the PnBA groups (Colombani et al., 2007a, 2007b). 1 H-NMR and GPC analysis confirmed the formation of welldefined triblock terpolymer with narrow molar mass distribution. The composition, calculated by 1H NMR analysis, corresponds to ¼ 26 800 g mol1, MGPC ¼ 38 100 PEO113PnBA163PAA12 (MNMR n n 1 g mol ; PDI ¼ 1.11). PEO113PnBA163PAA12 micelles comprising a hydrophobic PnBA core and a PEO/PAA mixed shell were obtained by the dialysis method. Since PEO chains are much longer than PAA chains, it is assumed that the outer layer of the shell is composed only from PEO segments (Petrov et al., 2013). It is well known that the size and surface morphology of nanoparticulate systems are of great importance for interactions between the mucosal surface and nanoparticles and subsequent transport. The main physicochemical properties of the micelles

In vitro release of prednisolone and budesonide from the micelles was examined in acid (pH ¼ 1.2, 0.1 N HCl) and phosphate buffers (pH ¼ 6.8 and pH ¼ 7.4, Eur. Ph.). The freshly prepared micellar dispersion was introduced into a dialysis membrane bag (MW ¼ 6000–8000) that further was placed into 100 mL buffer. The release medium was stirred (50 rpm), and the temperature was maintained constantly during the study (37 C). At predetermined time intervals, samples were withdrawn from the medium outside the dialysis bag, and the concentration of the released drugs was determined as described above. In vitro cytoprotective effect EOL-1 (ACC 386) cells originated from a patient with acute myeloid (eosinophilic) leukaemia. For this study, EOL-1 and HT-

Figure 1. Structural terpolymer.

formula

of

PEO113PnBA163PAA12

triblock

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Table 1. Physicochemical properties of prednisolone- and budesonideloaded PEO-PnBA-PAA micelles. Sample Empty micelles Prednisolone-loaded micelles Budesonide-loaded micelles

Size (nm)

PI

Zeta-potential (mV)

38 ± 3 39 ± 2 37 ± 4

0.203 0.145 0.153

15.7 ± 4.7 21.3 ± 5.3 15.0 ± 4.2

Table 2. Physicochemical properties of both drug molecules and calculated values for solubility parameter (s) and drug–polymer compatibility (sp).

Drug

log P

Water solubility (mg mL1)

s (MPa1/2)

sp (MPa1/2)

Budesonide Prednisolone

3.14 1.50

40* 223**

24.20 25.87

4.81 6.57

Note: Mean ± SD (n ¼ 3). Notes: *The value originated from the study of Piao et al. (2009). **The value originated from the study of Yalkowsky and Dannenfelser (1992).

Prednisolone micelles

100

EE, %

60 40 20 0 0.5 : 5

1:5

Figure 3. Influence of the ratio between initial concentrations of prednisolone/budesonide and the triblock terpolymer on the encapsulation efficiency. Mean ± SD (n ¼ 3).

(a) 50

Figure 2. Cryo-TEM micrograph of polymeric micelles formed by PEO113PnBA163PAA12 triblock terpolymer. Size, nm

and their shape are presented in Table 1 and Figure 2, respectively. No differences in the size of empty and drugloaded micelles were observed. The diameter was less than 40 nm, which was considered as prerequisite for facilitated penetration of such nanoparticles through the mucus layer. In addition, the micellar corona is consisted of flexible PEO and PAA chains that also could enable the penetration through the mucus (Yoncheva et al., 2005). The micelles possessed negatively charged surface due to the presence of poly(acrylic acid) block in the micellar corona. The values for zeta potential of prednisolone loaded micelles were more negative compared with that loaded with budesonide. This phenomenon could be explained with the different location of both drugs into the micellar structure. In particular, budesonide was predominantly incorporated into the micellar core due to the more hydrophobic properties comparing to prednisolone (Table 2). Thus, eventual distribution of prednisolone into the interface between the core and corona could provide the more negative zeta potential. Furthermore, the affinity of each drug to the PnBA segments that form the core was evaluated by calculation of Flory–Huggins parameter (sp). According to the Flory–Huggins theory, the closer sp is to zero, the better is the miscibility between the drug and the core forming segment of the copolymer. The values of solubility parameters and drug–polymer compatibility were calculated and listed in Table 2. As shown, higher value for sp was found for prednisolone compared with budesonide. The higher value for prednisolone suggested lower affinity to the micellar core that proved the hypothesis for its location not only in the core but also into the interface layer.

1h

4h

8h

40 30 20 10 0 SGF

SCF

SIF

(b) 0.25 1h Polydispersity index


Budesonide micelles

80

4h

8h

0.2 0.15 0.1 0.05 0 SGF

SIF

SCF

Figure 4. Evaluation of stability of the micelles by measurement their size (a) and polydispersity index (b) after incubation in simulated gastric fluid (SGF), simulated intestinal fluid (SIF) and simulated colonic fluid (SCF).

The influence of the quantitative ratio between drug and copolymer on the EE was evaluated. Two concentrations of budesonide/prednisolone with respect to the copolymer concentration were varied, in particular 0.5:5 and 1:5 (wt/wt). The results demonstrated that the increase of the initial amount of budesonide (1:5 ratio) slightly decreased the EE (Figure 3). In the case of


DOI: 10.3109/02652048.2014.995729

Drug release in pH=1.2


(b) 100

Budesonide micelles

80

Released, %

Released, %

Drug release in pH=7.4


(a) 100

5

60 40 20

Budesonide micelles

80 60 40 20

0

0 0

1

2

3

4

5

0

10

20

Time, h

30

40

50

Time, h Drug release in pH=6.8 Prednisolone micelles

(c) 100

Budesonide micelles

Released, %


80 60 40 20 0 0

10

20

30

40

50

Time, h

Figure 5. In vitro release of prednisolone and budesonide from the micelles in different buffers: pH ¼ 1.2 (a), pH ¼ 7.4 (b) and pH ¼ 6.8 (c). Mean ± SD (n ¼ 3).

prednisolone, such tendency was not observed that correlated with the suggestion for additional distribution of prednisolone into the interface between the core and corona. Evaluation of stability into simulated gastrointestinal fluids is required in order to ensure the appropriate drug release rate at desired region of the tract. In this study, stability of the micelles was evaluated by measurement of their size and polydispersity in three media, in particular simulated gastric fluid (SGF), simulated intestinal fluid (SIF) and simulated colonic fluid (SCF). The results showed that during eight hours, the size of the micelles did not change (Figure 4a). Regarding PDI, the measurements showed lower polydispersity for micelles in SGF and higher one for micelles incubated in SCF (Figure 4b). The higher polydispersity was more pronounced at longer incubation time, suggesting eventual slight agglomeration, which, however, did not influence the overall colloidal stability of systems. All these results indicated that the micelles maintained their original structure into the three media that was considered prerequisite for the achievement of appropriate budesonide delivery into gastrointestinal tract. In vitro release study The in vitro release was studied in acid and phosphate buffers (pH 1.2, 7.4 and 6.8) in order to mimic physiologically a stomach fluid, intestinal fluid in the distal parts of ileum and colonic fluid, respectively. Figure 5 represents the release profiles of budesonide and prednisolone into the selected media. For both drugs, faster release was observed in acidic medium compared with their profiles in phosphate buffers. The carboxylic groups of PAA (pKa ¼ 4.75) are protonated at pH 1.2, and there is no electrostatic repulsion between the chains. This resulted in decreased solubility of PAA chains as compared with pH 6.8 (Zhang et al., 2011). Most probably, the collapse of PAA macromolecules at pH 1.2 increased the permeability of PEO/PAA layer and, therefore, accelerates the diffusion of drug molecules. Comparing the profiles of the two drugs in acid medium, a slower release rate was registered for budesonide. This

observation correlated with the different location of both drugs suggested by zeta-potential measurements (Table 1). In particular, the location of budesonide was in the core, whereas part of prednisolone was located into the interface between the core and corona. The slower release of budesonide in acid medium was considered advantageous taking in account that for the treatment of distal segments of the ileum the drug might be delivered there instead of in the stomach segment.

Evaluation of cytoprotective effect Before evaluation of cytoprotective capacity of the drug-loaded polymeric micelles, the triblock terpolymer was examined for eventual cytotoxicity on HT-29 cell line. The results showed that the polymer did not provoke any cytotoxicity in the cellular population suggesting its safety (not shown). Furthermore, the estimation of the cytoprotective effect was based on the damage reduction of HT-29 mucosa monolayers co-cultured with PMA differentiated eosinophilic EOL-1 cells. PMA leads to differentiation of EOL-1 cells and to their functional activation, e.g. production of inflammatory cytokines. Such differentiated eosinophilic cells may damage mucosa epithelial HT-29 cell monolayers. In order to study the cytoprotective effect of drug-loaded micelles, such co-cultural model consisting of differentiated eosinophilic EOL-1 cells and HT-29 cells was applied. The addition of PMA-treated EOL-1 cells caused between 10 and 20% decrease of the cell viability of monolayer growing colon HT-29 cells (Figures 6 and 7). The pretreatment of HT-29 cells with the empty micelles did not show protective effect (not shown). Furthermore, the studies were performed with the drug-loaded micelles and respective free drugs as references. The pretreatment of HT-29 cells with free prednisolone (5 and 10 mM) did not protect cells from cytotoxic damage (Figure 6). The effect was studied even at higher concentrations (20 and 40 mM) but at these concentrations the cell viability additionally decreased due to the cytotoxic effect of prednisolone. The micellar formulations did not show protective effect against the damaging effect induced by

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120


Prednisolone

References


100 80 60 40 20 0 Untreated Damaged HT-29 cells HT-29 cells

5 µM

10 µM

20 µM

40 µM

Figure 6. Evaluation of cytoprotective effect of free prednisolone and micellar prednisolone on HT-29 cells damaged by co-cultivation with differentiated human EOL-1 cells. Mean ± SD (n ¼ 3).

% of untreated control


120

Budesonide

Budesonide micelles

100 80 60 40 20 0 Untreated HT-29 cells

Damaged HT-29 cells

5 µM

10 µM

Figure 7. Evaluation of cytoprotective effect of free budesonide and budesonide-loaded micelles on HT-29 cells damaged by co-cultivation with differentiated human EOL-1 cells. Mean ± SD (n ¼ 3).

co-cultivation of differentiated human EOL-1 cells and HT-29 cells. However, an important finding was the lower cytotoxicity of micellar prednisolone at 20 and 40 mM compared with the free prednisolone in the same concentrations. It seems that the binding of prednisolone to the micelles can significantly decrease its cytotoxicity. The pretreatment of the cell model with budesonide and budesonide-loaded micelles caused protection against the damage produced by PMA differentiated eosinophilic EOL-1 cells (Figure 7). Budesonide inclusion into micellar particles additionally increased the protective property of this locally used drug. As shown, even the lower concentration of micellar formulation (corresponding to 5 mM budesonide) resulted in statistically significant cytoprotective effect that was not observed with free drug. Thus, the important finding was that the cytoprotection could be achieved at lower concentration of budesonide when loaded in micelles.

Conclusion This study revealed the possibility to prepare polymeric micelles from PEO-PnBA-PAA triblock terpolymer possessing size less than 40 nm. The evaluation of the size and polydispersity of the micelles into simulated gastrointestinal fluids showed that the micelles maintained their structure, which is a prerequisite for the achievement of appropriate drug delivery into gastrointestinal tract. The investigation on cell model demonstrated that incorporation of budesonide into micelles provided higher cytoprotection against the induced cell damage than free drug. These results indicated the capacity of PEO-PnBA-PAA micelles as a platform for the development of oral nanoparticulate delivery systems.

Declaration of interest The authors report no conflict of interest. The financial support of the National Science Fund of Bulgaria (B01-25/2012) is gratefully acknowledged.

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