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Entrapment of capsaicin loaded nanoliposome in pH responsive hydrogel beads for colonic delivery
Journal of Drug Delivery Science and Technology 39 (2017) 417e422
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Entrapment of capsaicin loaded nanoliposome in pH responsive hydrogel beads for colonic delivery Tapan Kumar Giri*, Soumick Bhowmick, Subhasis Maity NSHM College of Pharmaceutical Technology, NSHM Knowledge Campus, Kolkata Group of Institutions, 124 BL Saha Road, Kolkata 700053, West Bengal, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 January 2017 Received in revised form 9 March 2017 Accepted 2 May 2017 Available online 4 May 2017
Liposomes loaded with capsaicin were encapsulated in alginate hydrogel beads followed by coating with Eudragit S-100 for efficient oral colonic delivery. Liposomes were prepared by thin film hydration method using soyalecithin and cholesterol. Hydrogel beads encapsulating liposome were prepared by iontropic gelation methods which were Eudragit coated. The liposomes and hydrogel beads were characterized for size, entrapment efficiency and in vitro drug release. Coated hydrogel bead has shown better drug release after 5 h and has shown 99.3% drug release till 24 h in a controlled manner in comparison to plain hydrogel beads, which released more than 65% of the drug before entering into the colonic region. We can conclude that coated alginate hydrogel beads containing liposome can be used as a potential carrier for colonic drug delivery. © 2017 Elsevier B.V. All rights reserved.
Keywords: Liposomes Capsaicin Hydrogel beads Alginate Colonic delivery
1. Introduction Capsaicin is an alkaloid responsible for the “hot and spicy” taste of chilli peppers and pepper extracts [1e3]. Capsaicin had strong effectiveness on relieving pain and initially been used to treat a diversity of neuropathic pain including diabetic neuropathy, rheumatoid arthritis, and herpes zoster [4]. Excluding above mentioned therapeutic value, a number of studies also have established chemopreventive activity of capsaicin in various human cancer models. It diminished the growth of gastric, leukemic, prostate, nasopharyngeal, hepatic, and colon carcinoma cells in vitro as it persuade cell apoptosis and mediate cell cycle arrest [5e10]. Capsaicin induces apoptosis of the human colorectal cells which respond to NAG-1 (nonsteroidal anti-inflammatory drug-activated gene-1) [11]. Another mechanism suggested for its anticancer activity is the reduced production of b-catenin gene which is responsible for coordination of cellecell adhesion and gene transcription [12]. The clinical use of capsaicin is handicapped through quick firstpass metabolism and a short half-life of less than 8 min following intravenous administration [13]. Poor aqueous solubility of
* Corresponding author. E-mail address: [email protected] (T.K. Giri). http://dx.doi.org/10.1016/j.jddst.2017.05.002 1773-2247/© 2017 Elsevier B.V. All rights reserved.
capsaicin led to low absorption and poor bioavailability through oral administration. It is extremely irritating compounds causing pain and burning on skin and mucosa in low concentrations. Additionally, a continuing use through the gastrointestinal tract is restricted owing to pungency of capsaicin [14]. Moreover, when administered orally, capsaicin causes increased salivation, gastric secretion, intense burning sensation, and sometimes gastrointestinal disorders depending on the dose [15]. When administered to human colorectal cell line, inhibitory concentration of capsaicin was found to be 15.25 mg/ml (50 mM), which indicates a fairly large dose will be required to inhibit the carcinogenic process in vivo [16]. A pharmacokinetic study in male adults have shown blood concentration of capsaicin after oral administration (0.4 mg capsaicin per kg body wt) is 2.47 ng/ml (equivalent to 8.1 nM) [17]. This indicates to be effective, the drug should be administered at the site of absorption at continual basis, which can be achieved by targeting the drug in colon. Colonic region of the gastrointestinal tract offers advantages than the stomach and small intestine, e.g. lower enzymatic activity, lower bile salt concentrations, milder pH, and prolonged residence time. Conversely, colonic drug delivery is allied with various obstacles including dosage form journey through regions of high acidity and digestive activity. In the past few years, liposomes have been used for the delivery of anticancer drug. Liposomes are phospholipid vesicles that have enormous prospective as drug delivery carriers and have been
418
T.K. Giri et al. / Journal of Drug Delivery Science and Technology 39 (2017) 417e422
widely investigated in the past [18]. Hydrophobic drugs can be entrapped into the bilayer of liposomes and hydrophilic drugs can be incorporated in the inner aqueous phase. They can interact with tissue of colon and get usefulness drug delivery in colonic region [19]. However, liposome degraded in the hostile milieu of the gastrointestinal tract which include enzymes (pancreatic lipases) and bile salts and that would dissolve the lipid bilayer [20e22]. However, there is minute information on how they could delivered the drug to the colon and susceptibility to liposome digestion while traveling through the gastrointestinal tract. Coating them with a polymer is one way that may protect them during transit, but very little work has been done on specifically targeting the colonic region. This work aimed to develop liposome encapsulated alginate beads coated with Eudragit S-100 as a carrier for colon targeting of capsaicin.
2.4. Preparation of coated beads containing liposome Eudragit S100 solution (2.0%, w/v) was prepared by dissolving 2.0 g of Eudragit S100 in 100 ml methanol. Then wet calcium alginate beads were transferred into Eudragit S100 solution and kept for 30 min under gentle magnetic stirring. The resulting coated beads were collected and rinsed with double distilled water and dried in an air overnight. Then the coated beads were dried in an oven at 45 C for 2e3 h to constant weight. 2.5. Measurement of particle size and zeta potential of liposome Particle size and zeta potential of the liposome was determined using particle size analyzer (Zetasizer nano ZS, Malvern, UK). 2.6. Determination of drug entrapment efficiency of liposome
2. Materials and methods 2.1. Materials Capsaicin was obtained from Naturite Agro Products Ltd. (Hyderabad, India). Soyalecithin and cholesterol were procured from HiMedia Laboratories Pvt. Ltd. (Mumbai, India). Eudrajit S-100 was purchased from Balaji Drugs, India and Sodium alginate was obtained from Loba chemie, India. All other ingredients used in the study were of analytical grade. 2.2. Preparation of liposome Liposomes were prepared by thin film hydration method reported earlier [23]. Weighted quantity of drug and lipids were taken into a round bottom flask and chloroform was used to dissolve drug and lipid. Thin lipid film was prepared by evaporating chloroform using rotary vacuum evaporator (PMTC-3040; Superfit, India). Then the flask was kept overnight in desiccator for complete elimination of the solvent. 20 ml of phosphate buffer (pH 7.4) was added to the flask and dispersion of the thin lipid film was done for 4 h at 55e60 C using rotary evaporator. The dispersion was sonicated using sonicator (UD200SH-6L; Takashi, Japan) for 30 min at 55e60 C and then the suspension was kept at room temperature for 1 h for the formation of vesicles. Liposomal suspension was centrifuged at 4 C and 15000 rpm using cooling centrifuge (C24BL; REMI, India) for 15 min to separate unentrapped capsaicin and liposomal vesicles were sediment as pellets. Then supernatant was separated from pellets and were resuspended with phosphate buffer solution pH 7.4 and centrifuged in same conditions. This process was repeated three times for complete removal of the unentrapped drug. Finally, the pellets were suspended in 20 ml phosphate buffer solutions pH 7.4 and stored in refrigerator at 4 C for further use.
The entrapped capsaicin containing liposomes are separated from unentrapped capsaicin by cooling centrifugation of a known aliquot (10 ml) of the prepared liposomal suspension at 15000 rpm for 60 min at 4 C. The supernatant was separated from the liposomal precipitate. Unentrapped drug was determined by diluting 1 ml of supernatant in 9 ml of methanol, followed by the measurement of absorbance of these solutions spectrometrically (UV1800, Shimadzu, Japan) at 279 nm wavelength. The entrapment efficacy percent was determined using the following equation:
Entrapment efficiency percent ¼ ½ðA BÞ=A 100 where A is the concentration of total capsaicin added and B is the concentration of free capsaicin detected. 2.7. Fourier transforms infrared spectroscopy study Fourier transform infrared spectra of pure capsaicin, physical mixture of capsaicin and excipients, and liposomal formulation were recorded in Fourier transform infrared spectroscopy (ALPHAT, Bruker, Germany) using KBr pellets. Spectra were recorded between 4000 and 500 cm1with a spectral resolution of 4 cm1 [24]. 2.8. Differential scanning calorimetry study Thermal behaviour of capsaicin, physical mixture of capsaicin and excipients, and liposomal formulation containing capsaicin were analyzed using differential scanning calorimeter (Pyris Diamond TG/DTA, Perkin Elmer, Singapore). The samples were heated at 15 C/min from 35 C to 250 C [25]. 2.9. Determination of particle size of beads The size of alginate and coated alginate beads were determined by optical microscopy.
2.3. Preparation of hydrogel beads containing liposome Required quantity of sodium alginate was taken and dissolves properly into 10 ml water to make a solution. Then 10 ml of liposomal suspension (approx 4 mg capsaicin) was mixed with sodium alginate solution such that final concentration of the solution was (2% w/v) of sodium alginate. Sodium alginate containing liposomal suspension was dropped through a syringe (22 gauge) into calcium chloride solution (2% w/v) with mild agitation for 15 min. The calcium chloride solutions were decanted and the beads were washed three times with doubled distilled water. The formed beads were air dried overnight and then dry in an oven at 45 C for 2e3 h to constant weight.
2.10. Determination of drug entrapment efficiency of hydrogel beads and coated hydrogel beads 20 mg of beads were taken in beaker containing 100 ml of pH 7.4 phosphate buffer solution and stirred 4 h in a magnetic stirrer. Similarly, coated hydrogel beads were taken in a beaker containing methanol instead of pH 7.4 phosphate buffer solutions. Then sonication was done for 30 min. After that, the absorbance of the buffer solution containing the extracted amount of capsaicin was taken at a wavelength of 279 nm in a UV spectrophotometer using pure phosphate buffer as a blank. The percentage of entrapment efficiency was then calculated as [26]:
T.K. Giri et al. / Journal of Drug Delivery Science and Technology 39 (2017) 417e422
Entrapment efficiency % ¼ ðActual drug content=theoretical drug contentÞ 100
2.11. In vitro drug release study The in vitro release profile of capsaicin from liposomal and hydrogel formulations were determined using the dialysis bag method [27]. The design of using the simulated fluids at different pH was as follows: simulated gastric fluid of pH 1.2 (1 h), mixture of simulated gastric and intestinal fluid of pH 4.5 (2e3 h), simulated intestinal fluid of pH 7.4 (4e5 h), and simulated colonic fluid pH (remaining time). 4 mL of liposomal suspension or 20 mg beads suspended in 4 ml water were taken into a dialysis bag (Himedia dialysis membrane-60, Mumbai, India) and hung inside the beaker containing simulated fluid. The content of beaker was rotated at 300 rpm with a magnetic bead at 37 C. The simulation of gastrointestinal tract conditions was achieved by altering the pH of the dissolution medium at various time intervals. Aliquots of the dissolution medium were withdrawn at predetermined time intervals and compensated with the same volume of fresh dissolution media and the amount of drug was quantified by UV spectrophotometer at 279 nm wavelength. 3. Results and discussion 3.1. Development and characterization of liposome Capsaicin loaded liposomes were prepared using soyalecithin and cholesterol by thin film hydration method. The average vesicle size of capsaicin loaded liposome was 284.1 nm (Fig. 1). Liposomal formulation showed the zeta potential value of 61 mV. High zeta potential (>±30 mV) can offer an electric repulsion which prevents aggregation of particles and give the stability of the colloidal system [28]. The capsaicin loaded liposomes were anticipated to have good physical stability as zeta potential (61 mV) was less than 30 mV. The entrapment efficiency of capsaicin in liposome was found to be 35 ± 5%. 3.2. Fourier transforms infrared spectroscopy study Fourier transform infrared spectrum of capsaicin, physical mixture of drug and excipients and drug loaded liposome were
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represented in Fig. 2. Capsaicin shows peak at 3313.58 cm1 which is associated with broadened O-H and N-H stretch (Fig. 2a). Stretching peak of aliphatic C-H bonds shown at 2861.38 cm1. The peak at 1630.18 cm1 was attributed to olefinic C¼O stretch, C¼C stretch, and amide II. N-H bends and C-N stretch peak of capsaicin shown at 1515.85 cm1. The peak at 1283.29 cm1 represents stretching of C-N bonds. Physical mixture of drug and excipients showed approximately analogous spectra of capsaicin (Table 1). Liposomal complex containing capsaicin shows the minor changing of a few peaks in comparison to pure capsaicin and physical mixture containing capsaicin (Table 1). The minor shifts of peaks may be due to the creation of hydrogen bonds or weak forces between drug and phospholipids. 3.3. Differential scanning calorimetry studies The differential scanning calorimetry thermograms of capsaicin, physical mixture containing capsaicin and liposomes containing capsaicin are represented in Fig. 3. Capsaicin showed a sharp melting endotherm at 69.72 C (Fig. 3a). The endothermic peak of capsaicin also was appeared in the graph of the physical mixture containing capsaicin with diminished intensity (Fig. 3b) may be due to dilution of drug with lipid. This suggests that there was no interaction between drug and lipid on simple mixing. The melting peak of capsaicin almost entirely disappeared in liposome loaded with capsaicin. This established that the crystalline nature of the drug was retained in the physical mixture but lost in the liposomal formulation. This indicates possible solid state transformation of the drug. 3.4. Development and characterization of alginate hydrogel beads containing capsaicin loaded liposome Liposomes are the most ideal carriers to colon as hydrophobic drug promotes absorption into colonic region. Nevertheless, liposomes often suffer from the problem of instability in harsh gastrointestinal environment. On the contrary polymeric hydrogel beads are more stable in harsh gastrointestinal environment. Therefore, hydrogel beads loaded with liposome are anticipated as an ideal drug delivery carrier. Alginate is a natural anionic polymer and has a distinctive property of gel formation in the existence of multivalent cations. However, fast release of drug occurs before reaching into the colon due to porosity of the alginate beads. So Eudragit S100 was selected as enteric coating material for coating of
Fig. 1. Particle size distribution of liposome.
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T.K. Giri et al. / Journal of Drug Delivery Science and Technology 39 (2017) 417e422
Fig. 2. Fourier transform infrared spectrum of a) capsaicin b) physical mixture of capsaicin and excipients c) Liposomal complex of capsaicin and excipients.
alginate hydrogel beads due to no release or less amount of release of encapsulated drug at the stomach as well as in the small intestine. When a dispersion of liposomal suspension containing capsaicin and alginate was extruded through the needle into a solution
containing calcium chloride, the beads were formed instantaneously. The produced alginate beads are represented in Fig. 4. The hydrogel beads are more or less spherical having smooth surface. The mean diameters of alginate hydrogel beads and coated alginate hydrogel beads were 1.213 ± 0.218 mm and 1.362 ± 0.180 mm,
T.K. Giri et al. / Journal of Drug Delivery Science and Technology 39 (2017) 417e422
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Table 1 Comparison of FTIR spectra of capsaicin functional groups. Functional groups
Capsaicin
Broadend O-H and N-H stretch Aliphatic C-H stretch Olefinic C¼C stretch, C¼O stretch, amide II N-H bend and C-N stretch, amide II C-N stretch
3313.58 2861.38 1630.18 1515.85 1283.29
cm1 cm1 cm1 cm1
Physical mixture containing capsaicin 3316.73 2863.48 1630.20 1516.02 1282.59
cm1 cm1 cm1 cm1 cm1
Liposome containing capsaicin 3320.31 2857.74 1631.13 1516.44 1282.14
cm1 cm1 cm1 cm1 cm1
Fig. 3. DSC thermo grams capsaicin (a), physical mixture (b), and drug loaded liposome (c).
maximum 2 h to complete release in simulated gastric fluid. It was observed that in case of capsaicin loaded liposomal formulation 100% drug released in 5 h. In the case of liposomal formulation entrapped in alginate beads, drug release was found to be 99.8% at the end of 8 h. In the case of the coated alginate beads containing liposomal formulation, drug release was found to be 99.3% at the end of 24 h. The values of t85% (i.e. time required for the release of 85% drug) were calculated as 190 min, 285 min, 900 min, respectively in the liposome, alginate beads containing liposome, and Eudragit coated alginate beads containing liposome. After 5 h of invitro study, in case of capsaicin loaded liposomal formulation 99% drug was released and for alginate hydrogel beads 88% drug was released. However, coated alginate hydrogel beads released about 30% drug before reaching colonic region.
4. Conclusion
Fig. 4. Alginate hydrogel beads.
respectively. The drug entrapment efficiency of alginate hydrogel beads and coated alginate hydrogel beads were 85 ± 1.15% and 82.9 ± 1.31%, respectively. 3.5. In vitro drug release study The cumulative percentage drug releases from different formulations in various simulated gastrointestinal fluids were represented in Fig. 5. in vitro release study of capsaicin shows that it takes
Capsaicin loaded liposome was successfully prepared and then incorporated into alginate hydrogel beads and finally coated with anionic polymer Eudragit S-100. Fourier transform infrared spectroscopy analysis showed that there was slight interaction of drug capsaicin with other excipients. Differential scanning calorimetry study showed that drug was molecularly dispersed into the matrix. Eudragit coated alginate beads allow a minimum drug release (<30%) in stomach and intestine and released maximum amount of drug (>70%) into colonic region. From in vitro release study we can conclude that Eudragit coated liposomal formulation can carry drug through gastrointestinal tract with very low release of drug in the stomach and intestine region and showed greater and control release of the drug into the colonic region for a longer period of time.
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Fig. 5. Cumulative percentage drug release profile from different formulations.
References [1] M. Almeida, J.M. Nadal, S. Grassiolli, K.S. Paludo, S.F. Zawadzki, L. Cruz, J.P. Paula, P.V. Farago, Enhanced gastric tolerability and improved anti-obesity effect of capsaicinoids-loaded PCL microparticles, Mater Sci. Eng. C 40 (2014) 345e356. [2] T.K. Giri, P. Mukherjee, T.K. Barman, S. Maity, Nano-encapsulation of capsaicin on lipid vesicle and evaluation of their hepatocellular protective effect, Int. J. Biol. Macromol. 88 (2016) 236e243. [3] T.K. Giri, A. Alexander, T. Ajazuddin, K. Barman, S. Maity, Infringement of the barriers of cancer via dietary phytoconstituents capsaicin through novel drug delivery system, Curr. Drug Deliv. 13 (2016) 27e39. [4] J. Jin, G. Lin, H. Huang, D. Xu, H. Yu, X. Ma, L. Zhu, D. Ma, H. Jiang, Capsaicin mediates cell cycle arrest and apoptosis in human colon cancer cells via stabilizing and activating p53, Int J. Biol. Sci. 10 (2014) 285e295. [5] K. Ito, T. Nakazato, K. Yamato, Y. Miyakawa, T. Yamada, N. Hozumi, K. Segawa, Y. Ikeda, M. Kizaki, Induction of apoptosis in leukemic cells by homovanillic acid derivative, capsaicin, through oxidative stress: implication of phosphorylation of p53 at Ser-15 residue by reactive oxygen species, Cancer Res. 64 (2004) 1071e1078. [6] H.C. Huh, S.Y. Lee, S.K. Lee, N.H. Park, I.S. Han, Capsaicin induces apoptosis of cisplatin-resistant stomach cancer cells by causing degradation of cisplatininducible Aurora-A protein, Nutr. Cancer 63 (2011) 1095e1103. [7] S.W. Ip, S.H. Lan, H.F. Lu, A.C. Huang, J.S. Yang, J.P. Lin, H.Y. Huang, J.C. Lien, C.C. Ho, C.F. Chiu, W. Wood, J.G. Chung, Capsaicin mediates apoptosis in human nasopharyngeal carcinoma NPC-TW 039 cells through mitochondrial depolarization and endoplasmic reticulum stress, Hum. Exp. Toxicol. 31 (2012) 539e549. [8] A. Mori, S. Lehmann, J. O'Kelly, T. Kumagai, J.C. Desmond, M. Pervan, W.H. McBride, M. Kizaki, H.P. Koeffler, Capsaicin, a component of red peppers, inhibits the growth of androgen-independent, p53 mutant prostate cancer cells, Cancer Res. 66 (2006) 3222e3229. [9] S.P. Huang, J.C. Chen, C. CWu, C.T. Chen, N.Y. Tang, Y.T. Ho, C. Lo, J.P. Lin, J.G. Chung, J.G. Lin, Capsaicin-induced apoptosis in human hepatoma HepG2 cells, Anticancer Res. 29 (2009) 165e174. [10] Y.M. Kim, J.T. Hwang, D.W. Kwak, Y.K. Lee, O.J. Park, Involvement of AMPK signaling cascade in capsaicin-induced apoptosis of HT-29 colon cancer cells, Ann. N. Y. Acad. Sci. 1095 (2007) 496e503. [11] S.H. Lee, C. Krisanapun, S.J. Baek, NSAID-activated gene-1 as a molecular target for capsaicin-induced apoptosis through a novel molecular mechanism involving GSK3b, C/EBPb ATF3, Carcinog. 31 (2010) 719e728. [12] S.H. Lee, R.L. Richardson, R.H. Dashwood, S.J. Baek, Capsaicin represses transcriptional activity of b-catenin in human colorectal cancer cells, J. Nutr. Biochem. 23 (2012) 646e655. [13] L. Tavano, P. Alfano, R. Muzzalupo, B. de Cindio, Niosomes vs microemulsions: new carriers for topical delivery of capsaicin, Colloids Surf. B Biointerfaces 87 (2011) 333e339.
[14] M. Hayman, P.C.A. Kam, Capsaicin: a review of its pharmacology and clinical applications, Curr. Anaesth. Crit. Care 19 (2008) 338e343. [15] V.S. Govindarajan, M.N. Sathyanarayana, Capsicum-production, technology, chemistry, and quality. Part V. Impact on physiology, pharmacology, nutrition, and metabolism; structure, pungency, pain, and desensitization sequences, Crit. Rev. Food Sci. Nutr. 29 (1991) 435e473. [16] S.H. Lee, C. Krisanapun, S.J. Baek, NSAID activated gene-1 as a molecular target for capsaicin induced apoptosis through a novel molecular mechanism involving GSK3b, C/EBP b ATF3, Carcinog. 4 (2010) 719e728. [17] K. Chaiyasit, W. Khovidhunkit, S. Wittayalertpanya, Pharmacokinetic and the effect of capsaicin in Capsicum frutescens on decreasing plasma glucose level, J. Med. Assoc. Thai 92 (2009) 108e113. [18] T.K. Giri, A. Giri, T.K. Barman, S. Maity, Nanoliposome is a promising carrier of protein and peptide biomolecule for the treatment of cancer, Anticancer Agents Med. Chem. 16 (2016) 816e831. [19] M.J. Barea, I.M.J. Jenkins, Y.S. Lee, I.P. Johnson, R.H. Bridson, Encapsulation of liposomes within pH responsive microspheres for oral colonic drug delivery, Int. J. Biomater. 2012 (2012) 1e8. [20] P.R. Karn, Z.P. Vanic, N. Skalko-Basnet, Mucoadhesive liposomal delivery systems: the choice of coating material, Drug Dev. Ind. Pharm. 37 (2011) 482e488. [21] K. Iwanaga, S. Ono, K. Narioka, M. Kakemi, K. Morimoto, S. Yamashita, Y. Namba, N. Oku, Application of surface-coated liposomes for oral delivery of peptide: effects of coating the liposome's surface on the GI transit of insulin, J. Pharm. Sci. 88 (1999) 248e252. [22] H. Takeuchi, Y. Matsui, H. Yamamoto, Y. Kawashima, Mucoadhesive properties of carbopol or chitosan-coated liposomes and their effectiveness in the oral administration of calcitonin to rats, J. Control Rel 86 (2003) 235e242. [23] B. Mukherjee, B. Patra, B. Layek, A. Mukherjee, Sustained release of acyclovir from nano-liposomes and nano-niosomes: an in vitro study, Int. J. Nanomed. 2 (2007) 213e225. [24] T.K. Giri, U. Verma, D.K. Tripathi, Effect of adsorption parameters on biosorption of Znþþ ions from aqueous solution by graft copolymer of locust bean gum and polyacrylamide, Indian J. Chem. Technol. 23 (2016) 93e103. [25] T.K. Giri, S. Vishwas, D.K. Tripathi, Synthesis of grafted locust bean gum using vinyl monomer and studies of physicochemical properties and acute toxicity, J Nat. Prod. 6 (2016) 1e9. [26] T.K. Giri, A. Thakur, D.K. Tripathi, Biodegradable hydrogel bead of casein and modified xanthan gum for controlled delivery of theophylline, Curr. Drug Ther. 11 (2016) 150e162. [27] D. Bansal, A. Gulbake, J. Tiwari, S.K. Jain, Development of liposomes entrapped in alginate beads for the treatment of colorectal cancer, Int. J. Biol. Macromol. 82 (2016) 687e695. [28] M.H. Subudhi, A. Jain, A. Jain, P. Hurkat, S. Shilpi, A. Gulbake, S.K. Jain, Eudragit S100 coated citrus pectin nanoparticles for colon targeting of 5-fluorouracil, Materials 8 (2015) 833e849.
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Entrapment of capsaicin loaded nanoliposome in pH responsive hydrogel beads for colonic delivery Tapan Kumar Giri*, Soumick Bhowmick, Subhasis Maity NSHM College of Pharmaceutical Technology, NSHM Knowledge Campus, Kolkata Group of Institutions, 124 BL Saha Road, Kolkata 700053, West Bengal, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 January 2017 Received in revised form 9 March 2017 Accepted 2 May 2017 Available online 4 May 2017
Liposomes loaded with capsaicin were encapsulated in alginate hydrogel beads followed by coating with Eudragit S-100 for efficient oral colonic delivery. Liposomes were prepared by thin film hydration method using soyalecithin and cholesterol. Hydrogel beads encapsulating liposome were prepared by iontropic gelation methods which were Eudragit coated. The liposomes and hydrogel beads were characterized for size, entrapment efficiency and in vitro drug release. Coated hydrogel bead has shown better drug release after 5 h and has shown 99.3% drug release till 24 h in a controlled manner in comparison to plain hydrogel beads, which released more than 65% of the drug before entering into the colonic region. We can conclude that coated alginate hydrogel beads containing liposome can be used as a potential carrier for colonic drug delivery. © 2017 Elsevier B.V. All rights reserved.
Keywords: Liposomes Capsaicin Hydrogel beads Alginate Colonic delivery
1. Introduction Capsaicin is an alkaloid responsible for the “hot and spicy” taste of chilli peppers and pepper extracts [1e3]. Capsaicin had strong effectiveness on relieving pain and initially been used to treat a diversity of neuropathic pain including diabetic neuropathy, rheumatoid arthritis, and herpes zoster [4]. Excluding above mentioned therapeutic value, a number of studies also have established chemopreventive activity of capsaicin in various human cancer models. It diminished the growth of gastric, leukemic, prostate, nasopharyngeal, hepatic, and colon carcinoma cells in vitro as it persuade cell apoptosis and mediate cell cycle arrest [5e10]. Capsaicin induces apoptosis of the human colorectal cells which respond to NAG-1 (nonsteroidal anti-inflammatory drug-activated gene-1) [11]. Another mechanism suggested for its anticancer activity is the reduced production of b-catenin gene which is responsible for coordination of cellecell adhesion and gene transcription [12]. The clinical use of capsaicin is handicapped through quick firstpass metabolism and a short half-life of less than 8 min following intravenous administration [13]. Poor aqueous solubility of
* Corresponding author. E-mail address: [email protected] (T.K. Giri). http://dx.doi.org/10.1016/j.jddst.2017.05.002 1773-2247/© 2017 Elsevier B.V. All rights reserved.
capsaicin led to low absorption and poor bioavailability through oral administration. It is extremely irritating compounds causing pain and burning on skin and mucosa in low concentrations. Additionally, a continuing use through the gastrointestinal tract is restricted owing to pungency of capsaicin [14]. Moreover, when administered orally, capsaicin causes increased salivation, gastric secretion, intense burning sensation, and sometimes gastrointestinal disorders depending on the dose [15]. When administered to human colorectal cell line, inhibitory concentration of capsaicin was found to be 15.25 mg/ml (50 mM), which indicates a fairly large dose will be required to inhibit the carcinogenic process in vivo [16]. A pharmacokinetic study in male adults have shown blood concentration of capsaicin after oral administration (0.4 mg capsaicin per kg body wt) is 2.47 ng/ml (equivalent to 8.1 nM) [17]. This indicates to be effective, the drug should be administered at the site of absorption at continual basis, which can be achieved by targeting the drug in colon. Colonic region of the gastrointestinal tract offers advantages than the stomach and small intestine, e.g. lower enzymatic activity, lower bile salt concentrations, milder pH, and prolonged residence time. Conversely, colonic drug delivery is allied with various obstacles including dosage form journey through regions of high acidity and digestive activity. In the past few years, liposomes have been used for the delivery of anticancer drug. Liposomes are phospholipid vesicles that have enormous prospective as drug delivery carriers and have been
418
T.K. Giri et al. / Journal of Drug Delivery Science and Technology 39 (2017) 417e422
widely investigated in the past [18]. Hydrophobic drugs can be entrapped into the bilayer of liposomes and hydrophilic drugs can be incorporated in the inner aqueous phase. They can interact with tissue of colon and get usefulness drug delivery in colonic region [19]. However, liposome degraded in the hostile milieu of the gastrointestinal tract which include enzymes (pancreatic lipases) and bile salts and that would dissolve the lipid bilayer [20e22]. However, there is minute information on how they could delivered the drug to the colon and susceptibility to liposome digestion while traveling through the gastrointestinal tract. Coating them with a polymer is one way that may protect them during transit, but very little work has been done on specifically targeting the colonic region. This work aimed to develop liposome encapsulated alginate beads coated with Eudragit S-100 as a carrier for colon targeting of capsaicin.
2.4. Preparation of coated beads containing liposome Eudragit S100 solution (2.0%, w/v) was prepared by dissolving 2.0 g of Eudragit S100 in 100 ml methanol. Then wet calcium alginate beads were transferred into Eudragit S100 solution and kept for 30 min under gentle magnetic stirring. The resulting coated beads were collected and rinsed with double distilled water and dried in an air overnight. Then the coated beads were dried in an oven at 45 C for 2e3 h to constant weight. 2.5. Measurement of particle size and zeta potential of liposome Particle size and zeta potential of the liposome was determined using particle size analyzer (Zetasizer nano ZS, Malvern, UK). 2.6. Determination of drug entrapment efficiency of liposome
2. Materials and methods 2.1. Materials Capsaicin was obtained from Naturite Agro Products Ltd. (Hyderabad, India). Soyalecithin and cholesterol were procured from HiMedia Laboratories Pvt. Ltd. (Mumbai, India). Eudrajit S-100 was purchased from Balaji Drugs, India and Sodium alginate was obtained from Loba chemie, India. All other ingredients used in the study were of analytical grade. 2.2. Preparation of liposome Liposomes were prepared by thin film hydration method reported earlier [23]. Weighted quantity of drug and lipids were taken into a round bottom flask and chloroform was used to dissolve drug and lipid. Thin lipid film was prepared by evaporating chloroform using rotary vacuum evaporator (PMTC-3040; Superfit, India). Then the flask was kept overnight in desiccator for complete elimination of the solvent. 20 ml of phosphate buffer (pH 7.4) was added to the flask and dispersion of the thin lipid film was done for 4 h at 55e60 C using rotary evaporator. The dispersion was sonicated using sonicator (UD200SH-6L; Takashi, Japan) for 30 min at 55e60 C and then the suspension was kept at room temperature for 1 h for the formation of vesicles. Liposomal suspension was centrifuged at 4 C and 15000 rpm using cooling centrifuge (C24BL; REMI, India) for 15 min to separate unentrapped capsaicin and liposomal vesicles were sediment as pellets. Then supernatant was separated from pellets and were resuspended with phosphate buffer solution pH 7.4 and centrifuged in same conditions. This process was repeated three times for complete removal of the unentrapped drug. Finally, the pellets were suspended in 20 ml phosphate buffer solutions pH 7.4 and stored in refrigerator at 4 C for further use.
The entrapped capsaicin containing liposomes are separated from unentrapped capsaicin by cooling centrifugation of a known aliquot (10 ml) of the prepared liposomal suspension at 15000 rpm for 60 min at 4 C. The supernatant was separated from the liposomal precipitate. Unentrapped drug was determined by diluting 1 ml of supernatant in 9 ml of methanol, followed by the measurement of absorbance of these solutions spectrometrically (UV1800, Shimadzu, Japan) at 279 nm wavelength. The entrapment efficacy percent was determined using the following equation:
Entrapment efficiency percent ¼ ½ðA BÞ=A 100 where A is the concentration of total capsaicin added and B is the concentration of free capsaicin detected. 2.7. Fourier transforms infrared spectroscopy study Fourier transform infrared spectra of pure capsaicin, physical mixture of capsaicin and excipients, and liposomal formulation were recorded in Fourier transform infrared spectroscopy (ALPHAT, Bruker, Germany) using KBr pellets. Spectra were recorded between 4000 and 500 cm1with a spectral resolution of 4 cm1 [24]. 2.8. Differential scanning calorimetry study Thermal behaviour of capsaicin, physical mixture of capsaicin and excipients, and liposomal formulation containing capsaicin were analyzed using differential scanning calorimeter (Pyris Diamond TG/DTA, Perkin Elmer, Singapore). The samples were heated at 15 C/min from 35 C to 250 C [25]. 2.9. Determination of particle size of beads The size of alginate and coated alginate beads were determined by optical microscopy.
2.3. Preparation of hydrogel beads containing liposome Required quantity of sodium alginate was taken and dissolves properly into 10 ml water to make a solution. Then 10 ml of liposomal suspension (approx 4 mg capsaicin) was mixed with sodium alginate solution such that final concentration of the solution was (2% w/v) of sodium alginate. Sodium alginate containing liposomal suspension was dropped through a syringe (22 gauge) into calcium chloride solution (2% w/v) with mild agitation for 15 min. The calcium chloride solutions were decanted and the beads were washed three times with doubled distilled water. The formed beads were air dried overnight and then dry in an oven at 45 C for 2e3 h to constant weight.
2.10. Determination of drug entrapment efficiency of hydrogel beads and coated hydrogel beads 20 mg of beads were taken in beaker containing 100 ml of pH 7.4 phosphate buffer solution and stirred 4 h in a magnetic stirrer. Similarly, coated hydrogel beads were taken in a beaker containing methanol instead of pH 7.4 phosphate buffer solutions. Then sonication was done for 30 min. After that, the absorbance of the buffer solution containing the extracted amount of capsaicin was taken at a wavelength of 279 nm in a UV spectrophotometer using pure phosphate buffer as a blank. The percentage of entrapment efficiency was then calculated as [26]:
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Entrapment efficiency % ¼ ðActual drug content=theoretical drug contentÞ 100
2.11. In vitro drug release study The in vitro release profile of capsaicin from liposomal and hydrogel formulations were determined using the dialysis bag method [27]. The design of using the simulated fluids at different pH was as follows: simulated gastric fluid of pH 1.2 (1 h), mixture of simulated gastric and intestinal fluid of pH 4.5 (2e3 h), simulated intestinal fluid of pH 7.4 (4e5 h), and simulated colonic fluid pH (remaining time). 4 mL of liposomal suspension or 20 mg beads suspended in 4 ml water were taken into a dialysis bag (Himedia dialysis membrane-60, Mumbai, India) and hung inside the beaker containing simulated fluid. The content of beaker was rotated at 300 rpm with a magnetic bead at 37 C. The simulation of gastrointestinal tract conditions was achieved by altering the pH of the dissolution medium at various time intervals. Aliquots of the dissolution medium were withdrawn at predetermined time intervals and compensated with the same volume of fresh dissolution media and the amount of drug was quantified by UV spectrophotometer at 279 nm wavelength. 3. Results and discussion 3.1. Development and characterization of liposome Capsaicin loaded liposomes were prepared using soyalecithin and cholesterol by thin film hydration method. The average vesicle size of capsaicin loaded liposome was 284.1 nm (Fig. 1). Liposomal formulation showed the zeta potential value of 61 mV. High zeta potential (>±30 mV) can offer an electric repulsion which prevents aggregation of particles and give the stability of the colloidal system [28]. The capsaicin loaded liposomes were anticipated to have good physical stability as zeta potential (61 mV) was less than 30 mV. The entrapment efficiency of capsaicin in liposome was found to be 35 ± 5%. 3.2. Fourier transforms infrared spectroscopy study Fourier transform infrared spectrum of capsaicin, physical mixture of drug and excipients and drug loaded liposome were
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represented in Fig. 2. Capsaicin shows peak at 3313.58 cm1 which is associated with broadened O-H and N-H stretch (Fig. 2a). Stretching peak of aliphatic C-H bonds shown at 2861.38 cm1. The peak at 1630.18 cm1 was attributed to olefinic C¼O stretch, C¼C stretch, and amide II. N-H bends and C-N stretch peak of capsaicin shown at 1515.85 cm1. The peak at 1283.29 cm1 represents stretching of C-N bonds. Physical mixture of drug and excipients showed approximately analogous spectra of capsaicin (Table 1). Liposomal complex containing capsaicin shows the minor changing of a few peaks in comparison to pure capsaicin and physical mixture containing capsaicin (Table 1). The minor shifts of peaks may be due to the creation of hydrogen bonds or weak forces between drug and phospholipids. 3.3. Differential scanning calorimetry studies The differential scanning calorimetry thermograms of capsaicin, physical mixture containing capsaicin and liposomes containing capsaicin are represented in Fig. 3. Capsaicin showed a sharp melting endotherm at 69.72 C (Fig. 3a). The endothermic peak of capsaicin also was appeared in the graph of the physical mixture containing capsaicin with diminished intensity (Fig. 3b) may be due to dilution of drug with lipid. This suggests that there was no interaction between drug and lipid on simple mixing. The melting peak of capsaicin almost entirely disappeared in liposome loaded with capsaicin. This established that the crystalline nature of the drug was retained in the physical mixture but lost in the liposomal formulation. This indicates possible solid state transformation of the drug. 3.4. Development and characterization of alginate hydrogel beads containing capsaicin loaded liposome Liposomes are the most ideal carriers to colon as hydrophobic drug promotes absorption into colonic region. Nevertheless, liposomes often suffer from the problem of instability in harsh gastrointestinal environment. On the contrary polymeric hydrogel beads are more stable in harsh gastrointestinal environment. Therefore, hydrogel beads loaded with liposome are anticipated as an ideal drug delivery carrier. Alginate is a natural anionic polymer and has a distinctive property of gel formation in the existence of multivalent cations. However, fast release of drug occurs before reaching into the colon due to porosity of the alginate beads. So Eudragit S100 was selected as enteric coating material for coating of
Fig. 1. Particle size distribution of liposome.
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Fig. 2. Fourier transform infrared spectrum of a) capsaicin b) physical mixture of capsaicin and excipients c) Liposomal complex of capsaicin and excipients.
alginate hydrogel beads due to no release or less amount of release of encapsulated drug at the stomach as well as in the small intestine. When a dispersion of liposomal suspension containing capsaicin and alginate was extruded through the needle into a solution
containing calcium chloride, the beads were formed instantaneously. The produced alginate beads are represented in Fig. 4. The hydrogel beads are more or less spherical having smooth surface. The mean diameters of alginate hydrogel beads and coated alginate hydrogel beads were 1.213 ± 0.218 mm and 1.362 ± 0.180 mm,
T.K. Giri et al. / Journal of Drug Delivery Science and Technology 39 (2017) 417e422
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Table 1 Comparison of FTIR spectra of capsaicin functional groups. Functional groups
Capsaicin
Broadend O-H and N-H stretch Aliphatic C-H stretch Olefinic C¼C stretch, C¼O stretch, amide II N-H bend and C-N stretch, amide II C-N stretch
3313.58 2861.38 1630.18 1515.85 1283.29
cm1 cm1 cm1 cm1
Physical mixture containing capsaicin 3316.73 2863.48 1630.20 1516.02 1282.59
cm1 cm1 cm1 cm1 cm1
Liposome containing capsaicin 3320.31 2857.74 1631.13 1516.44 1282.14
cm1 cm1 cm1 cm1 cm1
Fig. 3. DSC thermo grams capsaicin (a), physical mixture (b), and drug loaded liposome (c).
maximum 2 h to complete release in simulated gastric fluid. It was observed that in case of capsaicin loaded liposomal formulation 100% drug released in 5 h. In the case of liposomal formulation entrapped in alginate beads, drug release was found to be 99.8% at the end of 8 h. In the case of the coated alginate beads containing liposomal formulation, drug release was found to be 99.3% at the end of 24 h. The values of t85% (i.e. time required for the release of 85% drug) were calculated as 190 min, 285 min, 900 min, respectively in the liposome, alginate beads containing liposome, and Eudragit coated alginate beads containing liposome. After 5 h of invitro study, in case of capsaicin loaded liposomal formulation 99% drug was released and for alginate hydrogel beads 88% drug was released. However, coated alginate hydrogel beads released about 30% drug before reaching colonic region.
4. Conclusion
Fig. 4. Alginate hydrogel beads.
respectively. The drug entrapment efficiency of alginate hydrogel beads and coated alginate hydrogel beads were 85 ± 1.15% and 82.9 ± 1.31%, respectively. 3.5. In vitro drug release study The cumulative percentage drug releases from different formulations in various simulated gastrointestinal fluids were represented in Fig. 5. in vitro release study of capsaicin shows that it takes
Capsaicin loaded liposome was successfully prepared and then incorporated into alginate hydrogel beads and finally coated with anionic polymer Eudragit S-100. Fourier transform infrared spectroscopy analysis showed that there was slight interaction of drug capsaicin with other excipients. Differential scanning calorimetry study showed that drug was molecularly dispersed into the matrix. Eudragit coated alginate beads allow a minimum drug release (<30%) in stomach and intestine and released maximum amount of drug (>70%) into colonic region. From in vitro release study we can conclude that Eudragit coated liposomal formulation can carry drug through gastrointestinal tract with very low release of drug in the stomach and intestine region and showed greater and control release of the drug into the colonic region for a longer period of time.
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Fig. 5. Cumulative percentage drug release profile from different formulations.
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