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Novel mucoadhesion tests for polymers and polymer-coated particles to design optimal mucoadhesive drug delivery systems
Advanced Drug Delivery Reviews 57 (2005) 1583 – 1594 www.elsevier.com/locate/addr
Novel mucoadhesion tests for polymers and polymer-coated particles to design optimal mucoadhesive drug delivery systemsB Hirofumi Takeuchi *, Jringjai Thongborisute, Yuji Matsui, Hikaru Sugihara, Hiromitsu Yamamoto, Yoshiaki Kawashima Laboratory of Pharmaceutical Engineering, Gifu Pharmaceutical University, Japan 5-6-1 Mitahora-Higashi Gifu, 502-8585, Japan Received 27 August 2004; accepted 12 July 2005 Available online 16 September 2005
Abstract To design an effective particulate drug delivery system having mucoadhesive function, several mucoadhesion tests for polymers and the resultant particulate systems were developed. Mucin particle method is a simple mucoadhesion test for polymers, in which the commercial mucin particles are used. By measuring the change in particle size or zeta potential of the mucin particle in a certain concentration of polymer solution, we could estimate the extent of their mucoadhesive property. BIACORE method is also a novel mucoadhesion test for polymers. On passing through the mucin suspension on the polymerimmobilized chip of BIACORE instrument, the interaction was quantitatively evaluated with the change in its response diagram. By using these mucoadhesion tests, we detected a strong mucoadhesive property of several types of chitosan and Carbopol. Evaluation of mucoadhesive property of polymer-coated particulate systems was demonstrated with the particle counting method developed by us. To detect the mucoadhesive phenomena in the intestinal tract, we observed the rat intestine with the confocal laser scanning microscope (CLSM) after oral administration of the particulate systems. The resultant photographs clearly showed a longer retention of submicron-sized chitosan-coated liposomes (ssCS-Lip) in the intestinal tract than other liposomal particles tested such as non-coated liposomes and chitosan-coated multilamellar one. These observations explained well the superiority of the ssCS-Lip as drug carrier in oral administration of calcitonin in rats than other liposomal particles. D 2005 Elsevier B.V. All rights reserved. Keywords: Mucoadhesion; Mucin particle; BIACORE; Confocal laser scanning microscopy; Peptide drug; Oral administration
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mucin particle method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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B This review is part of the Advanced Drug Delivery Reviews theme issue on ‘‘Mucoadhesive Polymers: Strategies, Achievements and Future Challenges’’, Vol. 57/11, 2005. * Corresponding author. Tel.: +81 58 237 3931; fax: +81 58 237 5979. E-mail address: [email protected] (H. Takeuchi).
0169-409X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2005.07.008
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2.1. Interaction between mucin particles and polymer . . . . . . . . . . . . . . 2.2. Quantitative analysis of the interaction between mucin particle and chitosan 3. Mucoadhesion test with BIACORER . . . . . . . . . . . . . . . . . . . . . . . . 3.1. BIACORER system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Response of chitosan–mucin interaction . . . . . . . . . . . . . . . . . . . 3.2.1. Preparation of ss-mucin suspension for BIACORE experiment . . . 3.2.2. Immobilization of chitosan on the sensor chip . . . . . . . . . . . 3.2.3. Interaction between chitosan and ss-mucin . . . . . . . . . . . . . 3.3. Adhesion test for various polymers . . . . . . . . . . . . . . . . . . . . . 4. Mucoadhesion tests for fine particulate systems. . . . . . . . . . . . . . . . . . . 4.1. Mucoadhesion tests for coarse and fine particles. . . . . . . . . . . . . . . 4.2. Particle counting method for fine particles . . . . . . . . . . . . . . . . . . 4.3. Confocal laser scanning microscopy (CLSM) method . . . . . . . . . . . . 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Colloidal drug carriers, such as liposomes or nanoparticles of biodegradable polymers, have received much attention for their ability to improve the absorption of poorly absorbable drugs, including peptide drugs. It has been reported that the mucoadhesive properties of these particulate systems can prolong their retention in the gastrointestinal tract, thus further improving drug absorption [1]. We have demonstrated a novel mucoadhesive liposomal system prepared by coating the liposome surface with a mucoadhesive polymer, chitosan. The effectiveness of the chitosan-coated liposomes (CS-Lip) was confirmed by the enhanced and prolonged pharmacological effect of insulin, which was orally administered in the polymer-coated liposomal form in rats [2,3]. The effectiveness of mucoadhesive liposomes in drug absorption was also demonstrated by using calcitonin as a model peptide drug. Carbopol-coated liposomes, having a mucoadhesive property similar to that of CSLip, were as effective as CS-Lip [4]. A particulate bioadhesive system was also prepared by coating microspheres of poly-hydroxyethyl-methacrylate with mucoadhesive polymers using laboratoryscale equipment [5,6]. Akiyama et al. [7] prepared a polyglycerol ester of fatty acid-based microspheres coated with Carbopol934P (CP) and CP-dispersing microspheres to evaluate their mucoadhesive properties. In developing colloidal drug delivery systems, Lenaerts et al. [8] demonstrated the mucoadhesive
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property of polyalkylcyanoacrylate nanoparticles with autoradiographic studies and confirmed that the bioavailability of vincamine was improved in nanoparticulate systems. Pimienta et al. [9] investigated the bioadhesion of hydroxypropylmethacrylate nanoparticles or isohexylcyanoacrylate nanocapsules, the latter coated with poloxamers and poloxamine on rat ileal segments in vitro using a labeled compound. To design optimal mucoadhesive drug delivery systems such as polymer-coated liposomes, it is necessary to develop simple mucoadhesion tests that can evaluate the mucoadhesive properties of polymers. The development of mucoadhesion tests for particulate systems is also important. The most direct method is to measure the bioadhesive bond strength between a polymer and the mucosal layer of animals [10]. Ch’ng et al. [11] demonstrated the feasibility of this concept by using a polymer specimen and freshly excised stomach tissue from an animal. Ponchel et al. [12] developed a similar method to measure tensile force. Smart et al. [13] proposed the use of the Wilhelmy plate method, which is usually applied to determine surface tension. A rheological method proposed by Hassan and Gallo [14] is an alternative way to assess mucin–polymer adhesive bond strength. They evaluated bioadhesion forces by monitoring viscometric changes in a mixture of porcine gastric mucin and polymers in solution. In this paper we propose two types of novel mucoadhesion tests for polymers. We also report on tests we developed for mucoadhesive particulate systems.
H. Takeuchi et al. / Advanced Drug Delivery Reviews 57 (2005) 1583–1594
Conc. of Carbopol (%)
2. Mucin particle method -10
We tried to evaluate the mucoadhesion of polymers with commercially available porcine mucin particles [15]. In the test, mucin particles were suspended in suitable buffer solutions with a concentration of 1% w/v and then mixed with an appropriate amount of polymer solutions. It was expected that the surface property of the mucin particles might be changed by the adhesion of the polymer if the polymer has a mucoadhesive property. The occurrence of such change was detected by measuring the zeta potential with a Zetamaster (Malvern Instruments, UK). When the coarse mucin particle suspensions were mixed with the solutions of several types of chitosan (CS) and Carbopol (CP), the zeta potential of the mucin particles changed in each case (Figs. 1 and 2). On the other hand, when hydroxylpropylmethylcellulose (HPMC) solution was added to the mucin suspension instead of CS or CP solution, the zeta Conc. of chitosan (%) 0.5
1
-10 CS-2
-20
CS-5
(c)
Conc. of chitosan (%)
10 0 -10 -20 -30
0.8
1
-15 C-971P C-974P
-20
N-AA1
-25
Conc. of chitosan (%) 0
0.5
1
1.5
10 0 -10 -20
CS-15
-30
20
0.6
potential was unchanged. These results suggested that either CS or CP had a high affinity to mucin particles to cover their surfaces. The higher the concentration of CS and the greater its molecular weight, the more
(b)
0
0.4
Fig. 2. Zeta potential of coarse mucin particles in the solutions of Carbopol with various concentrations, pH 6.8. Molecular weigh of C-971P and C-974P are 1,250,000 and 3,000,000, respectively.
1.5
10
0.2
-35
0
0.5
1
Conc. of chitosan (%)
(d)
1.5
20
Zeta potential (mV)
Zeta potential (mV)
20
0
0
-30
Zeta potential (mV)
(a)
Zeta potential (mV)
2.1. Interaction between mucin particles and polymer
Zeta potential (mV)
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0
0.5
1
1.5
10 0 -10 -20 -30
Fig. 1. Zeta potential of coarse mucin particles in the solutions of chitosan having different molecular weight with various concentrations and different pH. (a) pH 5.0, (b) pH 6.8, (c) pH 7.4, (d) pH 9.0. Molecular weigh of chitosan: CS-2, 20,000; CS-5, 50,000; CS-15, 150,000.
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Relative size
80
Relative size Zeta potential (mV)
-10 -20
60 -30 40 -40 20
-50
Zeta potential (mV)
0
100
firmed that the adsorption of CS molecules on the mucin particles were responsible for the aggregation. The mucoadhesive properties of various types of CP were evaluated with this method, and the results suggested that the C-974-P was more adhesive than the other types of CP tested (data not shown). 2.2. Quantitative analysis of the interaction between mucin particle and chitosan
-60
0 Micronized mucin
CS-2
CS-5
CS-15
Fig. 3. Change in observed particle size of micronized mucin particles when mixed with the chitosan solutions. Concentration of chitosan solution: 1.5% w/v. pH of solution: 6.8.
extensive the changes were in the zeta potential (Fig. 1). Positive zeta potential was observed in the acidic solution (pH 5.0), possibly owing to the dissociated amino groups of CS on the surface of mucin particles (Fig. 1). In the case of Carbopol, the resultant zeta potential of mucin shifted to a higher negative value because of the negative charge of the polymer (Fig. 2). These results strongly supported what happened in the mixture and the feasibility of the mucin particle method for evaluating the mucoadhesive property of polymers. A modified mucin particle method was performed with the use of submicron-sized mucin particles (ssmucin, ca. 200–300 nm in particle diameter) and particle size analyzers (LPA 300, Otuska-Denshi or Cis-1, Galai Inc.). The ss-mucin were prepared by applying sonication to the coarse mucin suspension. When the suspension of ss-mucin particles was mixed with a polymer solution, the mucin particles may aggregate if the polymer has a strong affinity to them, i.e., a mucoadhesive property. When the ssmucin particles were mixed with CS solution, the particle size increased as expected. The extent of change in particle size was presented as the relative particle size, which was calculated by dividing the size of the aggregates by that of the original ss-mucin (Fig. 3). It was found that the aggregation tendency depended on the molecular weight of the CS, which corresponded well with the results observed in the coarse mucin particle test described above. The zeta potential of mucin particles shifted towards zero with increasing the aggregation tendency (Fig. 3). It con-
The quantitative analysis of the mucin particle method was carried out by controlling the amount of polymer added and then analyzing the zeta potential of the ss-mucin [16]. The particle size and zeta potential of the precisely size-controlled ss-mucin were 361 nm and 25.4 mV, respectively. The interaction between ss-mucin and the chitosan molecule was determined by mixing a known amount of ss-mucin with an appropriate 0.05% w/v of chitosan solution. Then the zeta potential value was measured in 10 mM acetate buffer solution, ABS, at pH 4.5. The indication of this electrostatic interaction is shown in the zeta potential value of ss-mucin after it was mixed with chitosan solution (Table 1). The zeta potential of ss-mucin was negative before the mixing. By increasing the amount of 0.05% w/v chitosan solution added, the zeta potential value of ss-mucin gradually changed from the negative to the positive until the critical ratio of 1:1 was reached. At that point, where the zeta potential value was nearly zero, the negative charge of the ss-mucin particles would be neutralized with the positive charge of the chitosan molecules adsorbed on their surface. Base on this hypothesis, we were able to calculate the occupying area of one chitosan molecule on the surface of a mucin particle. This area, calculated using
Table 1 Zeta potential of ss-mucin particles after mixing with various amounts of chitosan solution (n = 3) SS-mucin: 0.05% CS solution
Zeta potential in ABS, pH 4.5 (mV)
1 1 1 1 1
8.40 F 2.0 0.5 F 0.3 1.7 F 0.4 21.1 F 9.3 22.0 F 10.9
0 0.5 1 3 5
ss-mucin: submicron-sized mucin, ABS: acetate buffer solution.
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3. Mucoadhesion test with BIACORER 3.1. BIACORER system The BIACORE instrument is based on the principle underlying an optical phenomenon called Surface Plasmon Resonance (SPR). The SPR response is a measurement of the refractive index, which varies with the solute content in a solution that contacts a sensor chip. When a detected molecule is attached to the surface of the sensor chip, or when the analyte binds to the detected molecule, the solute concentration on the sensor chip surface increases, leading to an SPR response [17]. Quantitative measurements of the binding interaction between the chip surface and one or more molecules depend on the immobilization of target molecules that are in contact on the sensor surface. The sensor chip consists of a glass surface coated in a thin layer of gold. This forms the basis for a range of specialized surfaces designed to optimize the binding of a variety of molecules. The most widely used sensor chip is CM5 (BIACORER, Sweden), whose surface is modified with a carboxymethylated dextran layer. In general, the ligand can covalently bind to the sensor chip surface via carboxyl moieties on the dextran. Functional groups on the ligand that can be used for coupling include NH2, SH, CHO and COOH. The major advantages of the BIACORE instrument are its label-free detection of binding and the ability to monitor the change in response in real time [18,19]. In the detection of the mucoadhesive property of polymers using BIACORE, each polymer was immobilized on the surface of the sensor chip CM5 and the mucin suspension was passed through the sensor chip. When the analyte (mucin particle) binds to the ligand molecule (polymer) on the sensor chip surface, the solute concentration and the refractive index on that surface change, increasing the RU response; when
they dissociate, the RU response will fall. After that, the analyte can be removed from the ligand by using a regenerating reagent. The response will then turn back to the equilibrium state as the beginning step. 3.2. Response of chitosan–mucin interaction 3.2.1. Preparation of ss-mucin suspension for BIACORE experiment We prepared 1.0% w/v of mucin suspension by suspending and continuously stirring mucin powder in 10 mM acetate buffer solution (ABS) at pH 4.5 overnight. After incubation at 37 8C for one night, the suspension was ultrasonicated with a probe sonicator (Branson Sonifier 250) until the particle size was less than 1 Am. It was then centrifuged at 4000 rpm for 20 min to extract submicron-sized mucin particles in the supernatant portion, which was then diluted to a concentration of 0.01% w/v with 10 mM ABS, pH 4.5. 3.2.2. Immobilization of chitosan on the sensor chip Chitosan was immobilized on the CM5 chip surface by using amine-coupling chemistry. The immobilization was carried out at a flow rate of approximately 15 Al/min in 10 mM ABS, pH 4.5. Initially, the sensor chip surface was activated for 6 min with a mixture of 100 mM N-hydroxysuccinimide (NHS) and 400 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and then chitosan was injected at a concentration of 0.02% w/v across the activated surface for 10 min. Afterwards, the remaining reactive esters were transformed into amides by injection of 1 M of ethanolamine HCl, pH 8.5, for 6 min. Baseline data were
Resonance unit (RU)
the respective particle-size averages and the observed critical ratio, was about 66.5 Am2. This value was reasonable, considering CS’s molecular weight of 150,000. This method was also successfully applied to evaluate the coating phenomenon of the particulate systems with chitosan in developing the mucoadhesive systems.
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23000 22000 21000 20000 19000 18000 17000 CS baseline 16000 15000 0 500 1000 1500 2000 2500 3000 3500
Time (s) Fig. 4. The sensorgram for chitosan-immobilized sensor chip after passing the ss-mucin suspension. z start injection, A stop injection. Molecular weight of chitosan: 150,000.
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7000
interaction between submicron-sized mucin and chitosan on the chip because of the increase in the RU response after injection, which was the result of the change in the refractive index on the chip. The RU response decreased when the chitosan solution was injected again across the mucin-interacted sensor chip coated with chitosan (Fig. 5). This change suggested that a part of the mucin particle fixed to the chip surface was removed by the interaction with the newly injected chitosan molecules. In other words, this decrease in response may be an indicator of a polymer’s mucoadhesive property. To confirm this phenomenon, chitosan solutions with different molecular weights (MW 20,000 and 150,000) were injected after submicron-sized mucin (Fig. 5). As shown in these sensorgrams, the change in RU response depended on the molecular weight of the chitosan. These sensorgrams indicated that the chitosan with the higher molecular weight possessed the higher mucoadhesive property.
M
Resonance unit (RU)
6000 5000
CS2
4000
CS15
3000 CS2
2000 1000 0 0
CS15
M M 1000
2000
3000
4000
5000
6000
Time (s) Fig. 5. Overlay of the sensorgrams for the ss-mucin-immobilized sensor chip after injection of the chitosan solutions with MW 20,000 and MW 150,000. zM, AM: start and stop mucin injection, z CS2, and A CS2: start and stop chitosan MW 20,000 injection, z CS15, and A CS15: start and stop chitosan MW 150,000 injection.
collected at least 30 min before the experiment began. The immobilization process used 0.02% w/v of chitosan solutions with various molecular weights (dimer, tetramer, hexamer, and chitosan MW 150,000), and then 0.01% w/v of ss-mucin was passed over the sensor chip surface for 10 min. Afterwards, the sensorgram was collected until equilibrium was reached.
3.3. Adhesion test for various polymers To confirm the feasibility of this BIACORE-based mucoadhesion test, various polymers were applied to it. In each case, 0.02% w/v of mucin was injected for 10 min across the surface of the sensor chip after the chitosan immobilization process (chitosan MW 150,000). Then, 0.02% w/v of each polymer was passed over the sensor chip surface for 10 min. To change the polymer solution, mucin and the former
3.2.3. Interaction between chitosan and ss-mucin Fig. 4 shows the RU response detected as the ssmucin suspension was passed over the immobilized chitosan. The sensorgram in the figure shows the 18000
Chitosan baseline
Responses after injecting adhesive polymers
M
Arbitrary Units of RU
16000
P P
14000
P P
12000 10000
P
8000 6000 4000
Carbopol 971 PNF
P
Carbopol 974 PNF
P
PVA 205
P
P
PVP 25
P
P
TC-5-S
P
P
90SH-400
2000
65SH-50
P
0 0 -2000
M
1000
2000
3000
4000
Time (s) Fig. 6. Overlay of the sensorgrams for ss-mucin-immobilized sensor chip after injection of the various adhesive polymer solutions. zM, AM: start and stop mucin injection, zP, AP start and stop adhesive polymer injection.
H. Takeuchi et al. / Advanced Drug Delivery Reviews 57 (2005) 1583–1594
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50
Adhesive %
40
C
Chitosan
30 n
PAA-R 20 C
C
C n
C
10
PVA-R
PAA-R
Chitosan
Uncoated
C Cho
PVA-R 0
C
C
C
C n
Fig. 7. Adhesive % of polymer-coated liposomes to rat intestine evaluated with the particle-counting method. Lipid composition: DPPC/ DCP=8:2. Polymer concentration: 0.75%. Dispersion medium: phosphate buffer solution (pH 7.4).
polymer were removed with regenerating reagent as explained above. Fig. 6 summarizes the response changes after various polymers are injected. In this figure, the starting and stopping injection time point of each polymer were not the same time because each sensorgram was collected until re-equilibrium before injecting the new polymer. The decreasing in RU responses were calculated by using the following equation.
4. Mucoadhesion tests for fine particulate systems 4.1. Mucoadhesion tests for coarse and fine particles
% RU decrease RU from mucin baseline to polymer RU from chitosan baseline to mucin
¼
molecular weights. Like chitosan, Carbopol showed much larger decreases in RU response, whereas the responses of the other polymers were slight. These results correspond well with the results of other mucoadhesion tests.
100:
ðaÞ
Table 2 lists the values of decreases in RU response for these polymers, including chitosan at different
Table 2 Percentage of RU response decrement after injection of various adhesive polymer solutions Polymer
% RU decrease
Chitosan MW 20,000 Chitosan MW 150,000 Carbopol 971 PNF Carbopol 974 PNF PVA 205 PVP25 (Kollidon25) TC-5 S (HPMC 2910) 90SH-400 (HPMC 2208) 65SH-50 (HPMC 2906)
22.01 46.34 29.58 27.57 3.26 7.92 6.78 9.58 9.60
Various methods have been proposed to measure the mucoadhesive properties of particulate systems. Ranga Rao and Buri [20] demonstrated a simple method for coarse particulate systems, such as polymercoated glass beads and drug crystals. In their adhesion test, particles were placed on rat jejunum or stomach in a humid environment in vitro. The percent of particles retained on the tissue was considered an index of bioadhesion. Lehr et al. [21] used an in situ perfused ileal loop in the rat to study the mucoadhesion of coarse particles. In that study, polymercoated microspheres were perfused with isotonic saline, and the number of collected particles was counted. To evaluate the mucoadhesion of fine particles, Durrer et al. [22,23] investigated the adsorption isotherms of poly(styrene) latex delivered to rat intestinal mucosa under static conditions by use of turbidimetric and FTIR-ATR analyses. The use of labeled compounds is one way to estimate the mucoadhesion of
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particulate systems, including liposomes. The particle movement profiles in the mouse intestinal tract were determined after oral administration of polyhexyl[3-14C]-cyanoacrylate) nanoparticles; the recovery of radioactivity in those tissues was measured at appropriate intervals [23]. Ponchel et al. [24] also studied the intestinal mucoadhesion profiles of 14C-labeled polylactic acid (PLA) particles (mean diameter: 1.5 Am) as a function of time after PLA administration in rat. 4.2. Particle counting method for fine particles We tried to develop a simpler in vitro method (particle-counting method) to evaluate the mucoadhesion of fine particles, such as polymer-coated liposomes [2,3]. In this method, an intestinal tube was isolated from Wistar rats and cut into three parts, named as upper, middle, lower, from stomach side. The length of each intestinal tube was 15 cm. Each tube was washed inside with saline solution filled with liposomal suspensions diluted by 100 times with an appropriate buffer solution. After being sealed with closers, the tube was incubated in saline solution at 37 8C for more than 15 min. By measuring the number of liposomal particles before and after incubation with a Coulter counter, the mucoadhesive % was presented by the following equation. Adhesive % ¼ ðNo NsÞ=No 100
ðbÞ
where No and Ns are the number of liposomes before and after incubation, respectively. 80
As shown in Fig. 7, CS-coated liposomes showed the highest adhesive % among the three different polymer-coated liposomes tested. The mucoadhesive function was thought to be provided by the polymer layer fixed on the liposome surface, because uncoated liposomes showed no adhesive %. Interpenetration between the polymeric and the mucosal networks is considered to be responsible for the adhesion. The adhesive % of CS-coated liposomes depended on the CS concentration used for coating. Considering the combined results, it could be concluded that the more effective coating leads to the higher adhesive %. The mucoadhesive properties of positively and negatively charged liposomes were compared by the particle-counting method (Fig. 8) [4]. The positively charged liposomes showed a higher adhesive % to the mucosal layer than the negatively charged one; this was attributed to the negative charging of the surface of the mucosal layer of the intestine. However, the CP-coated and thus negatively charged liposomes, as well as the positively charged CS-coated liposomes, showed higher adhesive % than the positively charged uncoated liposomes. This result suggested that a physical entanglement between the polymer and mucosal layer is an important factor to facilitate the mucoadhesion of polymer-coated liposomes. 4.3. Confocal laser scanning microscopy (CLSM) method It is important to characterize the mucoadhesive properties of fine particulate systems in vivo. In a
In Saline (pH6.1)
70
Adhesive (%) =
Adhesive (%)
60
N0 – N15
×100
N0
50 N0: Number of initial liposomes. N15: Number of liposomes after 15 min
40 30 20
Negatively charged MLVs
10
Positively charged MLVs Carbopol coated MLVs
0 Upper
Middle
Lower
Chitosan coated MLVs
Fig. 8. Adhesive % of several liposomes to rat intestine evaluated with the particle-counting method. Lipid compositions; DPPC/DCP=8:2, DPPC/SA=40:1, Carbopol; C-971P, Chitosan; CS-15 (DSP: distealoylphosphatidylcholine, DCP: dicetyl phosphate, SA: stearylamine).
H. Takeuchi et al. / Advanced Drug Delivery Reviews 57 (2005) 1583–1594
(a)
CS-Lip 4.6µm
(b)
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ssCS-Lip (339nm)
Lumen
Mucosal layer
200µm
Jejunum
Ileum
Jejunum
Ileum
Fig. 9. Confocal laser scanning microscopy photographs of the jejunum and ileum of the rat intestine 2 h after intragastric administration of chitosan-coated liposomes: (a) CS-Lip and (b) ssCS-Lip. The measured mean diameters of the liposomes are 4.6 Am (a) and 339.2 nm (b), respectively. The formulation of liposomes: DSPC/DCP/Chol. = 8:2:1. The concentration of chitosan in the coating is 0.3% w/v (DSPC: distealoylphosphatidylcholine, DCP: dicetyl phosphate, Chol.: cholesterol).
previous study, we examined the mucosal layer of rat intestine in order to detect liposomes by using confocal laser scanning microscopy (CLSM) after administering these particulate systems [25]. For this purpose, a fluorescence marker, 1,1V-dioctadecyl-3, 3,3V,3V-tetramethylindo carbocyanine perchlorate (DiI, LAMBDA, Austria), was formulated into the liposome particles. The chitosan-coated liposomes (CSLip) was prepared by mixing the non-coated liposomal suspension (Lip) with the same volume of acetate buffer solution (pH 4.4) of CS (0.6%), followed by incubation at 10 8C for 1 h. The submicron-sized CSLip ssCS-Lip were prepared by using the submicronsized liposomes (ssLip), which were prepared by sonicating (Sonifier250, Branson) the multilamellar liposomes. The intestinal tubes were removed from the rats at the appropriate time after administration of the liposomes. Each tube was cut into five parts: the duodenum, upper jejunum, lower jejunum, upper ileum, and lower ileum. Each part was sliced with a cryostat (Leica, Germany) to generate sections for
confocal laser scanning microscopic observation. The thickness of the sections was 10 Am. Each sample was placed on a confocal laser scanning microscope (LSM510, Carl Zeiss, Germany) and observed at an excitation wavelength of 550 nm and an emission wavelength of 570 nm. The mucoadhesion profiles of the chitosan-coated liposomes (CS-Lip) and submicron-sized CS-Lip (ssCS-Lip) in the intestinal tube were evaluated by observing the residual liposomes on the mucosa. The higher mucoadhesive tendency of ssCS-Lip compared with CS-Lip was confirmed by comparing the resultant photographs in Fig. 9. At the jejunum, small amounts of CS-Lip particles were observed, while large amounts of ssCS-Lip were detected there. In the ileum, the retention of the liposomal particles seemed almost the same regardless of their size, but ssCS-Lip tended to penetrate deeply into the mucosal part of the intestine, whereas CS-Lip did not. A similar size dependency was observed when we measured the mucoadhesive properties of the
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Lip (4.1µm)
(a)
(b)
ssLip (182nm)
Lumen
Mucosal layer
200µm
Jejunum
Jejunum
Ileum
Ileum
Calcemia (% of the initial value)
Fig. 10. Confocal laser scanning microscopy photographs of the jejunum and ileum of the rat intestine 2 h after intragastric administration of non-coated liposomes: (a) Lip and (b) ssLip. The measured mean diameters of the liposomes are 4.1 Am (a) and 181.9 nm (b), respectively. The formulation of liposomes: DSPC/DCP/Chol. = 8:2:1 (DSPC: distealoylphosphatidylcholine, DCP: dicetyl phosphate, Chol.: cholesterol). Calcitonin solution (Ctrl) ssLip ssCS-Lip
110 105 100 95 90
*
85
*** * † *
*
80
* † †
†
*
*
75 0
24
48
72
96
120
Time (hr) Fig. 11. Profiles of plasma calcium levels after intragastric administration of submicron-sized liposomes, ssLip and ssCS-Lip, containing calcitonin. The measured mean particles sizes of ssLip and ssCS-Lip are 196.4 nm and 473.4 nm, respectively. The formulation of liposomes is DSPC/DCP/Chol. = 8:2:1. The concentration of chitosan for coating is 0.3%. *p b 0.05, **p b 0.01, ***p b 0.001: significantly different from the level for calcitonin solution; yp b 0.05, yyp b 0.01: significantly different from the level for ssLip (n = 3 in each case).
uncoated liposomes with different particle sizes, Lip and ssLip (Fig. 10). Although the retained amount of uncoated liposomes was lower than that of chitosancoated liposomes, ssLip showed a penetrative behavior similar to that of ssCS-Lip. It was confirmed that ssLip had lower retention than ssCS-Lip, since only a small amount of ssLip was observed at the jejunum. According to the retention profiles of these particles in the intestinal tract, CS-Lip was more retentive than Lip. In both cases, we also confirmed that the retention profile was enhanced by decreasing the particle size from the micron to the submicron (ca. 200–300 nm) range. It was also found that the submicron-sized liposomes penetrated the mucosa, whereas no such penetrative profile was observed for the larger liposomes. This penetrative property is what gives ssCS-Lip a much higher retentive property than CS-Lip. We previously demonstrated the effectiveness of CS-coated liposomes in the absorption of peptide drugs, such as insulin and calcitonin, in rats [2–4].
H. Takeuchi et al. / Advanced Drug Delivery Reviews 57 (2005) 1583–1594
Recently, we observed that the reduction in particle size of the liposomal system led to a large improvement in drug absorption. In the administration of calcitonin (500 IU/kg rat) with the liposomal formulation, the period of reduced calcium concentration in the blood was prolonged to 120 h after administration by reducing the particles to submicron size (ssCS-Lip) (Fig. 11). The period was much longer than that observed when CS-Lip were used as carriers of calcitonin under the same conditions. Comparison of these profiles confirms the particle size effect on calcitonin absorption. The uncoated ssLip were also found to be effective in reducing the calcium level under the same experimental conditions, although the extent was much less than that of ssCS-Lip, as shown in Fig. 12. These pharmacological effects of calcitonin administered with the liposomal formulations corresponded well with their retention profiles in the intestinal tract observed with the CLSM method. CLSM observation may be a promising method to characterize the mucoadhesive properties of fine particle drug delivery systems and to explain their effectiveness in the oral administration of drugs.
5. Conclusion In the present paper, novel mucoadhesion tests were proposed to design optimal particulate drug delivery systems having the mucoadhesive function. Mucin particle method is a simple in vitro test to detect the mucoadhesive property of polymers. BIACORE method is an alternative in vitro method to detect the mucoadhesive property of polymers. The advantage of BIACORE method is that the mucoadhesive function of the polymers can be detected by using a very small amount of the polymer. It was also reported that the mucoadhesive property of particulate systems was successfully evaluated by the particle counting method with Coulter counter and the CLSM observation. The latter method was very useful to confirm retention of the chitosan-coated liposomal systems having mucoadhesive function in the intestinal tract of the rat. This method was helpful to estimate the mechanism of oral delivery of peptide drugs such as insulin and calcitonin with the particulate systems.
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Acknowledgment This study was supported in part by a Grant-in-Aid for Scientific Research (B-14370731) from the Japan Society for the Promotion of Science.
References [1] P.K. Guputa, S.-H.S. Leung, J.R. Robinson, Bioadhesives/ mucoadhesives in drug delivery to the gastrointestinal tract, in: V. Lenaerts, R. Gurny (Eds.), Bioadhesive Drug Delivery Systems, CRC Press, Florida, 1990, pp. 65 – 92. [2] H. Takeuchi, H. Yamamoto, T. Niwa, T. Hino, Y. Kawashima, Mucoadhesion of polymer-coated liposomes to rat intestine in vitro, Chem. Pharm. Bull. 42 (1994) 1954 – 1956. [3] H. Takeuchi, H. Yamamoto, T. Niwa, T. Hino, Y. Kawashima, Enteral absorption of insulin in rats from mucoadhesive chitosan-coated liposomes, Pharm. Res. 13 (1996) 896 – 901. [4] 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. Release 86 (2003) 235 – 242. [5] C.M. Lehr, J.A. Bouwstra, J.J. Tukker, H.E. Junginger, Intestinal transit of bioadhesive microspheres in an in situ loop in the rat—a comparative study with copolymers and blends based on poly acrylic (acid), J. Control. Release 13 (1990) 51 – 62. [6] C.M. Lehr, J.A. Bouwstra, W. Kok, A.G. De Boer, J.J. Tukker, J.C. Verhoef, D.D. Breimer, H.E. Junginger, Effects of the mucoadhesive polymer polycarbophil on the intestinal absorption of a peptide drug in the rat, J. Pharm. Pharmacol. 44 (1992) 402 – 407. [7] Y. Akiyama, N. Nagahara, T. Kashihara, S. Hirai, H. Toguchi, In vitro and in vivo evaluation of mucoadhesive microspheres prepared for the gastrointestinal tract using polyglycerol esters of fatty acids and a poly(acrylic acid) derivative, Pharm. Res. 12 (1995) 397 – 405. [8] V. Lenaerts, P. Couvreur, L. Grislain, P. Maincent, Nanoparticles as a gastroadhesive drug delivery system, in: V. Lenaerts, R. Gurny (Eds.), Bioadhesive Drug Delivery Systems, CRC Press, Florida, 1990, pp. 93 – 104. [9] C. Pimienta, F. Chouinard, A. Labib, V. Lenaerts, Effect of various poloxamer coatings on in vitro adhesion of isohexylcyanoacrylate nanospheres to rat ileal segments under liquid flow, Int. J. Pharm. 80 (1992) 1 – 8. [10] N.A. Peppas, A.G. Mikos, Experimental methods for determination of bioadhesive bond strength of polymers with mucus, STP Pharma 5 (1989) 187 – 191. [11] H.S. Ch’ng, K. Park, P. Kelly, J.R. Robinson, Bioadhesive polymers as platforms, J. Pharm. Sci. 74 (1985) 399 – 406. [12] G. Ponchel, F. Touchard, D. Duchene, N.A. Peppas, Bioadhesive analysis of controlled-release systems, J. Control. Release 5 (1987) 129 – 141. [13] J.D. Smart, I.W. Kellaway, H.E.C. Worthington, An in-vitro investigation of mucosa-adhesive materials for use in con-
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H. Takeuchi et al. / Advanced Drug Delivery Reviews 57 (2005) 1583–1594 trolled drug delivery, J. Pharm. Pharmacol. 36 (1984) 295 – 299. E.E. Hassan, J.M. Gallo, A simple rheological method for the in vitro assessment of mucin–polymer bioadhesive bond strength, Pharm. Res. 7 (1990) 491 – 495. H. Takeuchi, Y. Matsui, H. Yamamoto, Y. Kawashima, A novel in vitro test for mucoadhesiveness of polymers using commercially available mucin particles for development of particulate mucoadhesive systems, Proceedings of Strategies of Optimizing Oral Drug Delivery, 1999, p. 164. J. Thongborisute, H. Yamamoto, H. Takeuchi, Y. Kawashima, Study on mucoadhesive property of chitosan coated liposomes, Proceedings of annual meeting of the 123rd Pharmaceutical Society of Japan, vol. 4, 2003, p. 78. BIACORE J instrument handbook. D.G. Myszka, M.D. Jonsen, B.J. Graves, Equilibrium analysis of high affinity interactions using BIACORE, Anal. Biochem. 265 (1998) 326 – 330. V. Sikavitsas, J.M. Nitsche, T.J. Mountziaris, Transport and kinetic processes underlying biomolecular interactions in the BIACORE optical biosensor, Biotechnol. Prog. 18 (2002) 885 – 897.
[20] K.V. Ranga Rao, P. Buri, A novel in situ method to test polymers and coated microparticles for bioadhesion, Int. J. Pharm. 52 (1989) 265 – 270. [21] C.M. Lehr, J.A. Bouwstra, J.J. Tukker, H.E. Junginger, Intestinal transit of bioadhesive microspheres in an in situ loop in the rat—a comparative study with copolymers and blends based on poly(acrylic acid), J. Control. Release 13 (1990) 51 – 62. [22] C. Durrer, J.M. Irache, F. Puisieux, D. Duchene, G. Ponchel, Mucoadhesion of latexes: I. Analytical methods and kinetic studies, Pharm. Res. 11 (1994) 674 – 679. [23] C. Durrer, J.M. Irache, F. Puisieux, D. Duchene, G. Ponchel, Mucoadhesion of latexes: II. Adsorption isotherms and desorption studies, Pharm. Res. 11 (1994) 680 – 683. [24] J. Kreuter, U. Muller, K. Munz, Quantitative and microautoradiographic study on mouse intestinal distribution of polycyanoacrylate nanoparticles, Int. J. Pharm. 55 (1989) 39 – 45. [25] H. Takeuchi, H. Sugihara Y. Matsui, H. Yamamoto, Y. Kawashima, Effectiveness of submicron-sized, chitosan-coated liposomes in oral administration of peptide drugs. Int. J. Pharm. (in press).
Novel mucoadhesion tests for polymers and polymer-coated particles to design optimal mucoadhesive drug delivery systemsB Hirofumi Takeuchi *, Jringjai Thongborisute, Yuji Matsui, Hikaru Sugihara, Hiromitsu Yamamoto, Yoshiaki Kawashima Laboratory of Pharmaceutical Engineering, Gifu Pharmaceutical University, Japan 5-6-1 Mitahora-Higashi Gifu, 502-8585, Japan Received 27 August 2004; accepted 12 July 2005 Available online 16 September 2005
Abstract To design an effective particulate drug delivery system having mucoadhesive function, several mucoadhesion tests for polymers and the resultant particulate systems were developed. Mucin particle method is a simple mucoadhesion test for polymers, in which the commercial mucin particles are used. By measuring the change in particle size or zeta potential of the mucin particle in a certain concentration of polymer solution, we could estimate the extent of their mucoadhesive property. BIACORE method is also a novel mucoadhesion test for polymers. On passing through the mucin suspension on the polymerimmobilized chip of BIACORE instrument, the interaction was quantitatively evaluated with the change in its response diagram. By using these mucoadhesion tests, we detected a strong mucoadhesive property of several types of chitosan and Carbopol. Evaluation of mucoadhesive property of polymer-coated particulate systems was demonstrated with the particle counting method developed by us. To detect the mucoadhesive phenomena in the intestinal tract, we observed the rat intestine with the confocal laser scanning microscope (CLSM) after oral administration of the particulate systems. The resultant photographs clearly showed a longer retention of submicron-sized chitosan-coated liposomes (ssCS-Lip) in the intestinal tract than other liposomal particles tested such as non-coated liposomes and chitosan-coated multilamellar one. These observations explained well the superiority of the ssCS-Lip as drug carrier in oral administration of calcitonin in rats than other liposomal particles. D 2005 Elsevier B.V. All rights reserved. Keywords: Mucoadhesion; Mucin particle; BIACORE; Confocal laser scanning microscopy; Peptide drug; Oral administration
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mucin particle method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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B This review is part of the Advanced Drug Delivery Reviews theme issue on ‘‘Mucoadhesive Polymers: Strategies, Achievements and Future Challenges’’, Vol. 57/11, 2005. * Corresponding author. Tel.: +81 58 237 3931; fax: +81 58 237 5979. E-mail address: [email protected] (H. Takeuchi).
0169-409X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2005.07.008
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2.1. Interaction between mucin particles and polymer . . . . . . . . . . . . . . 2.2. Quantitative analysis of the interaction between mucin particle and chitosan 3. Mucoadhesion test with BIACORER . . . . . . . . . . . . . . . . . . . . . . . . 3.1. BIACORER system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Response of chitosan–mucin interaction . . . . . . . . . . . . . . . . . . . 3.2.1. Preparation of ss-mucin suspension for BIACORE experiment . . . 3.2.2. Immobilization of chitosan on the sensor chip . . . . . . . . . . . 3.2.3. Interaction between chitosan and ss-mucin . . . . . . . . . . . . . 3.3. Adhesion test for various polymers . . . . . . . . . . . . . . . . . . . . . 4. Mucoadhesion tests for fine particulate systems. . . . . . . . . . . . . . . . . . . 4.1. Mucoadhesion tests for coarse and fine particles. . . . . . . . . . . . . . . 4.2. Particle counting method for fine particles . . . . . . . . . . . . . . . . . . 4.3. Confocal laser scanning microscopy (CLSM) method . . . . . . . . . . . . 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Colloidal drug carriers, such as liposomes or nanoparticles of biodegradable polymers, have received much attention for their ability to improve the absorption of poorly absorbable drugs, including peptide drugs. It has been reported that the mucoadhesive properties of these particulate systems can prolong their retention in the gastrointestinal tract, thus further improving drug absorption [1]. We have demonstrated a novel mucoadhesive liposomal system prepared by coating the liposome surface with a mucoadhesive polymer, chitosan. The effectiveness of the chitosan-coated liposomes (CS-Lip) was confirmed by the enhanced and prolonged pharmacological effect of insulin, which was orally administered in the polymer-coated liposomal form in rats [2,3]. The effectiveness of mucoadhesive liposomes in drug absorption was also demonstrated by using calcitonin as a model peptide drug. Carbopol-coated liposomes, having a mucoadhesive property similar to that of CSLip, were as effective as CS-Lip [4]. A particulate bioadhesive system was also prepared by coating microspheres of poly-hydroxyethyl-methacrylate with mucoadhesive polymers using laboratoryscale equipment [5,6]. Akiyama et al. [7] prepared a polyglycerol ester of fatty acid-based microspheres coated with Carbopol934P (CP) and CP-dispersing microspheres to evaluate their mucoadhesive properties. In developing colloidal drug delivery systems, Lenaerts et al. [8] demonstrated the mucoadhesive
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property of polyalkylcyanoacrylate nanoparticles with autoradiographic studies and confirmed that the bioavailability of vincamine was improved in nanoparticulate systems. Pimienta et al. [9] investigated the bioadhesion of hydroxypropylmethacrylate nanoparticles or isohexylcyanoacrylate nanocapsules, the latter coated with poloxamers and poloxamine on rat ileal segments in vitro using a labeled compound. To design optimal mucoadhesive drug delivery systems such as polymer-coated liposomes, it is necessary to develop simple mucoadhesion tests that can evaluate the mucoadhesive properties of polymers. The development of mucoadhesion tests for particulate systems is also important. The most direct method is to measure the bioadhesive bond strength between a polymer and the mucosal layer of animals [10]. Ch’ng et al. [11] demonstrated the feasibility of this concept by using a polymer specimen and freshly excised stomach tissue from an animal. Ponchel et al. [12] developed a similar method to measure tensile force. Smart et al. [13] proposed the use of the Wilhelmy plate method, which is usually applied to determine surface tension. A rheological method proposed by Hassan and Gallo [14] is an alternative way to assess mucin–polymer adhesive bond strength. They evaluated bioadhesion forces by monitoring viscometric changes in a mixture of porcine gastric mucin and polymers in solution. In this paper we propose two types of novel mucoadhesion tests for polymers. We also report on tests we developed for mucoadhesive particulate systems.
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Conc. of Carbopol (%)
2. Mucin particle method -10
We tried to evaluate the mucoadhesion of polymers with commercially available porcine mucin particles [15]. In the test, mucin particles were suspended in suitable buffer solutions with a concentration of 1% w/v and then mixed with an appropriate amount of polymer solutions. It was expected that the surface property of the mucin particles might be changed by the adhesion of the polymer if the polymer has a mucoadhesive property. The occurrence of such change was detected by measuring the zeta potential with a Zetamaster (Malvern Instruments, UK). When the coarse mucin particle suspensions were mixed with the solutions of several types of chitosan (CS) and Carbopol (CP), the zeta potential of the mucin particles changed in each case (Figs. 1 and 2). On the other hand, when hydroxylpropylmethylcellulose (HPMC) solution was added to the mucin suspension instead of CS or CP solution, the zeta Conc. of chitosan (%) 0.5
1
-10 CS-2
-20
CS-5
(c)
Conc. of chitosan (%)
10 0 -10 -20 -30
0.8
1
-15 C-971P C-974P
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N-AA1
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Conc. of chitosan (%) 0
0.5
1
1.5
10 0 -10 -20
CS-15
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20
0.6
potential was unchanged. These results suggested that either CS or CP had a high affinity to mucin particles to cover their surfaces. The higher the concentration of CS and the greater its molecular weight, the more
(b)
0
0.4
Fig. 2. Zeta potential of coarse mucin particles in the solutions of Carbopol with various concentrations, pH 6.8. Molecular weigh of C-971P and C-974P are 1,250,000 and 3,000,000, respectively.
1.5
10
0.2
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0.5
1
Conc. of chitosan (%)
(d)
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20
Zeta potential (mV)
Zeta potential (mV)
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Zeta potential (mV)
2.1. Interaction between mucin particles and polymer
Zeta potential (mV)
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10 0 -10 -20 -30
Fig. 1. Zeta potential of coarse mucin particles in the solutions of chitosan having different molecular weight with various concentrations and different pH. (a) pH 5.0, (b) pH 6.8, (c) pH 7.4, (d) pH 9.0. Molecular weigh of chitosan: CS-2, 20,000; CS-5, 50,000; CS-15, 150,000.
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Relative size
80
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firmed that the adsorption of CS molecules on the mucin particles were responsible for the aggregation. The mucoadhesive properties of various types of CP were evaluated with this method, and the results suggested that the C-974-P was more adhesive than the other types of CP tested (data not shown). 2.2. Quantitative analysis of the interaction between mucin particle and chitosan
-60
0 Micronized mucin
CS-2
CS-5
CS-15
Fig. 3. Change in observed particle size of micronized mucin particles when mixed with the chitosan solutions. Concentration of chitosan solution: 1.5% w/v. pH of solution: 6.8.
extensive the changes were in the zeta potential (Fig. 1). Positive zeta potential was observed in the acidic solution (pH 5.0), possibly owing to the dissociated amino groups of CS on the surface of mucin particles (Fig. 1). In the case of Carbopol, the resultant zeta potential of mucin shifted to a higher negative value because of the negative charge of the polymer (Fig. 2). These results strongly supported what happened in the mixture and the feasibility of the mucin particle method for evaluating the mucoadhesive property of polymers. A modified mucin particle method was performed with the use of submicron-sized mucin particles (ssmucin, ca. 200–300 nm in particle diameter) and particle size analyzers (LPA 300, Otuska-Denshi or Cis-1, Galai Inc.). The ss-mucin were prepared by applying sonication to the coarse mucin suspension. When the suspension of ss-mucin particles was mixed with a polymer solution, the mucin particles may aggregate if the polymer has a strong affinity to them, i.e., a mucoadhesive property. When the ssmucin particles were mixed with CS solution, the particle size increased as expected. The extent of change in particle size was presented as the relative particle size, which was calculated by dividing the size of the aggregates by that of the original ss-mucin (Fig. 3). It was found that the aggregation tendency depended on the molecular weight of the CS, which corresponded well with the results observed in the coarse mucin particle test described above. The zeta potential of mucin particles shifted towards zero with increasing the aggregation tendency (Fig. 3). It con-
The quantitative analysis of the mucin particle method was carried out by controlling the amount of polymer added and then analyzing the zeta potential of the ss-mucin [16]. The particle size and zeta potential of the precisely size-controlled ss-mucin were 361 nm and 25.4 mV, respectively. The interaction between ss-mucin and the chitosan molecule was determined by mixing a known amount of ss-mucin with an appropriate 0.05% w/v of chitosan solution. Then the zeta potential value was measured in 10 mM acetate buffer solution, ABS, at pH 4.5. The indication of this electrostatic interaction is shown in the zeta potential value of ss-mucin after it was mixed with chitosan solution (Table 1). The zeta potential of ss-mucin was negative before the mixing. By increasing the amount of 0.05% w/v chitosan solution added, the zeta potential value of ss-mucin gradually changed from the negative to the positive until the critical ratio of 1:1 was reached. At that point, where the zeta potential value was nearly zero, the negative charge of the ss-mucin particles would be neutralized with the positive charge of the chitosan molecules adsorbed on their surface. Base on this hypothesis, we were able to calculate the occupying area of one chitosan molecule on the surface of a mucin particle. This area, calculated using
Table 1 Zeta potential of ss-mucin particles after mixing with various amounts of chitosan solution (n = 3) SS-mucin: 0.05% CS solution
Zeta potential in ABS, pH 4.5 (mV)
1 1 1 1 1
8.40 F 2.0 0.5 F 0.3 1.7 F 0.4 21.1 F 9.3 22.0 F 10.9
0 0.5 1 3 5
ss-mucin: submicron-sized mucin, ABS: acetate buffer solution.
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3. Mucoadhesion test with BIACORER 3.1. BIACORER system The BIACORE instrument is based on the principle underlying an optical phenomenon called Surface Plasmon Resonance (SPR). The SPR response is a measurement of the refractive index, which varies with the solute content in a solution that contacts a sensor chip. When a detected molecule is attached to the surface of the sensor chip, or when the analyte binds to the detected molecule, the solute concentration on the sensor chip surface increases, leading to an SPR response [17]. Quantitative measurements of the binding interaction between the chip surface and one or more molecules depend on the immobilization of target molecules that are in contact on the sensor surface. The sensor chip consists of a glass surface coated in a thin layer of gold. This forms the basis for a range of specialized surfaces designed to optimize the binding of a variety of molecules. The most widely used sensor chip is CM5 (BIACORER, Sweden), whose surface is modified with a carboxymethylated dextran layer. In general, the ligand can covalently bind to the sensor chip surface via carboxyl moieties on the dextran. Functional groups on the ligand that can be used for coupling include NH2, SH, CHO and COOH. The major advantages of the BIACORE instrument are its label-free detection of binding and the ability to monitor the change in response in real time [18,19]. In the detection of the mucoadhesive property of polymers using BIACORE, each polymer was immobilized on the surface of the sensor chip CM5 and the mucin suspension was passed through the sensor chip. When the analyte (mucin particle) binds to the ligand molecule (polymer) on the sensor chip surface, the solute concentration and the refractive index on that surface change, increasing the RU response; when
they dissociate, the RU response will fall. After that, the analyte can be removed from the ligand by using a regenerating reagent. The response will then turn back to the equilibrium state as the beginning step. 3.2. Response of chitosan–mucin interaction 3.2.1. Preparation of ss-mucin suspension for BIACORE experiment We prepared 1.0% w/v of mucin suspension by suspending and continuously stirring mucin powder in 10 mM acetate buffer solution (ABS) at pH 4.5 overnight. After incubation at 37 8C for one night, the suspension was ultrasonicated with a probe sonicator (Branson Sonifier 250) until the particle size was less than 1 Am. It was then centrifuged at 4000 rpm for 20 min to extract submicron-sized mucin particles in the supernatant portion, which was then diluted to a concentration of 0.01% w/v with 10 mM ABS, pH 4.5. 3.2.2. Immobilization of chitosan on the sensor chip Chitosan was immobilized on the CM5 chip surface by using amine-coupling chemistry. The immobilization was carried out at a flow rate of approximately 15 Al/min in 10 mM ABS, pH 4.5. Initially, the sensor chip surface was activated for 6 min with a mixture of 100 mM N-hydroxysuccinimide (NHS) and 400 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and then chitosan was injected at a concentration of 0.02% w/v across the activated surface for 10 min. Afterwards, the remaining reactive esters were transformed into amides by injection of 1 M of ethanolamine HCl, pH 8.5, for 6 min. Baseline data were
Resonance unit (RU)
the respective particle-size averages and the observed critical ratio, was about 66.5 Am2. This value was reasonable, considering CS’s molecular weight of 150,000. This method was also successfully applied to evaluate the coating phenomenon of the particulate systems with chitosan in developing the mucoadhesive systems.
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23000 22000 21000 20000 19000 18000 17000 CS baseline 16000 15000 0 500 1000 1500 2000 2500 3000 3500
Time (s) Fig. 4. The sensorgram for chitosan-immobilized sensor chip after passing the ss-mucin suspension. z start injection, A stop injection. Molecular weight of chitosan: 150,000.
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7000
interaction between submicron-sized mucin and chitosan on the chip because of the increase in the RU response after injection, which was the result of the change in the refractive index on the chip. The RU response decreased when the chitosan solution was injected again across the mucin-interacted sensor chip coated with chitosan (Fig. 5). This change suggested that a part of the mucin particle fixed to the chip surface was removed by the interaction with the newly injected chitosan molecules. In other words, this decrease in response may be an indicator of a polymer’s mucoadhesive property. To confirm this phenomenon, chitosan solutions with different molecular weights (MW 20,000 and 150,000) were injected after submicron-sized mucin (Fig. 5). As shown in these sensorgrams, the change in RU response depended on the molecular weight of the chitosan. These sensorgrams indicated that the chitosan with the higher molecular weight possessed the higher mucoadhesive property.
M
Resonance unit (RU)
6000 5000
CS2
4000
CS15
3000 CS2
2000 1000 0 0
CS15
M M 1000
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3000
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Time (s) Fig. 5. Overlay of the sensorgrams for the ss-mucin-immobilized sensor chip after injection of the chitosan solutions with MW 20,000 and MW 150,000. zM, AM: start and stop mucin injection, z CS2, and A CS2: start and stop chitosan MW 20,000 injection, z CS15, and A CS15: start and stop chitosan MW 150,000 injection.
collected at least 30 min before the experiment began. The immobilization process used 0.02% w/v of chitosan solutions with various molecular weights (dimer, tetramer, hexamer, and chitosan MW 150,000), and then 0.01% w/v of ss-mucin was passed over the sensor chip surface for 10 min. Afterwards, the sensorgram was collected until equilibrium was reached.
3.3. Adhesion test for various polymers To confirm the feasibility of this BIACORE-based mucoadhesion test, various polymers were applied to it. In each case, 0.02% w/v of mucin was injected for 10 min across the surface of the sensor chip after the chitosan immobilization process (chitosan MW 150,000). Then, 0.02% w/v of each polymer was passed over the sensor chip surface for 10 min. To change the polymer solution, mucin and the former
3.2.3. Interaction between chitosan and ss-mucin Fig. 4 shows the RU response detected as the ssmucin suspension was passed over the immobilized chitosan. The sensorgram in the figure shows the 18000
Chitosan baseline
Responses after injecting adhesive polymers
M
Arbitrary Units of RU
16000
P P
14000
P P
12000 10000
P
8000 6000 4000
Carbopol 971 PNF
P
Carbopol 974 PNF
P
PVA 205
P
P
PVP 25
P
P
TC-5-S
P
P
90SH-400
2000
65SH-50
P
0 0 -2000
M
1000
2000
3000
4000
Time (s) Fig. 6. Overlay of the sensorgrams for ss-mucin-immobilized sensor chip after injection of the various adhesive polymer solutions. zM, AM: start and stop mucin injection, zP, AP start and stop adhesive polymer injection.
H. Takeuchi et al. / Advanced Drug Delivery Reviews 57 (2005) 1583–1594
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50
Adhesive %
40
C
Chitosan
30 n
PAA-R 20 C
C
C n
C
10
PVA-R
PAA-R
Chitosan
Uncoated
C Cho
PVA-R 0
C
C
C
C n
Fig. 7. Adhesive % of polymer-coated liposomes to rat intestine evaluated with the particle-counting method. Lipid composition: DPPC/ DCP=8:2. Polymer concentration: 0.75%. Dispersion medium: phosphate buffer solution (pH 7.4).
polymer were removed with regenerating reagent as explained above. Fig. 6 summarizes the response changes after various polymers are injected. In this figure, the starting and stopping injection time point of each polymer were not the same time because each sensorgram was collected until re-equilibrium before injecting the new polymer. The decreasing in RU responses were calculated by using the following equation.
4. Mucoadhesion tests for fine particulate systems 4.1. Mucoadhesion tests for coarse and fine particles
% RU decrease RU from mucin baseline to polymer RU from chitosan baseline to mucin
¼
molecular weights. Like chitosan, Carbopol showed much larger decreases in RU response, whereas the responses of the other polymers were slight. These results correspond well with the results of other mucoadhesion tests.
100:
ðaÞ
Table 2 lists the values of decreases in RU response for these polymers, including chitosan at different
Table 2 Percentage of RU response decrement after injection of various adhesive polymer solutions Polymer
% RU decrease
Chitosan MW 20,000 Chitosan MW 150,000 Carbopol 971 PNF Carbopol 974 PNF PVA 205 PVP25 (Kollidon25) TC-5 S (HPMC 2910) 90SH-400 (HPMC 2208) 65SH-50 (HPMC 2906)
22.01 46.34 29.58 27.57 3.26 7.92 6.78 9.58 9.60
Various methods have been proposed to measure the mucoadhesive properties of particulate systems. Ranga Rao and Buri [20] demonstrated a simple method for coarse particulate systems, such as polymercoated glass beads and drug crystals. In their adhesion test, particles were placed on rat jejunum or stomach in a humid environment in vitro. The percent of particles retained on the tissue was considered an index of bioadhesion. Lehr et al. [21] used an in situ perfused ileal loop in the rat to study the mucoadhesion of coarse particles. In that study, polymercoated microspheres were perfused with isotonic saline, and the number of collected particles was counted. To evaluate the mucoadhesion of fine particles, Durrer et al. [22,23] investigated the adsorption isotherms of poly(styrene) latex delivered to rat intestinal mucosa under static conditions by use of turbidimetric and FTIR-ATR analyses. The use of labeled compounds is one way to estimate the mucoadhesion of
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particulate systems, including liposomes. The particle movement profiles in the mouse intestinal tract were determined after oral administration of polyhexyl[3-14C]-cyanoacrylate) nanoparticles; the recovery of radioactivity in those tissues was measured at appropriate intervals [23]. Ponchel et al. [24] also studied the intestinal mucoadhesion profiles of 14C-labeled polylactic acid (PLA) particles (mean diameter: 1.5 Am) as a function of time after PLA administration in rat. 4.2. Particle counting method for fine particles We tried to develop a simpler in vitro method (particle-counting method) to evaluate the mucoadhesion of fine particles, such as polymer-coated liposomes [2,3]. In this method, an intestinal tube was isolated from Wistar rats and cut into three parts, named as upper, middle, lower, from stomach side. The length of each intestinal tube was 15 cm. Each tube was washed inside with saline solution filled with liposomal suspensions diluted by 100 times with an appropriate buffer solution. After being sealed with closers, the tube was incubated in saline solution at 37 8C for more than 15 min. By measuring the number of liposomal particles before and after incubation with a Coulter counter, the mucoadhesive % was presented by the following equation. Adhesive % ¼ ðNo NsÞ=No 100
ðbÞ
where No and Ns are the number of liposomes before and after incubation, respectively. 80
As shown in Fig. 7, CS-coated liposomes showed the highest adhesive % among the three different polymer-coated liposomes tested. The mucoadhesive function was thought to be provided by the polymer layer fixed on the liposome surface, because uncoated liposomes showed no adhesive %. Interpenetration between the polymeric and the mucosal networks is considered to be responsible for the adhesion. The adhesive % of CS-coated liposomes depended on the CS concentration used for coating. Considering the combined results, it could be concluded that the more effective coating leads to the higher adhesive %. The mucoadhesive properties of positively and negatively charged liposomes were compared by the particle-counting method (Fig. 8) [4]. The positively charged liposomes showed a higher adhesive % to the mucosal layer than the negatively charged one; this was attributed to the negative charging of the surface of the mucosal layer of the intestine. However, the CP-coated and thus negatively charged liposomes, as well as the positively charged CS-coated liposomes, showed higher adhesive % than the positively charged uncoated liposomes. This result suggested that a physical entanglement between the polymer and mucosal layer is an important factor to facilitate the mucoadhesion of polymer-coated liposomes. 4.3. Confocal laser scanning microscopy (CLSM) method It is important to characterize the mucoadhesive properties of fine particulate systems in vivo. In a
In Saline (pH6.1)
70
Adhesive (%) =
Adhesive (%)
60
N0 – N15
×100
N0
50 N0: Number of initial liposomes. N15: Number of liposomes after 15 min
40 30 20
Negatively charged MLVs
10
Positively charged MLVs Carbopol coated MLVs
0 Upper
Middle
Lower
Chitosan coated MLVs
Fig. 8. Adhesive % of several liposomes to rat intestine evaluated with the particle-counting method. Lipid compositions; DPPC/DCP=8:2, DPPC/SA=40:1, Carbopol; C-971P, Chitosan; CS-15 (DSP: distealoylphosphatidylcholine, DCP: dicetyl phosphate, SA: stearylamine).
H. Takeuchi et al. / Advanced Drug Delivery Reviews 57 (2005) 1583–1594
(a)
CS-Lip 4.6µm
(b)
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ssCS-Lip (339nm)
Lumen
Mucosal layer
200µm
Jejunum
Ileum
Jejunum
Ileum
Fig. 9. Confocal laser scanning microscopy photographs of the jejunum and ileum of the rat intestine 2 h after intragastric administration of chitosan-coated liposomes: (a) CS-Lip and (b) ssCS-Lip. The measured mean diameters of the liposomes are 4.6 Am (a) and 339.2 nm (b), respectively. The formulation of liposomes: DSPC/DCP/Chol. = 8:2:1. The concentration of chitosan in the coating is 0.3% w/v (DSPC: distealoylphosphatidylcholine, DCP: dicetyl phosphate, Chol.: cholesterol).
previous study, we examined the mucosal layer of rat intestine in order to detect liposomes by using confocal laser scanning microscopy (CLSM) after administering these particulate systems [25]. For this purpose, a fluorescence marker, 1,1V-dioctadecyl-3, 3,3V,3V-tetramethylindo carbocyanine perchlorate (DiI, LAMBDA, Austria), was formulated into the liposome particles. The chitosan-coated liposomes (CSLip) was prepared by mixing the non-coated liposomal suspension (Lip) with the same volume of acetate buffer solution (pH 4.4) of CS (0.6%), followed by incubation at 10 8C for 1 h. The submicron-sized CSLip ssCS-Lip were prepared by using the submicronsized liposomes (ssLip), which were prepared by sonicating (Sonifier250, Branson) the multilamellar liposomes. The intestinal tubes were removed from the rats at the appropriate time after administration of the liposomes. Each tube was cut into five parts: the duodenum, upper jejunum, lower jejunum, upper ileum, and lower ileum. Each part was sliced with a cryostat (Leica, Germany) to generate sections for
confocal laser scanning microscopic observation. The thickness of the sections was 10 Am. Each sample was placed on a confocal laser scanning microscope (LSM510, Carl Zeiss, Germany) and observed at an excitation wavelength of 550 nm and an emission wavelength of 570 nm. The mucoadhesion profiles of the chitosan-coated liposomes (CS-Lip) and submicron-sized CS-Lip (ssCS-Lip) in the intestinal tube were evaluated by observing the residual liposomes on the mucosa. The higher mucoadhesive tendency of ssCS-Lip compared with CS-Lip was confirmed by comparing the resultant photographs in Fig. 9. At the jejunum, small amounts of CS-Lip particles were observed, while large amounts of ssCS-Lip were detected there. In the ileum, the retention of the liposomal particles seemed almost the same regardless of their size, but ssCS-Lip tended to penetrate deeply into the mucosal part of the intestine, whereas CS-Lip did not. A similar size dependency was observed when we measured the mucoadhesive properties of the
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Lip (4.1µm)
(a)
(b)
ssLip (182nm)
Lumen
Mucosal layer
200µm
Jejunum
Jejunum
Ileum
Ileum
Calcemia (% of the initial value)
Fig. 10. Confocal laser scanning microscopy photographs of the jejunum and ileum of the rat intestine 2 h after intragastric administration of non-coated liposomes: (a) Lip and (b) ssLip. The measured mean diameters of the liposomes are 4.1 Am (a) and 181.9 nm (b), respectively. The formulation of liposomes: DSPC/DCP/Chol. = 8:2:1 (DSPC: distealoylphosphatidylcholine, DCP: dicetyl phosphate, Chol.: cholesterol). Calcitonin solution (Ctrl) ssLip ssCS-Lip
110 105 100 95 90
*
85
*** * † *
*
80
* † †
†
*
*
75 0
24
48
72
96
120
Time (hr) Fig. 11. Profiles of plasma calcium levels after intragastric administration of submicron-sized liposomes, ssLip and ssCS-Lip, containing calcitonin. The measured mean particles sizes of ssLip and ssCS-Lip are 196.4 nm and 473.4 nm, respectively. The formulation of liposomes is DSPC/DCP/Chol. = 8:2:1. The concentration of chitosan for coating is 0.3%. *p b 0.05, **p b 0.01, ***p b 0.001: significantly different from the level for calcitonin solution; yp b 0.05, yyp b 0.01: significantly different from the level for ssLip (n = 3 in each case).
uncoated liposomes with different particle sizes, Lip and ssLip (Fig. 10). Although the retained amount of uncoated liposomes was lower than that of chitosancoated liposomes, ssLip showed a penetrative behavior similar to that of ssCS-Lip. It was confirmed that ssLip had lower retention than ssCS-Lip, since only a small amount of ssLip was observed at the jejunum. According to the retention profiles of these particles in the intestinal tract, CS-Lip was more retentive than Lip. In both cases, we also confirmed that the retention profile was enhanced by decreasing the particle size from the micron to the submicron (ca. 200–300 nm) range. It was also found that the submicron-sized liposomes penetrated the mucosa, whereas no such penetrative profile was observed for the larger liposomes. This penetrative property is what gives ssCS-Lip a much higher retentive property than CS-Lip. We previously demonstrated the effectiveness of CS-coated liposomes in the absorption of peptide drugs, such as insulin and calcitonin, in rats [2–4].
H. Takeuchi et al. / Advanced Drug Delivery Reviews 57 (2005) 1583–1594
Recently, we observed that the reduction in particle size of the liposomal system led to a large improvement in drug absorption. In the administration of calcitonin (500 IU/kg rat) with the liposomal formulation, the period of reduced calcium concentration in the blood was prolonged to 120 h after administration by reducing the particles to submicron size (ssCS-Lip) (Fig. 11). The period was much longer than that observed when CS-Lip were used as carriers of calcitonin under the same conditions. Comparison of these profiles confirms the particle size effect on calcitonin absorption. The uncoated ssLip were also found to be effective in reducing the calcium level under the same experimental conditions, although the extent was much less than that of ssCS-Lip, as shown in Fig. 12. These pharmacological effects of calcitonin administered with the liposomal formulations corresponded well with their retention profiles in the intestinal tract observed with the CLSM method. CLSM observation may be a promising method to characterize the mucoadhesive properties of fine particle drug delivery systems and to explain their effectiveness in the oral administration of drugs.
5. Conclusion In the present paper, novel mucoadhesion tests were proposed to design optimal particulate drug delivery systems having the mucoadhesive function. Mucin particle method is a simple in vitro test to detect the mucoadhesive property of polymers. BIACORE method is an alternative in vitro method to detect the mucoadhesive property of polymers. The advantage of BIACORE method is that the mucoadhesive function of the polymers can be detected by using a very small amount of the polymer. It was also reported that the mucoadhesive property of particulate systems was successfully evaluated by the particle counting method with Coulter counter and the CLSM observation. The latter method was very useful to confirm retention of the chitosan-coated liposomal systems having mucoadhesive function in the intestinal tract of the rat. This method was helpful to estimate the mechanism of oral delivery of peptide drugs such as insulin and calcitonin with the particulate systems.
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Acknowledgment This study was supported in part by a Grant-in-Aid for Scientific Research (B-14370731) from the Japan Society for the Promotion of Science.
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