Complex Coacervation-Controlled Release from Monoolein Cubic

Dec 29, 2010 - Biomacromolecules , 2011, 12 (2), pp 466–471 ... The degree of coacervation dramatically decreased (from 581.2 to 5.2 nm in size and ...
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Biomacromolecules 2011, 12, 466–471

Complex Coacervation-Controlled Release from Monoolein Cubic Phase Containing Silk Fibroin and Alginate Taek Kwan Kwon and Jin-Chul Kim* Division of Biotechnology and Bioengineering and Institute of Bioscience and Biotechnology, Kangwon National University, 192-1, Hyoja 2 dong, Chunchon, Kangwon-do 200-701, Republic of Korea Received October 19, 2010; Revised Manuscript Received December 6, 2010

pH-dependent release from monoolein (MO) cubic phase was obtained by taking advantage of complex coacervation between hydrophobically modified alginate (HmAL) and hydrophobically modified silk fibroin (HmSF) in the water channels. The degree of coacervation was investigated at pH 3.0 by a light scattering method and the maximum coacervation was observed when the ratio of HmAL to HmSF was 1:15. The degree of coacervation dramatically decreased (from 581.2 to 5.2 nm in size and from 267.9 to 12.3 nm in Kcps) when the pH of medium increased from 3.0 to 5.0. The % release in 100 h of FITC-dextran increased from 2.42 to 7.20% when pH of release medium increased from 3.0 to 9.0. Under acidic conditions, coacervate will block the water channels of cubic phase, suppressing the release. As the pH of release medium increases, the coacervate will dissolve, resulting in a higher release. The cubic phase could be exploited as a pH-sensitive carrier for the oral delivery of an acid-labile drug.

Introduction Cubic phases have attracted much attention of scientists in the field of drug delivery systems for a few decades. Amphiphiles of which packing parameters are slightly less than 1 tend to form cubic phases in an aqueous phase,1-3 and a representative amphiphile constituting cubic phase is monoolein (1-monooleoyl glycerol, MO). Two intercrossing water channels pass through the cubic phase, they are separated by MO bilayers,4,5 and their sizes are 5-7 nm in diameter. The cubic phase is optically transparent due to its interconnected isotropic structure. It is known to be nontoxic to human,6,7 and it can accommodate not only water-soluble but also oil-soluble compounds.8-10 With the aid of a colloid stabilizer, the cubic phase is readily micronized into nanoparticles, so-called cubosome. Drugs loaded in the cubic phase diffuse through the water channels or the bilayers, so slow release, one of the aims of drug delivery system, can be achieved with the cubic phase.6,7,11,12 Insulin was entrapped in the water channel of cubosomes for the oral delivery.13,14 The cubosomes were proposed to stabilize insulin in the harsh conditions of stomach and to enhance the gastrointestinal absorption. A vaccine adjuvant was loaded in cubosomes for the subcutaneous injection.15 The cubosome was also reported to maintain the efficacious concentration for 6 h of somatostatin in the plasma of experimenat rabbits, when intravenously injected.16 In addition, cyclosporin A was included in the cubosome to enhance the bioavailability through the oral delivery.17 Recently, cubic phase was designed to exhibit stimuliresponsive release. Alginate was introduced to the water channel of cubic phase to obtain calcium ion-sensitive release.18 Diffusion through the water channel was hardly affected by the alginate when there was no calcium ion. However, the release from the cubic phase was significantly suppressed in the presence of the ion. The alginate in water channel became gel, and the rate of diffusions through the water channel could be * To whom correspondence should be addressed. Phone: +82-33-2506561. Fax: +82-33-253-6560. E-mail: [email protected].

slackened down. To obtain temperature-sensitive release, hydrophobically modified poly (N-isopropylacrylamide) was immobilized in the water channel.19 The release could be controlled owing to the thermal contraction and expansion of the thermosensitive polymer. To achieve pH-sensitive release, acidic protenoid20 and hydrophobically modified chitosan were immobilized in the water channel. The pH-dependent conformational change of the polymers or the pH-dependent electrostatic interaction of the polymers with model drug was reported to be responsible for the sensitive releases from the cubic phases. In this study, a novel drug delivery carrier which releases its water-soluble content in response to pH change was developed by including silk fibroin and alginate in the water channel of MO cubic phase. The release can be controlled in a pHdependent manner due to the complex coacervation between the protein and the negatively charged polysaccharide. For the immobilization of silk fibroin and alginate in the water channels of MO cubic phase, they were hydrophobically modified. The strategy to design a pH-sensitive cubic phase is based on the complex coacervation between hydrophobically modified silk fibroin (HmSF) and hydrophobically modified alginate (HmAL; Figure 1). The HmSF to HmAL ratio for the maximum coacervation was determined under an acidic condition through a light scattering method. HmSF and HmAL were loaded in MO cubic phase in the optimum ratio. Finally, the release of FITC-dextran from the cubic phase was observed with varying the pH of release medium.

Experimental Section Materials. Monoolein (1-monooleoyl glycerol, MO) was gifted from Danisco Ingredients A/S (Copenhagen, Denmark; monoglyceride content is approximately 95.7% and oleic acid content is approximately 90%). Alginic acid, sodium salt (MW 25600), stearylamine (SA, MW 269.5), (2-dodecen-1-yl) succinic anhydride (MW 266.38), pyrene, amino acid standard, Triama base, and FITC-dextran (MW 4000) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, U.S.A.). Silk fibroin (SF, Fine-Silk FD, fibroin >99%) was gifted from FINECO Ltd.(Chunchon, Korea). 2,4,6-Trinitrobenzene sulfonic acid (TNBS, 5%

10.1021/bm101249e  2011 American Chemical Society Published on Web 12/29/2010

Release from Monoolein Cubic Phase

Figure 1. Schematic diagram of cubic phase containing HmAL and HmSF. Below the isoelectric point of HmSF, the electrostatic interaction between positively charged HmSF and negatively charged HmAL will lead to the formation of complex coacervate in the water channel. Upon increasing the pH of medium across the isoelectric point, the HmSF will get negatively charged, so the coacervate will dissolve.

methanol solution; PIERCE, Rockford, IL, U.S.A.); glycine (Bio basic Inc. Markham, ON, Canada); N-(2-hydroxyethyl) piperazine-N′-(2ethanesulfonic acid) (HEPES; USB Corporation, Cleveland, OH, U.S.A.); and [2-(N-morpholino)-ethanesulfonic acid] (MES; Biopure Healing Products, LLC, Bellevue, WA, U.S.A.) were all used as received. All other reagents were of analytical grade. Preparations and Characterizations of HmAL. Alginate was hydrophobically modified by a method described elsewhere.21 In brief, 10 mL of SA solution in 2-propanol (0.026%, w/v) and 10 mL of alginate solution in distilled water (1.0%, w/v) were combined in a 50 mL round-bottom flask, and the mixture solution was stirred overnight around 50 °C. The reaction mixture was filtered through a filter paper (Whatman No. 2) and the cake was dried in a vacuum oven at 40 °C for 48 h. The FTIR spectrum of HmAL in KBr pellet was obtained on Perkin-Elmer Fourier Transformed Infrared spectrophotometer instrument (EXCALIBER series, U.S.A.). Nuclear magnetic resonance spectroscopy (1H NMR) of Hm AL was obtained on a Bruker Avance 600 (Karlsruhe, Germany) spectrometer using D2O as a solvent. The air/water interfacial activities of alginate and HmAL were determined by measuring the interfacial tensions of the solutions using a surface tension analyzer (SEO, DST 60, South Korea). The assembling phenomena of unmodified alginate and HmAL in PBS (pH 7.4) were observed by a method described in a previous work, where pyrene was used as a fluorescence probe.22-24 The intensity of the first emission peak (I1) and that of the third emission peak (I3) were determined at 379 and 384 nm, respectively, with an excitation of 338 nm. The intensity ratio (I1/I3) was plotted versus the concentrations of unmodified alginate and HmAL. Preparations and Characterizations of HmSF. SF was hydrophobically modified by a method described elsewhere.25 A total of 1 g SF was dissolved in 90 mL of distilled water and then 0.01 g (2-dodecen-1-yl) succinic anhydride was added to SF solution. The pH of reaction mixture was adjusted to 10 using 1 N NaOH solution, and the reaction was done for 5 h around 70 °C, while the reaction mixture was kept at the same pH. After the reaction mixture was cooled down to room temperature, the pH was adjusted to pH 7.0 using 1 N HCl solution. Unreacted unhydride was extracted from the reaction mixture using methyl tert-butyl ether. The extraction was performed three times. Residual methyl tert-butyl ether was removed from the raffinate phase in a rotary evaporator. The product was lyophilized in a freeze-drier (FD5508, Ilshin Lab., Yangju, Korea) for further use. The FTIR spectrum, the air/water interfacial activity, and the molecular assembling phenomena of HmSF were investigated by the same methods as used for the characterization of HmAL. The number of (2-dodecen-1-yl) succinic residues attached to one molecule of SF was assayed by TNBS method.26

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Figure 2. FTIR spectra of unmodified alginate (a) and Hm AL (b).

Observation of Complex Coacervation between HmAL and HmSF. HmAL and HmSF were separately dissolved in glycine buffer (pH 3.0) at room temperature, and the solutions were combined so that the concentration of the polymers is 1.0%, and the ratio of HmAL to HmSF was varied from 1:1 to 1:35.27 After the mixture was gently shaken at room temperature for 1 h, the size and the light scattering intensity of coacervates in the mixture were determined on a particle size analyzer (ZetaPlus 90, Brookhaven Instrument Co., U.S.A.). When the effect of pH on the degree of complex coacervation was observed, HmAL and HmSF were dissolved in buffer solution of different pH (glycine buffer (pH 3.0, 4.0, 4.5), MES buffer (pH 5.0), HEPES buffer (pH 7.0), Trizma base buffer (pH 9.0)). Preparations of Cubic Phases. MO, 2 g, was put into a vial and it was molten in a water bath and kept at 60 °C. HmAL and HmSF were codissolved in distilled water so that the concentrations were 0.077 and 1.924%, respectively. And then, FITC-dextran, a fluorescence dye, was dissolved in the solution for release experiment so that the concentration is 0.2%. A total of 0.858 g of the solution containing HmAL, HmSF, and the dye was heated up to 60 °C, and it was layered carefully over the molten MO. Then it was kept at room temperature until the buffer was completely adsorbed and clear gels were formed. The cubic phases containing FITC-dextran were prepared under dark conditions because the fluorescence dye is light-sensitive. pH-Dependent Release from Cubic Phases. Buffer solution, 5 mL, was layered carefully over the cubic phases contained in 20 mL glass vials and they were occasionally hand-shaken at room temperature under dark condition. Glycine buffer was used as a release medium for pH 3.0, 4.0, and 4.5, MES buffer was used for pH 5.0, HEPES buffer for pH 7.0, and Trizma base buffer for pH 9.0. The supernatant, 0.1 mL, was taken for the assay of dye at predetermined time intervals. In order to compensate for the reduction in the amount of release medium, the same amount of fresh buffer solution was added to each sample. FITCdextran released from the cubic phases was quantified by measuring its fluorescence intensity at 495 nm with excitation at 520 nm on a fluorescence spectrophotometer (F-2500, HITACHI, Tokyo, Japan).

Results and Discussion Preparations and Characterizations of HmAL. Figure 2 shows FTIR spectra of unmodified alginate and HmAL. In unmodified alginate spectrum, a peak found at 1040 cm-1 is due to -C-O-C- stretching, a peak at 1595 cm-1 is originated from -COOH stretching and broads peaks around 3233 cm-1 is the signal of -OH stretching. In HmAL spectrum, a peak found at 1245 cm-1 is ascribed to amide III (C-N) bending, a peak at 1380 cm-1 is the signal of -CH2- bending of alkyl chain, a strong peak at 1580 cm-1 is due to amide II (N-H) bending, and a sharp peak at 1650 cm-1 comes from amide I (CdO) stretching. The signal of methylene group of alkyl chain

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Figure 3. Designation of alginate protons for 1H NMR peak assignment.

and those of amide bonds indicate that stearylamine was successfully attached to the alginate through the formation of amide bonds. Figure 3 shows the designation of alginate protons. In NMR spectrum of HmAL, the signals can be assigned as follows: methyl protons of SA residues, 0.95; GH1′, 4.82; GH2′, 3.53; GH3′, 3.67; GH4′, 3.38; GH5′, 3.76; MH2, 3.71; MH3, 3.82; MH4, 3.96; MH5, 4.22 ppm. Based on the area of methyl signal of SA residues and that of HIG signal, the number of SA residues attached to one molecule of AL was calculated to be about 10.9. Figure 4 shows the interfacial tension of alginate solution and that of HmAL solution. As the concentration increased to 0.1%, the surface tension of alginate solution and that of HmAL solution decreased to around 50 and 40 dyn/cm, respectively. It means that the interfacial activity of HmAL is higher than that of alginate. The hydrophobic modifier, stearylamine residue, tends to bring alginate to air/water interface, due to its hydrophobicity. Thus, HmAL can be more surface-active than unmodified alginate. The critical micelle concentration of HmAL, determined by the intersection of two tangential lines, was around 0.020%. Figure 5 shows the I1/I3 ratio of pyrene in alginate solutions and HmAL solutions. I1/I3 ratio is a measure of the polarity of pyrene environment.28,29 I1/I3 ratios of the fluorescence probe in HmAL solutions were lower than those of pyrene in unmodified alginate solutions. For example, when the concentration of polymer was 0.1%, I1/I3 ratio of pyrene in alginate solutions and that of Hm alginate solution was about 1.0 and 0.8, respectively. This indicates that HmAL forms a less polar environment. Because HmAL has hydrophobic srearylamine

Figure 4. Interfacial tensions of alginate solution (b) and HmAL solution (O).

residues on their hydrophilic alginate chain, it was surfaceactive, as shown in Figure 4. Accordingly, it could readily surround pyrene molecules, giving a rise to less polar environment. This would account for why the I1/I3 ratios of pyrene in HmAL solutions were less than those of pyrene in unmodified alginate solutions. The critical micelle concentration of HmAL, determined by the intersection of two tangential lines, was found to be about 0.022% and the value is close to CMC obtained in the measurement of interfacial activity. Preparations and Characterizations of HmSF. Figure 6 shows FTIR spectra of unmodified SF and HmSF.30 In unmodified SF spectrum, strong peaks found at 1250, 1531, and 1644

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Figure 5. I1/I3 ratio of pyrene in alginate solution (b) and HmAL solution (O).

Figure 7. Interfacial tensions of SF solution (b) and HmSF solution (O).

Figure 6. FTIR spectra of unmodified SF (a) and HmSF (b).

Figure 8. I1/I3 ratio of pyrene in SF solution (b) and HmSF solution (O).

cm-1 are assigned to amide III (C-N) stretching, amide II (N-H) bending, and amide I (CdO) stretching, respectively. In the HmSF spectrum, characteristic peaks of the amide bond were observed. In addition, -CH3- and -CH2- bending of alkyl chain of (2-dodecen-1-yl) succinic acid residues was found at 1375 and 2921 cm-1, indicating that (2-dodecen-1-yl) succinic acid was successfully attached to SF. In TNBS method, the amino acid standard curve was set up and it was expressed as A335 ) 30.9970M + 0.0265 (R2 ) 0.9968), where A335 is the absorbance at 335 nm and M is the molar concentration of amino acids. The absorbance of enzyme solutions is proportional to the number of reactive lysine residues of the enzymes, because the TNBS method detects reactive amino groups.26,31 The absorbance of native SF solution was 0.174 and it was calculated to be 24.2 reactive amino groups per native SF molecule. The absorbance of HmSF solution was 0.102, corresponding to 12.4 reactive amino groups per HmSF molecule. This means that about 12 dodecenyl succinic acids were covalently attached to one SF molecule. Figure 7 shows the interfacial tensions of SF solution and HmSF solution. The surface tension of HmSF solution was much lower than that of SF solution at all the concentrations. For example, when the concentration was 0.2%, the interfacial tension of SF and that of HmSF were about 58 and 37 dyn/cm, respectively. HmSF is a kind of polymeric amphiphiles, because hydrophobic modifiers (dodecenyl succinic acid residues) were covalently attached to SF. This would account for why the interfacial activity of HmSF was much higher than unmodified SF. The CMC of HmSF, determined by the intersection of two tangential lines, was around 0.037%. Figure 8 shows the I1/I3

ratio of pyrene in SF solutions and HmSF solution. The ratio I1/I3 ratio in HmSF solutions was lower than that in SF solution at all the concentrations. It means that HmSF provides a more hydrophobic environment than the SF dose. This is possibly because HmSF has a hydrophobic modifier (dodecenyl succinic acid residue) on hydrophilic SF chain so it would act as a polymeric surfactant. The CMC of HmSF, determined by the intersection of two tangential lines, was close to CMC obtained in the measurement of interfacial activity. Determination of HmAL/HmSF Ratio for Maximum Complexation. Figure 9 shows the mean diameter and the light scattering intensity of HmAL/HmSF complexes formed at pH 3.0. The diameter was 300-400 nm at all the ratio of HmAL/ HmSF except the ratio of 1:25, where the diameter was about 581.2 nm. The light scattering intensity also exhibited its maximum value at the ratio of 1:25. The diameter is a measure of the number of polymers constituting one complex particle (so-called aggregation number), and the light scattering intensity is proportional to not only the size but also the number of complex particles (scattering centers). Accordingly, it is reasonable that maximum complexation occurs at the ratio of 1:25. The maximum complex coacervation will be obtained when the number of positive charge point is the same as the number of negative charge point. Because the ratio of sodium carboxylate to carboxylic group in alginate is about 7:3, the number of negative charge points were calculated to be about 46 per one molecule of HmAL (MW of alginate ) 25600, MW of glucopyranose unit ) 388, % salt form of carboxylic group ) 70%). On the other hand, the number of positively charged

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Figure 11. FITC-dextran release from polymer-free cubic phase at pH 3.0 (b), 4.0 (O), 4.5 (1), 5.0 (∆), 7.0 (9), and 9.0 (0). Figure 9. Mean diameter (bar) and light scattering intensity (b) of HmAL/HmSF complexes formed at pH 3.0 at various ratios of HmAL to HmSF. Mean diameter reads x-axis and light scattering intensity reads y-axis.

Figure 12. FITC-dextran release from HmAL/HmSF-loaded cubic phase at pH 3.0 (b), 4.0 (O), 4.5 (1), 5.0 (∆), 7.0 (9), and 9.0 (0). Figure 10. Mean diameter (bar) and light scattering intensity (b) of HmAL/HmSF (1:25) complexes at various pHs. Mean diameter reads x-axis and light scattering intensity reads y-axis.

amino acid residues was about 24 per one molecule of SF, and the number of dodecenyl succinic acid residues attached to one molecule of SF was about 12. Thus, the number of positive charge points was approximated to be about 12, assuming that all the amino groups are protonated at pH 3.0 (which is far below the pKb of amino groups),27 and the average molecular weight of amino acid residue is 200. Because the calculated molar ratio of negative charge point to positive charge point was 46:12, the molar ratio of HmAL to HmSF should be 1:3.8 to equalize the number of positive charge points to that of negative charge points. The HmAL to HmSF molar ratio of 1:3.8 corresponds to the weight ratio of 1:8.4. The calculated weight ratio for maximum complexation deviated from the observed weight ratio (1:25). SF could form R helix and β sheet, so the positively charged amino groups can be sequestered in the structures.32 This could be one of reasons why the observed amount of HmSF for maximum complexation was higher than the calculated amount. Figure 10 shows the mean diameter and the light scattering intensity of HmAL/HmSF (1:25) complexes at various pHs. The diameter and the light scattering intensity dramatically decreased when the pH of medium increased from 3.0 to 5.0. As the pH increases, HmSF will become negatively charged so the complex coacervate will be dissolved due to the disappearance of the intermolecular electrostatic interaction. This would be responsible for the decrease in the size and the light scattering intensity.

Release of FITC-Dextran. Figure 11 shows the degree of FITC-dextran release from polymer-free cubic phase at various pHs. The release steadily increased with time, but the release was so slow that the degree of release in 100 h was less than 10%. For the same period, the release of amaranth from MO cubic phase was about 30-50%.20 Molecular weight of diffusate is one of major factors to influence the release rate, because the diffusion coefficient is a function of the size of diffusate. The molecular weight of FITC-dextran, 4000, was about 8 times higher than that of amaranth, 600, and the high molecular weight may account for the reason the degree of release was so low. There was no significant difference in % release whatever the pH of release medium was. Monoolein cubic phase itself has no inoizable groups and it will hardly respond to change in the pH of release medium. Figure 12 shows the degree of FITC dextran release from HmAL/HmSF-loaded cubic phase at various pHs. The degree of release increased with increasing pH of release medium. For example, the % releases in 100 h at pH 3.0, 4.0, 4.5, 5.0, 7.0, and 9.0 were 2.42, 3.14, 3.62, 4.72, 6.63, and 7.20%, respectively. Below the isoelectric point of HmSF, the electrostatic interaction between positively charged HmSF and negatively charged HmAL will lead to the formation of complex coacervate (Figures 9 and 10) in the water channel. As a result, the water channel will be closed by the coacervate, resulting in a suppressed release. Upon increasing the pH of medium across the isoelectric point, the HmSF will get negatively charged, so the coacervate will dissolve (Figure 10). Mass transfer resistance in the water channel will decrease, leading to a higher release.

Release from Monoolein Cubic Phase

A reason why we chose FITC-dextran (MW 4000) as a model molecule for release experiment is that the cubic phase containing HmSF and HmAL could be used as a pH-sensitive vehicle for the oral delivery of acid-labile polymeric drugs.

Conclusions Under acidic condition, maximum complex coacervation was observed at the HmAL to HmSF ratio of 1:25. HmAL and HmSF were included in the water channel of the MO cubic phase. The release of FITC-dextran from the cubic phase increased with the pH of the release medium. The release was suppressed under acidic conditions (e.g., pH 3.0 and 4.0) possibly due to the formation of coacervate in the water channel. Higher degrees of release were observed under neutral and alkali conditions, possibly due to the dissolution of complex coacervation. The cubic phase containing HmSF and HmAL could be used as a pH-sensitive vehicle for the oral delivery of an acid-labile drug (e.g., insulin). Acknowledgment. Following are results of a study on the “Human Resource Development Center for Economic Region Leading Industry” Project, supported by the Ministry of Education, Science & Technology (MEST) and the National Research Foundation of Korea (NRF).

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