12Investigation of the interactions in complex of low molecular weight

J Solution Chem (2009) 38: 695–712 DOI 10.1007/s10953-009-9405-4

Investigation of the Interactions in Complexes of Low Molecular Weight Chitosan with Ibuprofen Amjad M. Qandil · Aiman A. Obaidat · Muaadh A. Mohammed Ali · Bashar M. Al-Taani · Bassam M. Tashtoush · Nawzat D. Al-Jbour · Mayyas M. Al Remawi · Khaldoun A. Al-Sou’od · Adnan A. Badwan Received: 18 November 2008 / Accepted: 21 January 2009 / Published online: 9 April 2009 © Springer Science+Business Media, LLC 2009

Abstract Complexation between ibuprofen and low molecular weight chitosan (LMWC) was studied. LMWC was prepared from high molecular weight chitosan using the acid hydrolysis method. The complexes were investigated by using DSC, FT-IR and liquid-state 1 H-NMR. Molecular mechanics (MM) calculations were used to give insight into the stoichiometry of the interaction of chitosan with ibuprofen. The results showed that complexation of ibuprofen with LMWC involves ionic interaction between the ammonium group of LMWC and the carboxylate anion of ibuprofen. It was also shown that it is more efficient to prepare the complexes using lower concentration solutions of the polymer. These results were supported by molecular mechanics calculations. The experimental results may explain the discrepancies in the literature where, in many studies, the concentration of chitosan and its low average molecular weight were not considered to be important factors in the complexation process. Keywords Low molecular weight chitosan (LMWC) · Ibuprofen · NMR · FT-IR · DSC · Molecular mechanics

A.M. Qandil () Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan e-mail: [email protected] A.A. Obaidat · M.A.M. Ali · B.M. Al-Taani · B.M. Tashtoush Department of Pharmaceutical Technology, Faculty of Pharmacy, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan N.D. Al-Jbour · M.M. Al Remawi · K.A. Al-Sou’od · A.A. Badwan Jordanian Pharmaceutical Manufacturing Company, P.O. Box 94, Naor 1170, Jordan K.A. Al-Sou’od Department of Chemistry, Faculty of Science, Al al-Bayt University, P.O. Box 130040, Mafraq 25113, Jordan

696

J Solution Chem (2009) 38: 695–712

1 Introduction Chitosan is a hydrophilic polymer that is composed of (1 → 4)-2-acetamido-2-deoxy-β-Dglucopyrasonyl and (1 → 4)-2-amino-2-deoxy-β-D-glucopyrasonyl units, and is produced by the chemical or enzymatic depolymerization of chitin (which is among the most abundant natural polymers) [1–3]. Chitosan has been investigated for various potential applications in the food, cosmetic, and pharmaceutical industries [4–6]. It has been widely investigated in drug delivery systems for pharmaceutical applications. For example, high molecular weight chitosan (HMWC) polymers are used either alone, or with other hydrophilic polymers, as matrices for controlled release drug delivery systems [7]. Low molecular weight chitosan (LMWC) polymers were used as mucosal absorption enhancers for small polar molecules and proteinateous drugs, and it was shown that chitosan can be used as a stabilizer in some of these products [8, 9]. Commercially, LMWC is obtained by the depolymerization of HMWC and the resultant polymers exhibit various degrees of deacetylation (DDA), which results in significant changes in its physicochemical properties such as solubility and viscosity as well as affecting their biological activities [6, 10]. For example, LMWC polymers with high DDA are much less viscous and are soluble over a much wider pH range when compared to HMWC polymers [2]. The interactions between anionic drugs and chitosan samples of different molecular weights have been reported in the literature. The majority of these reports used HMWC with molecular weights ranging from 50 to 2000 kDa, whereas there are a few investigations that described similar interactions with LMWC ranging from glucosamine up to 50 kDa. For example, Imai et al. [11] studied the interactions between four kinds of LMWC polymers and indomethacin in solutions and in the solid state. The polymers differed both in their molecular weights (3.8–25 kDa) and degrees of deacetylation (60–90%). The solubility of the drug was enhanced with increasing concentration of LMWC, The results also showed that the acetyl group and amino group of chitosan played important roles in complexation [11]. In another study, the efficacies of chitosan of different molecular weights (110–260 kDa) and its glutamate and hydrochloride salts were investigated for improving the dissolution behavior of naproxen [12]. In addition, transport studies showed that chitosan salts allowed a significant improvement in the apparent permeability for drugs [3]. It is important to mention that in many studies, chitosan polymers were obtained from commercial sources and were used without any purification, and sometimes without any verification of their molecular weight and degree of deacetylation [11, 13–17]. Complexation of LMWC with other anionic drugs has been studied using spectroscopy. For example, the authors of a 1 H-NMR study on a liquid solution, made by adding benzoic acid to a chitosan solution, reported that there was no complex formation [15]. Most of the drug-chitosan complexes studied by FT-IR spectra did not show clear characteristic bands for the complexed drug; instead these spectra showed broadened bands for chitosan [14, 16]. Most of the above reports concluded that there are insignificant or no ionic interactions between HMWC and the acidic drugs. On the other hand, there are some reports that contradicted such findings, especially when LMWC having molecular weights up to 50 kDa were used [11, 17]. These contradictions prompted us to conduct the present systemic study where a LMWC sample with a wellcharacterized molecular weight and degree of deacetylation was used in order to investigate its interaction with the anionic drug ibuprofen, which contains a carboxylic acid group and is here treated as a model drug.

J Solution Chem (2009) 38: 695–712

697

2 Materials and Methods 2.1 Materials Hydrochloric acid (HCl) 37%, deuterium oxide (D2 O), and sodium deuteroxide (NaOD) were all obtained from Acros Organics (Geels, Belgium). Water used was double distilled. High molecular weight chitosan with a viscosity average molecular weight of 250 kDa and degree of deacetylation of 93% was obtained from Hongjo Chemical Company Ltd. (China). Ibuprofen was obtained from Dr. Reddy’s Company (Hyderabad, India). 2.2 Instruments The prepared polymers and complexes were dried using a Heto Power Dry PL9000 freeze dryer (Thermo Fisher Scientific-Inc, Waltham-MA, USA). Viscosity measurements were performed using a Sine-wave Vibro SV-10/SV-100 viscometer (KSV Instruments, Helsinki, Finland). Centrifugation was carried out using a Sorvall Super Speed RC2-B centrifuge (Ivan Sorvall-Inc, Norwalk-CT, USA). Mnova 5.2 was used to process the 1 H-NMR spectra (Mestrelab Research, Spain). ChemDraw Ultra 10 was used for chemical structure drawing (Cambridgesoft, USA). Hyperchem® release 7.52 update was used for molecular mechanics computations (Hypercube Inc., USA). 2.3 Preparation of LMWC Polymers The LMWC polymer was prepared by the acid hydrolysis method. A 10 g sample of high molecular weight chitosan (250 kDa) was dissolved in 830 mL 0.1 mol·L−1 HCl and then 170 mL of concentrated HCl (37%) was added, leading to a concentration of HCl equal to 2 mol·L−1 and a concentration of chitosan equal to 10 g·L−1 . The resultant solution was stirred at a speed of 1000 rpm and maintained under reflux for 2 h. The reaction mixture was then allowed to cool, and 96% ethanol was added to precipitate the LMWC hydrochloride salt. Centrifugation followed by freeze-drying afforded the desired product. The obtained LMWC polymer was stored in glass bottles at 25 ◦ C. 2.4 Preparation of Ibuprofen-LMWC Complexes For the preparation of each complex, a solution of LMWC, as the hydrochloride salt, with a specific composition (solution 1) and a solution of ibuprofen (in aqueous sodium hydroxide) with a specific concentration (solution 2) were prepared, and then both were mixed as described below. The compositions of these solutions and the resultant complex, expressed as the mass composition of ibuprofen relative to the total mass of ibuprofen and LMWC, are reported in Tables 1 to 3. In general, solution 2 was added to solution 1 with efficient stirring by a magnetic stirrer. During the addition, the pH was always controlled in the range of 2–4. After complete addition, the resultant mixture was stirred for 0.5 hour and was then freeze dried. Finally, the resulting dry powder was crushed and stored in glass bottles at 25 ◦ C. 2.5 Characterization of the LMWC Polymers 2.5.1 Molecular Weight Determination of LMWC A 100 g·L−1 stock solution of the synthesized LMWC was prepared by dissolving 5 g of the polymer in a 50 mL volumetric flask containing water, and then concentrations of 70, 50,

698

J Solution Chem (2009) 38: 695–712

Table 1 The complexes of LMWC (17 kDa) and ibuprofen prepared using 25 g·L−1 LMWC solutions. The compositions are expressed in terms of mass percent of ibuprofen, which is defined as 100 times the mass composition of ibuprofen relative to the total mass of ibuprofen + LMWC

Table 2 The complexes of LMWC (17 kDa) and ibuprofen prepared using 0.45 g·L−1 LMWC solutions. The compositions are expressed in terms of mass percent of ibuprofen, which is defined as 100 times the mass composition of ibuprofen relative to the total mass of ibuprofen + LMWC

Solution 1

Solution 2

Ibuprofen

(LMWC

(ibuprofen in sodium

(mass percent)

hydrochloride in

hydroxide solution)/g·L−1

water)/g·L−1 1

50

5.0

9%

2

50

12.5

20%

3

50

25.0

33%

4

50

37.5

43%

5

50

50.0

50%

6

50

75.0

60%

Solution 1

Solution 2

Ibuprofen

(LMWC

(ibuprofen in sodium

(weight percent)

hydrochloride in

hydroxide solution)/g·L−1

water)/g·L−1 1

5.0

0.50

9%

2

5.0

1.25

20%

3

5.0

2.50

33%

4

5.0

3.75

43%

5

5.0

5.00

50%

6

5.0

7.50

60%

Table 3 The five complexes with 33% weight-% of ibuprofen were prepared using LMWC solutions with variable concentrations of 0.45–50 g·L−1 Solution 1

Solution 2

Chitosan concentration/

(LMWC

(ibuprofen in sodium

g·L−1

hydrochloride in

hydroxide solution)/g·L−1

water)/g·L−1 1

50.0

50

2

50.0

25.0

25

3

20.0

10.0

10

4

11.1

5

100

0.50

5.55

5.55

0.25

0.45

30 and 10 g·L−1 were prepared by serial dilution of the stock solution. Before carrying out the measurements, the Sine-wave Vibro viscometer was calibrated and verified at 25 ◦ C using viscosity standards of 3.51 and 7.43 mPa·s. Then, the viscosity of a solution at each concentration was measured in (at least) triplicate at 25 ◦ C. The relative and reduced viscosities were calculated, and were then used in determining the viscosity average molecular weight of the polymer using the Mark-Houwink equation [η] = KMra , where [η] is the intrinsic viscosity, M r is the molecular weight, and K and a are the Mark-Houwink constants that are equal to 0.0005820 and 0.693295, respectively.

J Solution Chem (2009) 38: 695–712

699

2.5.2 Determination of the Degree of Deacetylation (DDA) of LMWC The first derivative UV spectroscopic method was used for the DDA determination according to the British Pharmacopoeia 2004 [18]. The experiments were done in triplicate. 2.6 Differential Scanning Calorimetry (DSC) A differential scanning calorimetry (DSC) was connected to a thermal analysis operating system (TA-50WS, Shimadzu, Kyoto, Japan). The sample to be analyzed (10 to 15 mg) was placed in an aluminum pan. An empty aluminum pan was used as reference and the runs were performed by heating the samples from 25 to 350 ◦ C at a rate of 15 ◦ C·min−1 under nitrogen purge (50–100 mL·min−1 ). Indium metal (99.99%) was used to calibrate the DSC modulus in relation to temperature and enthalpy. 2.7 FT-IR Spectrometry The FT-IR spectra were recorded on a Perkin-Elmer Spectrum One Spectrophotometer (Waltham-MA, USA) using KBr discs. One milligram of the analyte was physically mixed with 100 mg of dry KBr and an appropriate amount of the mixture were pressed to prepare the disks. All spectra were recorded at ambient temperature in the wave number region from 4000 and 400 cm−1 . In all cases, the IR spectra were recorded by accumulation of at least 10 scans with an optical path velocity (OPD) velocity of 2 cm−1 . 2.8 1 H-Nuclear Magnetic Resonance Spectrometry 1

H-NMR spectra were acquired using a Bruker Advance UltraShield 400 MHz spectrometer. The experiments were run at ambient temperature. Fifteen milligrams (15 mg) of chitosan, ibuprofen + LMWC mixture, or ibuprofen sodium were added to 1 mL of deuterium oxide (D2 O) and the solution or the supernatant (when insoluble matter was present) was placed in a 5 mm NMR tube. Processing of the spectra was done using Mnova 5.2. 2.9 Molecular Mechanics Computations in vacuum were performed with Hyperchem® (release 7.52 update) using the Amber Force field implemented in Hyperchem. Partial atomic charges were obtained by performing AM1 semi-empirical calculations. Energy minimizations were obtained using the conjugate gradient algorithm (0.01 kcal·mol−1 ·Å−1 gradient). Chitosan monomer, i.e. glucosamine, and the ibuprofen salt were built up with natural bond angles as defined in the Hyperchem software. The structures were then minimized with the Amber force field and further optimized at the HF-ab initio level using the 3-21G* basis set.

3 Results and Discussion 3.1 Characterization of LMWC Polymers The structure of the prepared LMWC polymer was confirmed by 1 H-NMR and FT-IR and the resulting spectra are shown in Figs. 1 and 2. As seen in Fig. 1, the triplet at δ = 3.18 corresponds to the C-2 proton of glucosamine in the chitosan skeleton, whereas the doublet

700

J Solution Chem (2009) 38: 695–712

Fig. 1 The general chemical structure of the LMWC polymer and its 1 H-NMR spectrum

at δ = 4.88 corresponds to the anomeric proton at C-1. The multiplets from δ = 3.6 to 4.1 correspond to the protons at C-3, C-4, C-5 and C-6. The obtained LMWC structure was used to prepare the complexes detailed in Tables 1, 2 and 3. With regard to molecular weight, the standard deviation between the three viscosity replicate measurements at each concentration was less than 1%, which means that the viscosity method is reproducible for determining the average molecular weight. Under the conditions used here, the average molecular weight of the prepared LMWC polymer was 17.15 ±0.13 kDa and it will be referred to as 17 kDa in the rest of the discussion. The degree of deacetylation (DDA) values of the prepared chitosan hydrochloride samples was approximately 100% according to the first derivative UV spectroscopic method [18]. In addition, the 1 H-NMR spectrum of the prepared LMWC showed no acetyl peak in the spectrum, indicating total deacetylation of this polymer, see Fig. 1. 3.2 Differential Scanning Calorimetry (DSC) In an in-house study (results unpublished), the interactions of ibuprofen with different molecular weight samples of LMWC (6, 13, 17 and 30 kDa) were studied using thermal analysis. The DSC results showed that the maximum interaction occurred with a LMWC having an average molecular weight of 17 kDa, where the peak for ibuprofen disappeared completely. In this work, a thorough testing of the LMWC (17 kDa)-ibuprofen complexes of variable composition was made, using LMWC solutions of different concentrations. In the thermogram of chitosan (Fig. 3a) the first broad endothermic peak that falls between 40 and 110 ◦ C can be ascribed to the evaporation of adsorbed water and residual water, and the second is an exothermic decomposition peak in the range of 270–290 ◦ C. This decomposition occurs at a lower temperature than reported in the literature for high molecular

J Solution Chem (2009) 38: 695–712

701

Fig. 2 The FT-IR spectra of ibuprofen sodium (a), ibuprofen (b), chitosan (c), the 33.3% ibuprofen-LMWC(25) complex (d), the 33.3% ibuprofen-LMWC(25) complex (e) and the 33.3% ibuprofen + LMWC physical mixture (f)

Fig. 3 DSC thermogram of (a) LMWC, 17 kDa, (b) ibuprofen, (c) the 33.3% ibuprofen-LMWC physical mixture, and (d) the 33.3% ibuprofen-LMWC complex, all in the temperature range of 50–350 ◦ C

702

J Solution Chem (2009) 38: 695–712

weight chitosan, which falls in the range of (300–400) ◦ C [19]. The area and height of the second exothermic peak increase as the degree of deacetylation (DDA) increases [20]. The DSC thermogram of ibuprofen (Fig. 3b) shows two obvious endothermic peaks; the first sharp endothermic peak corresponds to the melting of ibuprofen at 79.08 ◦ C, and the second peak occurs at around 235.62 ◦ C which is due to the endothermic weight reduction and evaporation of ibuprofen [21]. The DSC thermogram of the 33% ibuprofen + LMWC physical mixture is shown in Fig. 3c. In this thermogram, the peaks due to free ibuprofen as well as to LMWC appear, but with some slight changes as compared with those of the individual components because of dilution. The endothermic peak representing free ibuprofen has a reduced intensity and was shifted to a lower temperature (77.85 ◦ C). At the same time, the second peak representing LMWC was shifted to 274.72 ◦ C with some change in intensity. Finally, in the DSC thermogram for the 33% ibuprofen-LMWC complex (Fig. 3d), the peak for free ibuprofen was absent from the thermogram indicating that the ibuprofen molecules are completely contained in the complex. Furthermore, there is no sharp exothermic peak for the complex. Rather, a gradual thermal degradation behavior was observed. This allows one to conclude that some kind of interaction appears when the DSC technique is used to study the prepared complex. 3.3 FT-IR Spectrometry In all the upcoming discussion, the complexes of ibuprofen with chitosan, that were prepared using a LMWC solution with the concentration of 25 g·L−1 , will be designated as ibuprofenLMWC(25) complexes, whereas those prepared using a LMWC solution with the concentration 0.45 g·L−1 will be designated-LMWC(0.45) complexes. Figure 2 shows the FT-IR spectra of ibuprofen sodium (a), ibuprofen (b), 17 kDa LMWC (c), the 33% ibuprofen-LMWC(25) complex (d), 33% the ibuprofen-LMWC(0.45) complex (e), and the 33% ibuprofen + LMWC physical mixture (f). It is evident that complexation using the 0.45 g·L−1 chitosan solution is more efficient. Furthermore, comparing spectrum (e) to (a) shows a very significant shift in the asymmetrical stretching carbonyl band, which changes from 1584.38 cm−1 in the IR spectra of ibuprofen sodium to a lower value of 1550.1 cm−1 , and a less prominent shift occurs in the symmetric stretching band from 1410.51 cm−1 to a higher value of 1413.37 cm−1 . These changes are a result of the formation of the 33% ibuprofen-LMWC(0.45) complex. The shifts can be attributed to the transformation of the sodium-carboxylate ion pair to an ammonium-carboxylate ion pair [11, 22, 23]. This clear presence of ionic interaction between ibuprofen and LMWC comes in contrast to recent reports that there is very weak or even no electrostatic interaction between high molecular weight chitosan (150 KDa and 85% deacetylation) and benzoic acid [15], salicylic acid and diclofenac [14]. In the case of the 33% ibuprofen-LMWC(25) complex, spectrum Fig. 2d, incomplete complexation is evident by the appearance of a carbonyl peak at 1720.65 cm−1 that is due to the carbonyl peak of the ibuprofen free acid, which is formed by the pH adjustment to 2–4 that converts the excess uncomplexed ibuprofen sodium to the ibuprofen acid. In addition, in spectrum 2d, it can be noticed that the LMWC band at 1625.66 cm−1 is clear, which also means that complexation is not complete. These two bands at 1720.65 and 1625.66 cm−1 are not present in the FT-IR spectrum of the 33% ibuprofen-LMWC(0.45) complex, spectrum 2e. It is assumed that in diluted solutions the LMWC molecules will assume a conformation that allows more amino groups to interact with the carboxylic groups of ibuprofen. It is worth mentioning that the effect of the concentration of the LMWC solution used in the complexation procedure has never been explicitly studied previously. Finally, spectrum 2f of

J Solution Chem (2009) 38: 695–712

703

the physical mixture was clearly distinct from those of chitosan and the LMWC-ibuprofen complexes, but showed a high similarity to the spectrum of uncomplexed ibuprofen sodium with essentially unchanged carbonyl bands. The absence of chitosan bands, especially those in range 1250–890 cm−1 , from the spectrum of the physical mixture means that there is no complexation between LMWC and ibuprofen sodium in the solid state. It is obvious that the complexation process is more evident when low concentrations of LMWC are used. 3.4 1 H-Nuclear Magnetic Resonance (1 H-NMR) Spectrometry The 1 H-NMR spectrum of the LMWC hydrochloride polymer, 17 kDa, in D2 O, is shown with the peak assignment in Fig. 1 [24]. Also, the chemical structure and 1 H-NMR spectrum of ibuprofen sodium in D2 O is shown in Fig. 4. With regard to the complexes, it was noticed that those prepared using solutions of 25 g·mL−1 or higher LMWC concentrations formed a visible precipitate when added to D2 O. Hence, the 1 H-NMR spectrum of the supernatant was measured. The precipitate was dissolved in D2 O/NaOD, analyzed by 1 H-NMR, and was found to be ibuprofen, which formed from the excess uncomplexed ibuprofen sodium upon the pH adjustment to 2–4. In general, it can be seen that upon complexation of ibuprofen with chitosan, the peak corresponding to the H-2 proton of chitosan was shifted up field from 3.18 ppm to 2.92– 3.03 ppm, and the peak corresponding to the H-1 proton of chitosan was shifted up field from 4.88 to 4.65–4.73 ppm. In addition, peaks in the range from 3.6 to 3.95 ppm became better resolved to reveal a doublet at 3.89 ppm. To see the effects on complexation from using different amounts of ibuprofen, analysis of the 1 H-NMR spectra of the complexes was based on comparison of the ratio of the integral

Fig. 4 The chemical structure and 1 H-NMR spectrum of ibuprofen sodium in HOD

704

J Solution Chem (2009) 38: 695–712

Fig. 5a The 1 H-NMR spectra of ibuprofen-LMWC(0.45) complexes: (a) 60% ibuprofen, (b) 50% ibuprofen, (c) 42.9% ibuprofen, (d) 33.3% ibuprofen, (e) 20% ibuprofen, and (f) 9.1% ibuprofen

area of the peak corresponding to the H-2 proton of chitosan to the integral area of the peak corresponding to the benzylic methylene group, Ar-CH2 , in ibuprofen that is denoted (R ibu/chit ). The peak corresponding to the H-2 proton of chitosan was chosen because it is the only chitosan related peak that showed no interference from other peaks in the spectrum and is also the furthest away from the solvent peak. The 1 H-NMR spectra of the ibuprofen-LMWC(0.45) complexes are shown in Fig. 5a. These complexes contain ibuprofen in weight percents ranging from 9% up to 60%. Analysis of the spectra showed that R ibu/chit increases as the ratio of ibuprofen to LMWC increases, Fig. 6. The ratio of integral areas for the 60% ibuprofen-LMWC(0.45) complex are significantly higher than the rest, which may indicate the presence of more than one kind of interaction at this composition. The 1 H-NMR spectra of the ibuprofen-LMWC(25) complexes are shown in Fig. 5b. These complexes have similar ibuprofen weight percents ranging from 9% to 60%. In contrast to the spectra in Fig. 5a, these spectra showed that R ibu/chit stays almost constant regardless of the concentration of ibuprofen, Fig. 6. It can be concluded that the limiting factor for complex formation is the concentration of LMWC rather than the ratio of the drug to the polymer. This dependency on the concentration of the LMWC solution may explain the discrepancies reported in the literature with regard to the presence or absence of interactions between anionic drugs and chitosan. It is evident that it is more efficient to prepare these complexes at lower chitosan concentrations. Again, this may be due to the fact that chitosan at lower concentrations can assume a more accessible conformation and/or it will be less aggregated, hence allowing a higher number of drug molecules to interact with the same molecule of chitosan.

J Solution Chem (2009) 38: 695–712

705

Fig. 5b The 1 H-NMR spectra of ibuprofen-LMWC(25) : (a) 60% ibuprofen, (b) 50% ibuprofen, (c) 42.9% ibuprofen, (d) 33.3% ibuprofen, (e) 20% ibuprofen, and (f) 9.1% ibuprofen

Figure 7 shows the 1 H-NMR spectra of the 33% ibuprofen-LMWC complexes that were prepared with different concentrations of LMWC solution: 0.45, 5.55, 10, 25, and 50 g·L−1 . Analysis of the spectra revealed that as the concentration of LMWC in the solution decreases, R ibu/chit increases, despite the fact that the weight percent of ibuprofen in the complexes remained constant, Fig. 8. This is an added confirmation of the idea that complexation occurs to a higher extent when a low concentration of chitosan is used. Another feature that can be noted from these spectra of the ibuprofen-LMWC complexes is the changes in chemical shifts of some peaks. In the spectra of complexes prepared with different concentrations of LMWC, Fig. 7, it can be seen the largest up field shift of the peaks corresponding to the H-1 and H-2 protons is in spectrum 7b, which is for the complex prepared with the most dilute LMWC. Figure 9 shows the differences in the chemical shifts in Hz, δ, for peaks corresponding to H-1 and H-2 in uncomplexed LMWC from those in complexed LMWC, and their relation to the concentration of the LMWC solutions used in preparing the complex. It can be seen that δ decreases as the concentration of LMWC increases. It was interesting to see that the spectra of the complexes containing different concentrations of ibuprofen, that were prepared with solutions of the same concentration of LMWC, did not show a clear trend in δ, Fig. 10. This means that the concentration of LMWC used for preparing the complexes is more important than the amount of ibuprofen. This implies that the flexible LMWC molecule can assume different conformations at different concentrations and hence, the final complexes, which are expected to be more rigid than the free LMWC, will also adopt different conformations resulting in visible differences in the respective 1 H-NMR spectra.

706

J Solution Chem (2009) 38: 695–712

Fig. 6 A plot of R ibu/chit versus the weight percent of ibuprofen for complexes prepared with two different concentrations of LMWC stock solutions: (2) 0.45 g·L−1 and (Q) 25 g·L−1

The results from 1 H-NMR, FT-IR spectroscopy, and DSC prove the existence of ionic interactions between the amine group of chitosan and the carboxyl group of ibuprofen. 3.5 Polymer Buildup To gain some insight into the above findings, simulation of such interactions was attempted based on information obtained from the glucosamine unit structure and other physicochemical properties including the extent of deacetylation. The LMWC monomer (Structure I), i.e. glucosamine, and the ibuprofen salt (Structure II), are shown in Fig. 11. The resulting chitosan monomer (Structure I) is defined in the HyperChem workspace. A set of named selections has been made to define the HEAD (name is “a”) and TAIL (name is “b”) for building the polymer (Fig. 12). An additional set of named selections is made to define the atoms involved in the torsion angle that will be used when putting the monomers together. The HEAD is assumed to be connected to x (name is “x”), which in turn is connected to x (name is “x ”). The TAIL is assumed to be connected to y (name is “y”), which in turn is connected to y (name is “y ”). The torsional angle connecting monomers is then the x -xy-y angle, with retention of the internal structure of the monomer. The torsion angle used to put the monomers together could be specified to have a random value rather than the constant value described above. The chitosan polymer was constructed with a suitable length from the monomer unit (Structure I). Two different structures of LMWC polymer with the same number of units (108 units) were built. The first, Chit I, is longer (length ∼300 Å) and the other is shorter, Chit II (length ∼111 Å).

J Solution Chem (2009) 38: 695–712

707

Fig. 7 The 1 H-NMR spectra of ibuprofen-LMWC complexes prepared using 33.3 weight % ibuprofen and different stock solutions of chitosan: (a) LMWC, 17 kDa, (b) 0.45 g·L−1 LMWC, (c) 5.0 g·L−1 LMWC, (d) 10 g·L−1 LMWC, (e) 25 g·L−1 LMWC, and (f) 50 g·L−1 LMWC

Fig. 8 A plot of the concentrations of LMWC stock solutions (0.45–50 g·L−1 ) versus R ibu/chit

708

J Solution Chem (2009) 38: 695–712

Fig. 9 A graph of δ of the peaks H-1 and H-2 of LMWC in Hz versus the concentration of the stock solutions of LMWC used in preparation of the complexes

Fig. 10 A graph of δ of the peaks H-1 and H-2 of LMWC in Hz versus the weight percent of ibuprofen used in the preparation of the complexes

3.6 Drug-Chitosan Complex Formation The ibuprofen salt was positioned manually close to (and in front of) the amino groups of the LMWC polymer and allowed to be optimized, leading to an ibuprofen-LMWC complex that was further optimized by using the Amber force field (0.1 gradient).

J Solution Chem (2009) 38: 695–712

709

Fig. 11

Fig. 12 The three-dimensional structures of glucosamine (Structure I) and the ibuprofen salt (Structure II)

It is expected that at low concentration (0.45 g·L−1 ), LMWC will be more extended as compared to the structure at high concentration (25 g·L−1 ), and therefore it is more available to interact with the ibuprofen sodium salt or with any other molecule. This possibility was examined by selecting two polymers (both have the same number of building units but different lengths; Chit I and Chit II). The results of MM calculations show that Chit I can accommodate one ibuprofen molecule for every two chitosan monomers (Fig. 13), whereas Chit II interacts to a much lesser extent with ibuprofen which might be attributed to steric hindrance (Fig. 14). In Fig. 13, red balls represent the Ar-CH2 protons and the gray balls represent the H-2 proton.

4 Conclusions From the analysis of the ibuprofen-LMWC complexes by liquid 1 H-NMR, FT-IR spectrometry and molecular mechanics calculations, it is evident that complexation of LMWC with ibuprofen involves ionic interaction between the ammonium groups of LMWC and the carboxylate anion of ibuprofen, and also that it is more efficient to prepare ibuprofen-LMWC complexes using dilute solutions of the polymer. The latter observation leads to the assumptions that LMWC assumes a more accessible conformation in dilute solutions or that dilute solutions of LMWC might be less aggregated, both of which will lead to more efficient drug-LMWC interactions. These results were confirmed using MM calculations. This may explain the discrepancies in the literature where, in many studies, the dilution of chitosan and its average molecular weight was not considered to be important factors in the complexation process. These results warrant further investigation by other techniques to fully understand the nature of the interaction between LMWC and anionic drug molecules.

710

J Solution Chem (2009) 38: 695–712

Fig. 13 Complexation of one ibuprofen molecule to each two glucosamine monomers (50% substitution) of Chit I (H-2 and Ar-CH2 are denoted by white and red balls, respectively)

J Solution Chem (2009) 38: 695–712

711

Fig. 14 Complexation of ibuprofen molecules to glucosamine monomers of Chit II

Acknowledgement This work was funded by a grant from the Deanship of Scientific Research at Jordan University of Science and Technology.

References 1. Kumar, M.N.V.R., Muzzarelli, R.A.A., Muzzarelli, C., Sashiwa, H., Domb, A.J.: Chitosan chemistry and pharmaceutical perspectives. Chem. Rev. 104, 6017–6084 (2004). doi:10.1021/cr030441b 2. Rinaudo, M.: Chitin and chitosan: properties and applications. Prog. Polym. Sci. 31, 603–632 (2006). doi:10.1016/j.progpolymsci.2006.06.001 3. Maoa, S., Shuai, X., Unger, F., Simona, M., Bi, D., Kissel, T.: The depolymerization of chitosan: effects on physicochemical and biological properties. Int. J. Pharm. 281, 45–54 (2004). doi:10.1016/j. ijpharm.2004.05.019 4. Majeti, N.V., Kumar, R.: A review of chitin and chitosan applications. React. Funct. Polym. 46, 1–27 (2000). doi:10.1016/S1381-5148(00)00038-9 5. Shahidi, F., Kamil, J., Arachchi, V., Jeon, Y.-J.: Food applications of chitin and chitosans. Trends Food Sci. Technol. 10, 37–51 (1999). doi:10.1016/S0924-2244(99)00017-5 6. Li, J., Du, Y., Yang, J., Feng, T., Li, A., Chen, P.: Preparation and characterisation of low molecular weight chitosan and chito-oligomers by a commercial enzyme. Polym. Degrad. Stabil. 87, 441–448 (2005). doi:10.1016/j.polymdegradstab.2004.09.008 7. Prabaharan, M., Mano, J.F.: Chitosan-based particles as controlled drug delivery systems. Drug Deliv. 12, 41–57 (2004). doi:10.1080/10717540590889781 8. Thanou, M., Verhoef, J.C., Junginger, H.E.: Oral drug absorption enhancement by chitosan and its derivatives. Adv. Drug Deliv. Rev. 52, 117–126 (2001). doi:10.1016/S0169-409X(01)00231-9

712

J Solution Chem (2009) 38: 695–712

9. Senel, S., Kremer, M.J., Kas, S., Wertz, P.W., Hincal, A.A., Squier, C.A.: Enhancing effect of chitosan on peptide drug delivery across buccal mucosa. Biomaterials 21, 2067–2071 (2000). doi:10.1016/S01429612(00)00134-4 10. Jeon, Y.-J., Shahidi, F., Kim, S.-K.: Preparation of chitin and chitosan oligomers and their applications in physiological functional foods. Food Rev. Int. 16, 159–176 (2000). doi:10.1081/FRI-100100286 11. Imai, T., Shiraishi, S., Saito, H., Otagiri, M.: Interaction of indomethacin with low molecular weight chitosan and improvements of some pharmaceutical properties of indomethacin by low molecular weight chitosans. Int. J. Pharm. 67, 11–20 (1991). doi:10.1016/0378-5173(91)90260-U 12. Mura, P., Zerrouk, N., Mennini, N., Maestrelli, F., Chemtob, C.: Development and characterization of naproxen-chitosan solid systems with improved drug dissolution properties. Eur. J. Pharm. Sci. 19, 67–75 (2003). doi:10.1016/S0928-0987(03)00068-X 13. Berthold, A., Cremer, K., Kreuter, J.: Preparation and characterization of chitosan microspheres as drug carrier for prednisolone sodium phosphate as model for anti-inflammatory drugs. J. Control. Release 39, 17–25 (1996). doi:10.1016/0168-3659(95)00129-8 14. Boonsongrit, Y., Mitrevej, A., Mueller, B.W.: Chitosan drug binding by ionic interaction. Eur. J. Pharm. Biopharm. 62, 267–274 (2006). doi:10.1016/j.ejpb.2005.09.002 15. Boonsongrit, Y., Mueller, B.W., Mitrevej, A.: Characterization of drug–chitosan interaction by 1 H-NMR FTIR and isothermal titration calorimetry. Eur. J. Pharm. Biopharm. 69, 388–395 (2008). doi:10.1016/j. ejpb.2007.11.008 16. Kweon, D.-K., Song, S.-B., Park, Y.-Y.: Preparation of water-soluble chitosan/heparin complex and its application as wound healing accelerator. Biomaterials 24, 1595–1601 (2003). doi:10.1016/S01429612(02)00566-5 17. Puttipipatkhachorn, S., Nunthanid, J.J., Yamamoto, K., Peck, G.E.: Drug physical state and drugpolymer interaction on drug release from chitosan matrix films. J. Control. Release 75, 143–153 (2001). doi:10.1016/S0168-3659(01)00389-3 18. BPC: British Pharmacopoeia 2004. Stationery Office Books, Norwich (2004) 19. Paulino, A.T., Simionato, J.I., Garcia, J.C., Nozaki, J.: Characterization of chitosan and chitin produced from silkworm chrysalids. Carbohydr. Polym. 64, 98–103 (2006). doi:10.1016/j.carbpol.2005.10.032 20. Guinesi, L.S., Cavalheiro, E.T.G.: The use of DSC curves to determine the acetylation degree of chitin/chitosan samples. Thermochim. Acta 444, 128–133 (2006). doi:10.1016/j.tca.2006.03.003 21. Xu, F., Sun, L.-X., Tan, Z.-C., Liang, J.-G., Li, R.-L.: Thermodynamic study of ibuprofen by adiabatic calorimetry and thermal analysis. Thermochim. Acta 412, 33–37 (2004). doi:10.1016/j.tca.2003.08.021 22. Lee, D.H., Sr, R.A.C., Reed, J.S.: Infrared spectral investigation of polyacrylate adsorption on alumina. J. Mater. Sci. 31, 471–478 (1996). doi:10.1007/BF01139166 23. Silverstein, R.M., Webster, F.X., Kiemle, D.J.: Spectroscopic Identification of Organic Compounds, 7th edn., p. 502. Wiley, New Jersey (2005) 24. Kittur, F.S., Vishu Kumar, A.B., Tharanathan, R.N.: A validated 1 H-NMR method for the determination of the degree of deacetylation of chitosan. J. Pharm. Biomed. Anal. 32, 1149–1158 (2003). doi:10.1016/S0731-7085(03)00155-9