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Quaternized chitosan–organic rectorite intercalated composites based nanoparticles for protein controlled release
International Journal of Pharmaceutics 438 (2012) 258–265
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Quaternized chitosan–organic rectorite intercalated composites based nanoparticles for protein controlled release Ruifen Xu a,b,1 , Shangjing Xin c,1 , Xue Zhou d,1 , Wei Li c , Feng Cao e , Xuyang Feng e,∗ , Hongbing Deng b,c,∗∗ a
Department of Anesthesiology, School of Stomatology, Fourth Military Medical University, Xi’an 710032, China Department of Environmental Science, College of Resource and Environmental Science, Wuhan University, Wuhan 430079, China College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China d State Key Laboratory of Environment Health (Incubation); Key Laboratory of Environment and Health, Ministry of Education; Key Laboratory of Environment and Health (Wuhan), Ministry of Environmental Protection; Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China e Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China b c
a r t i c l e
i n f o
Article history: Received 25 July 2012 Received in revised form 27 August 2012 Accepted 6 September 2012 Available online 12 September 2012 Keywords: Quaternized chitosan Organic rectorite Intercalation Nanoparticles Drug delivery
a b s t r a c t Organic rectorite (OREC) was added in the quaternized chitosan (QC)/alginate (ALG) nanoparticles using an ionic gelation method to fabricate a controllable release system for proteins for the first time. The morphology of nanoparticles, the intercalated structure of OREC, bovine serum albumin encapsulation efficiency and in vitro release properties were investigated. Fourier transform infrared spectra, energy dispersive X-ray, X-ray photoelectron spectroscopy, small angle X-ray diffraction and size distribution analysis were performed to characterize the composite nanoparticles. With the addition of OREC, the encapsulation efficiency and the loading capacity of nanoparticles had increased from 21.2% to 44.9% and from 13.7% to 25.0%, respectively. In addition, the rapid initial release was inhibited successfully from 20.15% to 11.07% in stimulated gastric fluid and from 14.69% to 4.52% in stimulated intestinal fluid. The results verified that the addition of OREC could make these nanoparticles effective carriers to encapsulate drug and slow the drug controlled release of nanoparticles. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Nowadays, proteins and peptides have drawn many attentions in biotechnology because of their effective properties for disease treatment (Castro et al., 2005). However, they are hard to be absorbed in stomach and intestinal tracts as their hydrophilic property. In order to overcome this disadvantage, colloidal materials such as nanoparticles (NPs) have been used to protect proteins and peptides from quick dissolving and fabricate controlled release systems for the enduring utilization of drugs (Khalil and Mainardes, 2009; Mora-Huertas et al., 2010). Chitosan (CS) is not only naturally abundant, but also nontoxic, biodegradable, and regenerable. Because of these properties, CS has been used in a variety of application areas such as bacterial inhibition (Deng et al., 2011a), cell culture (Deng et al., 2010), cosmetic
∗ Corresponding author. Tel.: +86 29 84775183; fax: +86 29 84775183. ∗∗ Corresponding author at: Department of Environmental Science, College of Resource and Environmental Science, Wuhan University, Wuhan 430079, China. Tel.: +86 27 68778501; fax: +86 27 68778501. E-mail addresses: [email protected] (X. Feng), [email protected] (H. Deng). 1 Co-first authors with the same contribution to this work. 0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2012.09.010
skin masks (Huang et al., 2003), tissue engineering (Thein-Han et al., 2008) and drug delivery (Yuan et al., 2008). Some researchers have reported that CS NPs was applied as drug controlled release systems for hydrophilic drugs delivery by utilizing the hydrophobic property of CS (Wu et al., 2005; Xu and Du, 2003). Quaternized chitosan (QC), a water soluble derivative of CS, has been found that it is with better properties of permeability and mucoadhesion than CS (Kotze et al., 1999; Thanou et al., 2000), when used as an absorption enhancer transporting across the intestinal epithelia. In addition, QC NPs were with smaller particle size and remarkable advantages such as weakly alkaline values in drug delivery system (Bayat et al., 2008). Moreover, some reports have covered that QC NPs were used as a protein carrier (Xu et al., 2003) and that NPs cross-linking with alginate (ALG) were with better controlled release properties (Wang et al., 2011). However, the encapsulation efficiency of drug in the NPs is too low because the superabundant negative charged ALG could compete for the positive charged QC with drugs (Li et al., 2007). In order to solve this problem, inorganic materials will be added for preparing QC/ALG organic–inorganic hybrid NPs. So far, CS based organic–inorganic hybrid has been reported as a nontoxic and biodegradable scaffold for tissue engineering and drug delivery response (Depan et al., 2011a; Thein-Han and
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Scheme 1. Schematic diagram illustrating the fabrication process of QC–OREC nanoparticles.
Misra, 2009; Thein-Han et al., 2009; Yuan et al., 2010a) and the structure–process–property relationship has been studied to show the effect of adding inorganic component (Depan et al., 2011b). Based on these previous researches, the organic–inorganic composites might form a novel potential system with both of high encapsulation efficiency and loading capacity of drugs. Layered silicate, such as montmorillonite (MMT) and rectorite (REC), can be easily intercalated into layered structure by nature polymer chains and has been used in drug delivery recently (Burgentzle et al., 2004). CS-layered silicate nanocomposite carriers have been determined to be effective in cancer therapy. In the carriers, the positively charged chemotherapeutic drug was strongly bounded to the negatively charged layered silicate and release of the drug was slow (Yuan et al., 2010b). REC, especially organic rectorite (OREC) organic modified from REC, has larger interlayer distance, better separable layer thickness and larger aspect ratio than MMT (Wang et al., 2006). REC has been intercalated by QC chains and fabricated to QC–REC nanocomposites for antibacterial application (Wang et al., 2009) and gene delivery (Wang et al., 2008). In addition, layered silicate has been verified safe enough as food additives by European Food Safety Authority (ESFA) last year so that it is good for drug delivery (European Food Safety Authority, 2011). While CS-layered silicate composites are biocompatible and become a potential candidate to drug controlled release, the mechanical properties and biological response of these composites are inadequate to qualify them for drug delivery or facilitate transfer of the applied load at the implant site. Furthermore, when considered intercalation between polymer chains and layered silicate, larger interlayer distance and better electrostatic force still need to be achieved. In the present study, novel QC–OREC/ALG composite NPs with intercalated structure and controllable OREC interlayer distance were fabricated and used as drug carrier for the first time. Bovine Serum Albumin (BSA) was selected as hydrophilic drug model for investigating the drug delivery properties of NPs. Transmission scanning electron microscope (TEM), field emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray (EDX), X-ray elemental spectroscopy (XPS), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) and NPs size distribution analysis are performed to characterize the properties of NPs. Encapsulation efficiency, loading capacity and in vitro release properties of BSA will be evaluated.
2. Materials and methods 2.1. Materials Chitosan (Mw = 2.1 × 105 kDa) was provided by Yuhuan Ocean Biochemical Co. (Taizhou, China) and the degree of deacetylation was 92%. Alginate (ALG) was purchased from Aladdin reagent Inc. (Shanghai, China). BSA (Mw = 6.8 × 104 kDa) was supplied by Amresco Inc., USA. Calcium rectorite (Ca2+ –REC) was provided by Hubei Mingliu Inc. Co. (Wuhan, China). QC and organic rectorite (OREC) were prepared in our lab as previously reports (Wang et al., 2006; Xu et al., 2003). The degree of substitution of QC was 71%. The average diameter of OREC particles was about 100 nm. All other chemicals were of analytical grade. 2.2. Preparation of QC/ALG NPs QC–ALG NPs were labeled with NP0. QC–OREC/ALG NPs were prepared in mass ratios of QC:OREC were at 12:1, 6:1 and 3:1, which were labeled with NP121, NP61 and NP31, respectively. NPs were prepared as shown in Scheme 1. QC–OREC nanocomposites were moderately stirred for 24 h in 60 ◦ C water bath. Aqueous calcium chloride (0.5 mg/mL) was dispersed by ultrasonic for 5 min and then added into aqueous sodium alginate (1.0 mg/mL) to form the pregel. The QC solution or the QC–OREC composites was distilled into the pre-gel with stirring and continued stirring for another 30 min. The solution volume ratio of CaCl2 , ALG and QC or QC–OREC composites was at 2:6:1. The obtained opalescent emulsion was stored overnight and centrifuged at 15,000 × g for 30 min at 4 ◦ C. The sediment lyophilized to produce solid NPs aggregates. The BSA-loaded QC/ALG NPs were prepared by using the same way as above by adding BSA (1.0 mg/mL) into ALG solutions, and the pH value of BSA/ALG mixture solution was determined at 6.78. 2.3. Morphology and characterization of QC/ALG NPs The morphology of the NPs was observed by transmission electron microscope (TEM, JEM-2100, JEOL, Japan). QC/ALG NPs were stained with phosphotungstic acid solution (2%, w/v) and other samples were observed on a micro grid mesh scaffold. All samples were dried on copper grill at room temperature. The
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Fig. 1. TEM images of QC/ALG NPs (a, b) and QC–OREC/ALG NPs (c); FE-SEM image of QC/ALG NPs (d).
surface morphology of NPs was performed by field emission scanning electron microscopy (FE-SEM, JSM-6700F, JEOL, Japan). The NPs emulsion was dropped on aluminum foil and dried at room temperature. Energy-dispersive X-ray (EDX) spectroscopy images were obtained by Sirion 200 (FEI, Netherlands). The composition of the NPs was examined by X-ray elemental spectroscopy (XPS) using an axis ultra DLD apparatus (Kratos, UK). The small angle X-ray diffraction (SAXRD) was performed on type D/max-rA diffractometer (Rigaku Co., Japan) with Cu target and K␣ radiation ( = 0.154 nm). Fourier transform infrared (FT-IR) spectra were recorded by Nicolet170-SX (Thermo Nicolet Ltd., USA). The size distribution and zeta potential of the NPs were determined by Nano 3690 (Malvern, UK). The wavelength for the size measurement was 532.0 nm and the angle detection was 90◦ at 25 ◦ C. The pH value of NPs was also measured by pH meter.
2.4. Encapsulation efficiency and loading capacity The encapsulation efficiency (EE) and loading capacity (LC) were calculated by determining the concentration of BSA of the initial solution and that after loading. The concentration was examined by UV spectrophotometry at 595 nm using Coomassie Brilliant Blue protein assay. The EE and LC were calculated from: EE =
C0 − C1 × 100% C0
(1)
LC =
C0 − C1 × 100% M
(2)
where C0 (mol/L) is the concentration of BSA in initial solution, C1 (mol/L) is the concentration of free BSA in the supernatant, and M (g) is the total mass of the NPs.
Table 1 Zeta potential and pH value of NPs emulsions. Samples
-Potential (mV)
pH value
NP0 NP121 NP61 NP31
−38.2 −40.8 −42.5 −43.9
6.31 6.32 6.34 6.23
± ± ± ±
3.1 5.2 2.3 4.2
± ± ± ±
0.02 0.06 0.03 0.14
2.5. In vitro release tests 9 mL BSA-loaded NPs opalescent emulsion was centrifuged and then the sediment was suspended into 6 mL 0.1 M PBS buffer solution (pH = 7.4) or 0.1 M HCl solution (pH = 1.2). The mixtures were stirred in a mechanical vortexer at 100 rpm at 37 ◦ C. All the solutions were centrifuged before examining the amount of BSA released. 1 mL supernatant was taken out for the determination of the BSA concentration and another 1 mL fresh PBS buffer or HCl medium was supplied into the samples to ensure the total volume was 6 mL. The amount of BSA was examined by UV spectrophotometry at 595 nm using Coomassie Brilliant Blue protein assay. 3. Results and discussion 3.1. Zeta potential and pH value of NPs Table 1 shows the zeta potential and pH value of samples. A slight variation of zeta potential of NPs was detected. The pH results indicated that adding OREC had little remarkable effect on the pH value of NPs emulsions. During the process of NPs formation, the amount of ALG was excessive which resulted in the negative potential of NPs. But the consistent increase of the zeta potential verified that OREC, a negative material (Deng et al., 2011a), had been successfully added into NPs and might form some electrostatic interactions with QC and ALG. The uniform pH value of NPs showed the solubility of BSA was identical in each sample.
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Fig. 2. EDX spectrum of QC–OREC/ALG NPs sediments after centrifugation.
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Fig. 4. SAXRD patterns of OREC powder, NP121, NP61 and NP31.
3.2. Morphology of the NPs TEM was performed to investigate the morphology and the intercalation structure between polymer and OREC (Fig. 1). Fig. 1a shows the morphology of dispersed NP0 after negative staining. It could be seen that an ideal model of about 20–50 nm size NPs with widely distribution had been produced successfully. In addition, the NPs were ball-like and agglomerate beads (Fig. 1b). As we know, OREC was a kind of layered silicate and the primary elements in OREC was silicon which could exhibit dark color in TEM. Fig. 1c gave the images of QC–OREC/ALG NPs with the addition of OREC, which could be observed without negative stain which indicated that NPs and OREC clay particles could interact with each other. Their size of QC–OREC/NPs was about 100–120 nm and larger than that of NP0. The possible reason was that OREC particles were encapsulated into the NPs during their formation and a few QC NPs might interact with a single OREC particle at the same time. FE-SEM image (Fig. 1d) exhibited the spherical shape of NPs. The average size was 20–50 nm and identical with the TEM results. 3.3. Elements analysis and SAXRD patterns of the NPs The NPs composition analysis results were shown in Figs. 2 and 3. EDX spectrum (Fig. 2) was recorded to identify
Fig. 5. FT-IR spectra of OREC, ALG, HTCC and the NPs: (a) NP0 and (b) NP31.
the expected compositions on a selected rectangle area of NP31 sediment. The characteristic peaks of Si and Ca elements in the spectrum were identified. It proved that OREC had successfully been introduced into NPs. Fig. 3 shows the XPS narrow scans with
Fig. 3. XPS narrow scans with the curve fit of QC–OREC/ALG NPs: (a) N 1s and (b) Si 2p.
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Fig. 6. The size distribution of NPs: (a) NP0, (b) NP121, (c) NP61, (d) NP31, (e) BSA-loaded NP0 and (f) BSA-loaded NP31. Each sample was measured for three times.
the curve fit of NP31 sediment centrifuged from 9 mL opalescent emulsion. In Fig. 3a, N1s peak was observed around 397.8 eV, assigned to the binding energy of Si N structure (Ono et al., 1999). It strongly attested that the interaction was occurred between QC and OREC. Fig. 3b showed the XPS narrow scan result of silicon and the peak at 99.8 eV observed was corresponding to Si Si bond, which also indicated Si was existed in NP31 (Ma et al., 2004). SAXRD was utilized to calculate the interlayer distance between OREC and the composite NPs: NP121, NP61 and NP31. The interlayer distances were calculated by Bragg’s equation. In Fig. 4,
the increasing trend for interlayer distance of OREC was measured. The bulk OREC powder was with the interlayer distance of 2.45 nm and the largest interlayer distance of OREC in composite NPs was 3.75 nm in NP31, which indicated that the QC chains had successfully intercalated into the interlayer of OREC. The disappearance of features diffraction peak revealed that the crystallization of both OREC and QC were destroyed. The reason might be that the molecular movement of QC chains was limited greatly because of the formation of the interaction structure (Wang et al., 2008).
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3.4. FT-IR spectrum of NPs As shown in Fig. 5, the FT-IR spectrum of bulk OREC had the dominant peaks around 467 and 546 cm−1 , representing Si O bending vibration (Deng et al., 2011a,b). In the FT-IR spectrum of ALG, the bands around 1030 cm−1 (C O C stretching) and 950 cm−1 (C O stretching) were assigned to its saccharine structure. In addition, the bands at 1620 and 1416 cm−1 are attributed to asymmetric and symmetric stretching peaks of carboxylate salt groups (Li et al., 2007). In QC FT-IR spectrum, bands around 1657 and 1609 cm−1 commonly assigned to the C O stretching of the secondary amide and NH2 bending of the primary amine, respectively. The band at 1085 cm−1 was known as the unsymmetric stretching of C O C. New band at 1484 cm−1 was attributed to the C H bending of trimethylammonium group. In the spectra of NPs (Fig. 5a and b), the 3643 cm−1 band of OREC disappeared, which indicated that the OH of OREC had reacted with QC or ALG. The asymmetrical stretching of COO groups shifted to 1611 cm−1 and the symmetrical stretching of COO groups shifted to 1420 cm−1 , which revealed that the carboxylic groups of ALG had interacted with QC. The dominant peaks of OREC at 467 and 546 cm−1 in Fig. 5b verified that OREC was in the NPs. Besides, hydrogen bonding might generate between and inside the QC chains, and a previous research verified that OH groups in OREC could interact immensely with QC or ALG to form new kind of interface layer or partly network structure (Wang et al., 2006). The above analysis suggested that there were strong interactions between OREC and polymers. 3.5. Size distribution of NPs Fig. 6 displays the size distribution results of NP0, NP121, NP61, NP31, BSA-loaded NP0 and BSA-loaded NP31. Fig. 6a indicated that most of NP0 was with the mean size of 43.82 nm, which was identical with the results of TEM (Fig. 1a). In addition, NP121, NP61 and NP31 were with the mean size of 122.4, 105.7 and 91.28 nm, respectively. The results indicated that OREC had the mean size of around 90 nm and which verified our previous prediction in the TEM study. Furthermore, the mean size of NP121 was much bigger than that of NP61 and NP31. In NP121, the weight ratio of QC:OREC was 12:1, which could cause more QC chains had the chance to intercalate into the interlayer of OREC than in NP61 and NP31. Larger QC chains to OREC particles ratio, more gelation reaction would occur outside the interlayers of OREC and thus bigger size would be formed. Moreover, when BSA was loaded in the NPs, the mean size would be increased. The result was caused by crosslinking via electrostatic interaction between BSA and the quaternary ammonium salt of QC. The above results were attributed to the presence of OREC in the system of NPs, known for its remarkable size and controllable interlayer, which can enable polymer chains intercalate into the interlayer, resulting in an obvious different size distribution between the NP0 and QC–OREC/ALG NPs. 3.6. Encapsulation efficiency (EE) and loading capacity (LC) of BSA Fig. 7 shows the encapsulation efficiency and loading capacity of BSA in the NPs, respectively. It could be noted that with the addition of OREC (Fig. 7b–d), the EE and LC were both larger than those in NP0 (Fig. 7a), because OREC had controllable interlayer and large surface area. When the amount of OREC increased (Fig. 7b–d), the EE decreased slightly. It was attributed to two results as follow: first, some of the QC chains were intercalated into the interlayer of OREC, so the gelation reaction between QC and ALG in the NPs was partly occurred between the interlayer of OREC as well as outside the OREC. The interlayer distance of the OREC was about 2–4 nm, thus
Fig. 7. The encapsulation efficiency (EE) and loading capacity (LC) of NPs: (a) NP0, (b) NP121, (c) NP61 and (d) NP31.
the space was limited for gelation. When the mass ratio of QC: OREC increased from 3:1 to 12:1, the interlayer distance decreased (Fig. 4). In addition, the interlayer distance was different but the difference was not obvious in NP121, NP61 and NP31, respectively, herein the distance was not the main factor for the BSA entrapment. Second, OREC was with negative charge (Deng et al., 2011a), which could cause an electrostatic repulsive force among OREC, BSA and ALG. Furthermore, the electrostatic interactions generated between the free NH3+ of QC and the negative charge of BSA were weakened because the increased amount of OREC could entrap more QC. In this case, a little amount of OREC might help BSA to compete with ALG for the entrapment space, but much of them would cause EE decreased. This was the main reason for the BSA encapsulation variety. The combined effect of two above factors indicated that the NP121 had best EE of 44.9% and the LC was also highest than those of any others. More significantly, the EE increased almost 10% compared with the results of the previous report with the addition of OREC, and few LC loss was occurred (Li et al., 2007). We can draw a conclusion that OREC had a positive effect on both EE and LC of BSA in the composite NPs. 3.7. In vitro release tests In order to investigate the controlled release properties of BSAloaded NPs, in vitro release tests were performed in 0.1 M PBS buffer (pH = 7.4) and 0.1 M HCl (pH = 1.2), which were simulated to the gastric and intestinal fluid, respectively. The release results were shown in Fig. 8. Obviously, rapid initial drug release was exhibited in all samples, followed by slow release. In the first release period, the rapid release of BSA was due to the desorption behavior of BSA on the surface of the NPs. After that the amount of released BSA decreased, especially in HCl solution, because some of the free BSA in the solution was reabsorbed onto the surface of the NPs. The result was identical with previous reports (Bouillot et al., 1999). In high pH solution, the electrostatic interactions between QC and ALG were reduced so that the erosion of the NPs was easy to be occurred and more BSA could be released. In addition, the solubility of BSA in pH = 7.4 was larger than that in pH = 1.2 so BSA was easier to be released. With the addition of OREC, the rapid initial release was depressed obviously both in PBS buffer and HCl. In addition, the OREC had little effect on the following release. In pH = 7.4, BSA-loaded NP31 had the lowest initial release of 11.07% among the samples, which was caused by the reduction of electrostatic
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Acknowledgement This work was supported by the National Natural Science Fund (Nos. 81000597, 81070248, 81100579, and 81090274), the Critical Project Foundation on Social Development of ShanXi province (No. 2010K16-04-05) and Natural Science Fund of ShanXi province (No. 2009JM4023).
References
Fig. 8. The cumulative release profiles of BSA from NPs in (a) 0.1 M HCl (pH = 1.2) and (b) 0.1 M PBS (pH = 7.4).
repulsive force among OREC, BSA and ALG in high pH value. Furthermore, according to the previous report, NP31 had larger distance between OREC interlayer which could entrap more BSA, leading to hard initial release of BSA (Wang et al., 2008). While in pH = 1.2, BSA-loaded NP61 had the lowest initial release of 4.52% compared with other samples. In this condition, the factor of electrostatic repulsive force was as important as the factor of increased interlayer distance of OREC, and led the BSA located between the interlayer of OREC to release. 4. Conclusion With the addition of OREC, the novel NPs with intercalated structure were successfully obtained according to our predesigned architecture. The variety of the mass ratio of QC–OREC could obviously affect the encapsulation efficiency and loading capacity of BSA in the NPs and especially in NP121, both of which were higher than those in previous report. In addition, OREC could avoid the burst release phenomenon in the first period. NP61 and NP31 were suitable for drug release in stomach and intestine, respectively. Furthermore, the established method for BSA delivery may also be extended to tailor the size and composition of other NPs with tunable release speed for various applications such as catalysis, enzyme immobilization, etc.
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Quaternized chitosan–organic rectorite intercalated composites based nanoparticles for protein controlled release Ruifen Xu a,b,1 , Shangjing Xin c,1 , Xue Zhou d,1 , Wei Li c , Feng Cao e , Xuyang Feng e,∗ , Hongbing Deng b,c,∗∗ a
Department of Anesthesiology, School of Stomatology, Fourth Military Medical University, Xi’an 710032, China Department of Environmental Science, College of Resource and Environmental Science, Wuhan University, Wuhan 430079, China College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China d State Key Laboratory of Environment Health (Incubation); Key Laboratory of Environment and Health, Ministry of Education; Key Laboratory of Environment and Health (Wuhan), Ministry of Environmental Protection; Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China e Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China b c
a r t i c l e
i n f o
Article history: Received 25 July 2012 Received in revised form 27 August 2012 Accepted 6 September 2012 Available online 12 September 2012 Keywords: Quaternized chitosan Organic rectorite Intercalation Nanoparticles Drug delivery
a b s t r a c t Organic rectorite (OREC) was added in the quaternized chitosan (QC)/alginate (ALG) nanoparticles using an ionic gelation method to fabricate a controllable release system for proteins for the first time. The morphology of nanoparticles, the intercalated structure of OREC, bovine serum albumin encapsulation efficiency and in vitro release properties were investigated. Fourier transform infrared spectra, energy dispersive X-ray, X-ray photoelectron spectroscopy, small angle X-ray diffraction and size distribution analysis were performed to characterize the composite nanoparticles. With the addition of OREC, the encapsulation efficiency and the loading capacity of nanoparticles had increased from 21.2% to 44.9% and from 13.7% to 25.0%, respectively. In addition, the rapid initial release was inhibited successfully from 20.15% to 11.07% in stimulated gastric fluid and from 14.69% to 4.52% in stimulated intestinal fluid. The results verified that the addition of OREC could make these nanoparticles effective carriers to encapsulate drug and slow the drug controlled release of nanoparticles. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Nowadays, proteins and peptides have drawn many attentions in biotechnology because of their effective properties for disease treatment (Castro et al., 2005). However, they are hard to be absorbed in stomach and intestinal tracts as their hydrophilic property. In order to overcome this disadvantage, colloidal materials such as nanoparticles (NPs) have been used to protect proteins and peptides from quick dissolving and fabricate controlled release systems for the enduring utilization of drugs (Khalil and Mainardes, 2009; Mora-Huertas et al., 2010). Chitosan (CS) is not only naturally abundant, but also nontoxic, biodegradable, and regenerable. Because of these properties, CS has been used in a variety of application areas such as bacterial inhibition (Deng et al., 2011a), cell culture (Deng et al., 2010), cosmetic
∗ Corresponding author. Tel.: +86 29 84775183; fax: +86 29 84775183. ∗∗ Corresponding author at: Department of Environmental Science, College of Resource and Environmental Science, Wuhan University, Wuhan 430079, China. Tel.: +86 27 68778501; fax: +86 27 68778501. E-mail addresses: [email protected] (X. Feng), [email protected] (H. Deng). 1 Co-first authors with the same contribution to this work. 0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2012.09.010
skin masks (Huang et al., 2003), tissue engineering (Thein-Han et al., 2008) and drug delivery (Yuan et al., 2008). Some researchers have reported that CS NPs was applied as drug controlled release systems for hydrophilic drugs delivery by utilizing the hydrophobic property of CS (Wu et al., 2005; Xu and Du, 2003). Quaternized chitosan (QC), a water soluble derivative of CS, has been found that it is with better properties of permeability and mucoadhesion than CS (Kotze et al., 1999; Thanou et al., 2000), when used as an absorption enhancer transporting across the intestinal epithelia. In addition, QC NPs were with smaller particle size and remarkable advantages such as weakly alkaline values in drug delivery system (Bayat et al., 2008). Moreover, some reports have covered that QC NPs were used as a protein carrier (Xu et al., 2003) and that NPs cross-linking with alginate (ALG) were with better controlled release properties (Wang et al., 2011). However, the encapsulation efficiency of drug in the NPs is too low because the superabundant negative charged ALG could compete for the positive charged QC with drugs (Li et al., 2007). In order to solve this problem, inorganic materials will be added for preparing QC/ALG organic–inorganic hybrid NPs. So far, CS based organic–inorganic hybrid has been reported as a nontoxic and biodegradable scaffold for tissue engineering and drug delivery response (Depan et al., 2011a; Thein-Han and
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Scheme 1. Schematic diagram illustrating the fabrication process of QC–OREC nanoparticles.
Misra, 2009; Thein-Han et al., 2009; Yuan et al., 2010a) and the structure–process–property relationship has been studied to show the effect of adding inorganic component (Depan et al., 2011b). Based on these previous researches, the organic–inorganic composites might form a novel potential system with both of high encapsulation efficiency and loading capacity of drugs. Layered silicate, such as montmorillonite (MMT) and rectorite (REC), can be easily intercalated into layered structure by nature polymer chains and has been used in drug delivery recently (Burgentzle et al., 2004). CS-layered silicate nanocomposite carriers have been determined to be effective in cancer therapy. In the carriers, the positively charged chemotherapeutic drug was strongly bounded to the negatively charged layered silicate and release of the drug was slow (Yuan et al., 2010b). REC, especially organic rectorite (OREC) organic modified from REC, has larger interlayer distance, better separable layer thickness and larger aspect ratio than MMT (Wang et al., 2006). REC has been intercalated by QC chains and fabricated to QC–REC nanocomposites for antibacterial application (Wang et al., 2009) and gene delivery (Wang et al., 2008). In addition, layered silicate has been verified safe enough as food additives by European Food Safety Authority (ESFA) last year so that it is good for drug delivery (European Food Safety Authority, 2011). While CS-layered silicate composites are biocompatible and become a potential candidate to drug controlled release, the mechanical properties and biological response of these composites are inadequate to qualify them for drug delivery or facilitate transfer of the applied load at the implant site. Furthermore, when considered intercalation between polymer chains and layered silicate, larger interlayer distance and better electrostatic force still need to be achieved. In the present study, novel QC–OREC/ALG composite NPs with intercalated structure and controllable OREC interlayer distance were fabricated and used as drug carrier for the first time. Bovine Serum Albumin (BSA) was selected as hydrophilic drug model for investigating the drug delivery properties of NPs. Transmission scanning electron microscope (TEM), field emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray (EDX), X-ray elemental spectroscopy (XPS), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) and NPs size distribution analysis are performed to characterize the properties of NPs. Encapsulation efficiency, loading capacity and in vitro release properties of BSA will be evaluated.
2. Materials and methods 2.1. Materials Chitosan (Mw = 2.1 × 105 kDa) was provided by Yuhuan Ocean Biochemical Co. (Taizhou, China) and the degree of deacetylation was 92%. Alginate (ALG) was purchased from Aladdin reagent Inc. (Shanghai, China). BSA (Mw = 6.8 × 104 kDa) was supplied by Amresco Inc., USA. Calcium rectorite (Ca2+ –REC) was provided by Hubei Mingliu Inc. Co. (Wuhan, China). QC and organic rectorite (OREC) were prepared in our lab as previously reports (Wang et al., 2006; Xu et al., 2003). The degree of substitution of QC was 71%. The average diameter of OREC particles was about 100 nm. All other chemicals were of analytical grade. 2.2. Preparation of QC/ALG NPs QC–ALG NPs were labeled with NP0. QC–OREC/ALG NPs were prepared in mass ratios of QC:OREC were at 12:1, 6:1 and 3:1, which were labeled with NP121, NP61 and NP31, respectively. NPs were prepared as shown in Scheme 1. QC–OREC nanocomposites were moderately stirred for 24 h in 60 ◦ C water bath. Aqueous calcium chloride (0.5 mg/mL) was dispersed by ultrasonic for 5 min and then added into aqueous sodium alginate (1.0 mg/mL) to form the pregel. The QC solution or the QC–OREC composites was distilled into the pre-gel with stirring and continued stirring for another 30 min. The solution volume ratio of CaCl2 , ALG and QC or QC–OREC composites was at 2:6:1. The obtained opalescent emulsion was stored overnight and centrifuged at 15,000 × g for 30 min at 4 ◦ C. The sediment lyophilized to produce solid NPs aggregates. The BSA-loaded QC/ALG NPs were prepared by using the same way as above by adding BSA (1.0 mg/mL) into ALG solutions, and the pH value of BSA/ALG mixture solution was determined at 6.78. 2.3. Morphology and characterization of QC/ALG NPs The morphology of the NPs was observed by transmission electron microscope (TEM, JEM-2100, JEOL, Japan). QC/ALG NPs were stained with phosphotungstic acid solution (2%, w/v) and other samples were observed on a micro grid mesh scaffold. All samples were dried on copper grill at room temperature. The
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Fig. 1. TEM images of QC/ALG NPs (a, b) and QC–OREC/ALG NPs (c); FE-SEM image of QC/ALG NPs (d).
surface morphology of NPs was performed by field emission scanning electron microscopy (FE-SEM, JSM-6700F, JEOL, Japan). The NPs emulsion was dropped on aluminum foil and dried at room temperature. Energy-dispersive X-ray (EDX) spectroscopy images were obtained by Sirion 200 (FEI, Netherlands). The composition of the NPs was examined by X-ray elemental spectroscopy (XPS) using an axis ultra DLD apparatus (Kratos, UK). The small angle X-ray diffraction (SAXRD) was performed on type D/max-rA diffractometer (Rigaku Co., Japan) with Cu target and K␣ radiation ( = 0.154 nm). Fourier transform infrared (FT-IR) spectra were recorded by Nicolet170-SX (Thermo Nicolet Ltd., USA). The size distribution and zeta potential of the NPs were determined by Nano 3690 (Malvern, UK). The wavelength for the size measurement was 532.0 nm and the angle detection was 90◦ at 25 ◦ C. The pH value of NPs was also measured by pH meter.
2.4. Encapsulation efficiency and loading capacity The encapsulation efficiency (EE) and loading capacity (LC) were calculated by determining the concentration of BSA of the initial solution and that after loading. The concentration was examined by UV spectrophotometry at 595 nm using Coomassie Brilliant Blue protein assay. The EE and LC were calculated from: EE =
C0 − C1 × 100% C0
(1)
LC =
C0 − C1 × 100% M
(2)
where C0 (mol/L) is the concentration of BSA in initial solution, C1 (mol/L) is the concentration of free BSA in the supernatant, and M (g) is the total mass of the NPs.
Table 1 Zeta potential and pH value of NPs emulsions. Samples
-Potential (mV)
pH value
NP0 NP121 NP61 NP31
−38.2 −40.8 −42.5 −43.9
6.31 6.32 6.34 6.23
± ± ± ±
3.1 5.2 2.3 4.2
± ± ± ±
0.02 0.06 0.03 0.14
2.5. In vitro release tests 9 mL BSA-loaded NPs opalescent emulsion was centrifuged and then the sediment was suspended into 6 mL 0.1 M PBS buffer solution (pH = 7.4) or 0.1 M HCl solution (pH = 1.2). The mixtures were stirred in a mechanical vortexer at 100 rpm at 37 ◦ C. All the solutions were centrifuged before examining the amount of BSA released. 1 mL supernatant was taken out for the determination of the BSA concentration and another 1 mL fresh PBS buffer or HCl medium was supplied into the samples to ensure the total volume was 6 mL. The amount of BSA was examined by UV spectrophotometry at 595 nm using Coomassie Brilliant Blue protein assay. 3. Results and discussion 3.1. Zeta potential and pH value of NPs Table 1 shows the zeta potential and pH value of samples. A slight variation of zeta potential of NPs was detected. The pH results indicated that adding OREC had little remarkable effect on the pH value of NPs emulsions. During the process of NPs formation, the amount of ALG was excessive which resulted in the negative potential of NPs. But the consistent increase of the zeta potential verified that OREC, a negative material (Deng et al., 2011a), had been successfully added into NPs and might form some electrostatic interactions with QC and ALG. The uniform pH value of NPs showed the solubility of BSA was identical in each sample.
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Fig. 2. EDX spectrum of QC–OREC/ALG NPs sediments after centrifugation.
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Fig. 4. SAXRD patterns of OREC powder, NP121, NP61 and NP31.
3.2. Morphology of the NPs TEM was performed to investigate the morphology and the intercalation structure between polymer and OREC (Fig. 1). Fig. 1a shows the morphology of dispersed NP0 after negative staining. It could be seen that an ideal model of about 20–50 nm size NPs with widely distribution had been produced successfully. In addition, the NPs were ball-like and agglomerate beads (Fig. 1b). As we know, OREC was a kind of layered silicate and the primary elements in OREC was silicon which could exhibit dark color in TEM. Fig. 1c gave the images of QC–OREC/ALG NPs with the addition of OREC, which could be observed without negative stain which indicated that NPs and OREC clay particles could interact with each other. Their size of QC–OREC/NPs was about 100–120 nm and larger than that of NP0. The possible reason was that OREC particles were encapsulated into the NPs during their formation and a few QC NPs might interact with a single OREC particle at the same time. FE-SEM image (Fig. 1d) exhibited the spherical shape of NPs. The average size was 20–50 nm and identical with the TEM results. 3.3. Elements analysis and SAXRD patterns of the NPs The NPs composition analysis results were shown in Figs. 2 and 3. EDX spectrum (Fig. 2) was recorded to identify
Fig. 5. FT-IR spectra of OREC, ALG, HTCC and the NPs: (a) NP0 and (b) NP31.
the expected compositions on a selected rectangle area of NP31 sediment. The characteristic peaks of Si and Ca elements in the spectrum were identified. It proved that OREC had successfully been introduced into NPs. Fig. 3 shows the XPS narrow scans with
Fig. 3. XPS narrow scans with the curve fit of QC–OREC/ALG NPs: (a) N 1s and (b) Si 2p.
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Fig. 6. The size distribution of NPs: (a) NP0, (b) NP121, (c) NP61, (d) NP31, (e) BSA-loaded NP0 and (f) BSA-loaded NP31. Each sample was measured for three times.
the curve fit of NP31 sediment centrifuged from 9 mL opalescent emulsion. In Fig. 3a, N1s peak was observed around 397.8 eV, assigned to the binding energy of Si N structure (Ono et al., 1999). It strongly attested that the interaction was occurred between QC and OREC. Fig. 3b showed the XPS narrow scan result of silicon and the peak at 99.8 eV observed was corresponding to Si Si bond, which also indicated Si was existed in NP31 (Ma et al., 2004). SAXRD was utilized to calculate the interlayer distance between OREC and the composite NPs: NP121, NP61 and NP31. The interlayer distances were calculated by Bragg’s equation. In Fig. 4,
the increasing trend for interlayer distance of OREC was measured. The bulk OREC powder was with the interlayer distance of 2.45 nm and the largest interlayer distance of OREC in composite NPs was 3.75 nm in NP31, which indicated that the QC chains had successfully intercalated into the interlayer of OREC. The disappearance of features diffraction peak revealed that the crystallization of both OREC and QC were destroyed. The reason might be that the molecular movement of QC chains was limited greatly because of the formation of the interaction structure (Wang et al., 2008).
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3.4. FT-IR spectrum of NPs As shown in Fig. 5, the FT-IR spectrum of bulk OREC had the dominant peaks around 467 and 546 cm−1 , representing Si O bending vibration (Deng et al., 2011a,b). In the FT-IR spectrum of ALG, the bands around 1030 cm−1 (C O C stretching) and 950 cm−1 (C O stretching) were assigned to its saccharine structure. In addition, the bands at 1620 and 1416 cm−1 are attributed to asymmetric and symmetric stretching peaks of carboxylate salt groups (Li et al., 2007). In QC FT-IR spectrum, bands around 1657 and 1609 cm−1 commonly assigned to the C O stretching of the secondary amide and NH2 bending of the primary amine, respectively. The band at 1085 cm−1 was known as the unsymmetric stretching of C O C. New band at 1484 cm−1 was attributed to the C H bending of trimethylammonium group. In the spectra of NPs (Fig. 5a and b), the 3643 cm−1 band of OREC disappeared, which indicated that the OH of OREC had reacted with QC or ALG. The asymmetrical stretching of COO groups shifted to 1611 cm−1 and the symmetrical stretching of COO groups shifted to 1420 cm−1 , which revealed that the carboxylic groups of ALG had interacted with QC. The dominant peaks of OREC at 467 and 546 cm−1 in Fig. 5b verified that OREC was in the NPs. Besides, hydrogen bonding might generate between and inside the QC chains, and a previous research verified that OH groups in OREC could interact immensely with QC or ALG to form new kind of interface layer or partly network structure (Wang et al., 2006). The above analysis suggested that there were strong interactions between OREC and polymers. 3.5. Size distribution of NPs Fig. 6 displays the size distribution results of NP0, NP121, NP61, NP31, BSA-loaded NP0 and BSA-loaded NP31. Fig. 6a indicated that most of NP0 was with the mean size of 43.82 nm, which was identical with the results of TEM (Fig. 1a). In addition, NP121, NP61 and NP31 were with the mean size of 122.4, 105.7 and 91.28 nm, respectively. The results indicated that OREC had the mean size of around 90 nm and which verified our previous prediction in the TEM study. Furthermore, the mean size of NP121 was much bigger than that of NP61 and NP31. In NP121, the weight ratio of QC:OREC was 12:1, which could cause more QC chains had the chance to intercalate into the interlayer of OREC than in NP61 and NP31. Larger QC chains to OREC particles ratio, more gelation reaction would occur outside the interlayers of OREC and thus bigger size would be formed. Moreover, when BSA was loaded in the NPs, the mean size would be increased. The result was caused by crosslinking via electrostatic interaction between BSA and the quaternary ammonium salt of QC. The above results were attributed to the presence of OREC in the system of NPs, known for its remarkable size and controllable interlayer, which can enable polymer chains intercalate into the interlayer, resulting in an obvious different size distribution between the NP0 and QC–OREC/ALG NPs. 3.6. Encapsulation efficiency (EE) and loading capacity (LC) of BSA Fig. 7 shows the encapsulation efficiency and loading capacity of BSA in the NPs, respectively. It could be noted that with the addition of OREC (Fig. 7b–d), the EE and LC were both larger than those in NP0 (Fig. 7a), because OREC had controllable interlayer and large surface area. When the amount of OREC increased (Fig. 7b–d), the EE decreased slightly. It was attributed to two results as follow: first, some of the QC chains were intercalated into the interlayer of OREC, so the gelation reaction between QC and ALG in the NPs was partly occurred between the interlayer of OREC as well as outside the OREC. The interlayer distance of the OREC was about 2–4 nm, thus
Fig. 7. The encapsulation efficiency (EE) and loading capacity (LC) of NPs: (a) NP0, (b) NP121, (c) NP61 and (d) NP31.
the space was limited for gelation. When the mass ratio of QC: OREC increased from 3:1 to 12:1, the interlayer distance decreased (Fig. 4). In addition, the interlayer distance was different but the difference was not obvious in NP121, NP61 and NP31, respectively, herein the distance was not the main factor for the BSA entrapment. Second, OREC was with negative charge (Deng et al., 2011a), which could cause an electrostatic repulsive force among OREC, BSA and ALG. Furthermore, the electrostatic interactions generated between the free NH3+ of QC and the negative charge of BSA were weakened because the increased amount of OREC could entrap more QC. In this case, a little amount of OREC might help BSA to compete with ALG for the entrapment space, but much of them would cause EE decreased. This was the main reason for the BSA encapsulation variety. The combined effect of two above factors indicated that the NP121 had best EE of 44.9% and the LC was also highest than those of any others. More significantly, the EE increased almost 10% compared with the results of the previous report with the addition of OREC, and few LC loss was occurred (Li et al., 2007). We can draw a conclusion that OREC had a positive effect on both EE and LC of BSA in the composite NPs. 3.7. In vitro release tests In order to investigate the controlled release properties of BSAloaded NPs, in vitro release tests were performed in 0.1 M PBS buffer (pH = 7.4) and 0.1 M HCl (pH = 1.2), which were simulated to the gastric and intestinal fluid, respectively. The release results were shown in Fig. 8. Obviously, rapid initial drug release was exhibited in all samples, followed by slow release. In the first release period, the rapid release of BSA was due to the desorption behavior of BSA on the surface of the NPs. After that the amount of released BSA decreased, especially in HCl solution, because some of the free BSA in the solution was reabsorbed onto the surface of the NPs. The result was identical with previous reports (Bouillot et al., 1999). In high pH solution, the electrostatic interactions between QC and ALG were reduced so that the erosion of the NPs was easy to be occurred and more BSA could be released. In addition, the solubility of BSA in pH = 7.4 was larger than that in pH = 1.2 so BSA was easier to be released. With the addition of OREC, the rapid initial release was depressed obviously both in PBS buffer and HCl. In addition, the OREC had little effect on the following release. In pH = 7.4, BSA-loaded NP31 had the lowest initial release of 11.07% among the samples, which was caused by the reduction of electrostatic
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Acknowledgement This work was supported by the National Natural Science Fund (Nos. 81000597, 81070248, 81100579, and 81090274), the Critical Project Foundation on Social Development of ShanXi province (No. 2010K16-04-05) and Natural Science Fund of ShanXi province (No. 2009JM4023).
References
Fig. 8. The cumulative release profiles of BSA from NPs in (a) 0.1 M HCl (pH = 1.2) and (b) 0.1 M PBS (pH = 7.4).
repulsive force among OREC, BSA and ALG in high pH value. Furthermore, according to the previous report, NP31 had larger distance between OREC interlayer which could entrap more BSA, leading to hard initial release of BSA (Wang et al., 2008). While in pH = 1.2, BSA-loaded NP61 had the lowest initial release of 4.52% compared with other samples. In this condition, the factor of electrostatic repulsive force was as important as the factor of increased interlayer distance of OREC, and led the BSA located between the interlayer of OREC to release. 4. Conclusion With the addition of OREC, the novel NPs with intercalated structure were successfully obtained according to our predesigned architecture. The variety of the mass ratio of QC–OREC could obviously affect the encapsulation efficiency and loading capacity of BSA in the NPs and especially in NP121, both of which were higher than those in previous report. In addition, OREC could avoid the burst release phenomenon in the first period. NP61 and NP31 were suitable for drug release in stomach and intestine, respectively. Furthermore, the established method for BSA delivery may also be extended to tailor the size and composition of other NPs with tunable release speed for various applications such as catalysis, enzyme immobilization, etc.
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