- Email: [email protected]
Synthesis and characterization of folate conjugated chitosan and cellular uptake of its nanoparticles in HT-29 cells
Carbohydrate Research 346 (2011) 801–806
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Synthesis and characterization of folate conjugated chitosan and cellular uptake of its nanoparticles in HT-29 cells Puwang Li a,b, Yichao Wang a, Fanbo Zeng c, Lijue Chen c, Zheng Peng b, Ling Xue Kong a,⇑ a
Centre for Material and Fiber Innovation, Deakin University, Geelong Vic 3217, Australia Agricultural Product Processing Research Institute, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang 524001, PR China c Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, PR China b
a r t i c l e
i n f o
Article history: Received 15 September 2010 Received in revised form 21 January 2011 Accepted 27 January 2011
Keywords: Folate Chitosan Chemical modification Cell uptake HT-29 cells
a b s t r a c t Folate–chitosan (FA–CS) conjugates synthesized by coupling FA with CS render new and improved functions because the original properties of CS are maintained and the targeting ligand of FA is incorporated. In this work, FA–CS conjugates were synthesized based on chemical linking of carboxylic group of FA with amino group of CS as confirmed by Fourier transform spectroscopy (FTIR) and nuclear magnetic resonance (1H NMR). FA–CS conjugates displayed less crystal nature when compared to CS. The FA–CS nanoparticles (NPs) were prepared by crosslinking FA–CS conjugates with sodium tripolyphosphate (STPP). Positively charged FA–CS nanoparticles were spherical in shape with a particle size of about 100 nm. Cellular uptake of CS or FA–CS nanoparticles was assayed by fluorescent microscopy using calcein as fluorescent marker in colon cancer cells (HT-29). The FA–CS nanoparticles exhibited improved uptake of HT-29 and could become a potential targeted drug delivery system for colorectal cancer. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Colon cancer is one of the leading causes of deaths in the world, which accounts for 655,000 deaths worldwide every year.1 Various drugs or drug combinations2,3 have been employed for the chemotherapeutic treatment of colon cancer. To achieve efficient chemotherapy, anticancer drug concentration in the blood should be maintained between the minimum effective therapeutic level and the maximum tolerable level for prolonged time interval.4 However, in many cases, the drug concentration exceeds the maximum tolerable level immediately after being administrated, which leads to serious side effects, and then declines quickly below the minimum effective therapeutic level, which makes therapy inefficient. Frequent dosing is required to maintain the therapeutic level, which cause serious side effect due to the presence of anticancer drugs in normal cells.5,6 Therefore, an ideal drug delivery system should be able to control the release rate of drugs.7 Nanoparticle drug delivery systems8 are believed to provide sustained and controlled release of drugs which leads to improved therapeutic index. However, those systems were still limited by the lack of selectivity9 toward tumor cells. Chitosan is attracting more and more attention in the applications as biomaterials due to its inherent biocompatibility and biodegradability.10,11 The abundant functional groups existing in CS monomers12 make CS amenable to various chemical ⇑ Corresponding author. Tel.: +61 3 5227 2087, fax: +61 3 5227 1103. E-mail address: [email protected] (L.X. Kong). 0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2011.01.027
modifications.13,14 The incorporation of targeting ligand with CS can not only keep the original physicochemical and biochemical properties without changing the original fundamental structure of CS but also introduce targeting ligand.15,16 Tumor-targeted drug delivery using CS nanoparticles could be achieved by surface decoration of CS nanoparticles with targeting ligand such as antibodies, hormones, peptides, and small compounds like FA and hyaluronic acid (HA).9,17,18 When compared to other targeting ligands, FA is less expensive, more easily conjugated to drug delivery carriers, and more stable in processing, storage and application.7 Therefore, it is widely used for targeting cell membrane and enhancing endocytosis of nanoparticles. FA has been covalently incorporated to CS nanoparticles as targeting ligand to selectively deliver anticancer drugs to cancer cells.19–21 However, there were only a few reports22 about targeting colon cancer cells by using FA incorporated CS. In addition, the incorporation of FA with CS is affected by many factors such as FA to CS ratio, reaction temperature and time. Nevertheless, up to now, no systemic investigation has been conducted in literature to optimize the preparation parameters and systematically investigate the properties of FA incorporated CS. Working on this rationale, the parameters for preparing FA–CS conjugates was optimized using orthogonal experimental design. FA–CS nanoparticles were prepared by ionic gelation technology, the resultant FA–CS conjugates were characterized with FTIR, 1H NMR and X-ray diffraction (XRD), and their physicochemical properties were examined in terms of surface morphology, particle size and zeta potential. Moreover, their cellular uptake was assayed in HT-29 cancerous cells by fluorescence microscope technology.
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P. Li et al. / Carbohydrate Research 346 (2011) 801–806
Figure 1. The preparation of FA–CS conjugates.
2. Experimental 2.1. Materials Chitosan (medium viscosity, deacetylation degree >84%) was purchased from Zhanjiang Xinmao Chemical & Glass Company (Zhanjiang, China). FA (cell culture tested), and 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Sigma Chemicals (Sydney, Australia). All other reagents were of analytical grade and obtained from the chemical store of Deakin University (Waurn Ponds Campus, Australia). 2.2. Synthesis of FA–CS conjugates The synthesis of FA–CS conjugates was based on the mechanism of carbodiimide chemistry.23 CS (1 g) was dissolved in 100 mL acetic acid solution (1%). A mixture solution of FA and EDC was prepared by dissolving FA and EDC simultaneously in anhydrous dimethyl sulfoxide (DMSO). Then, the mixture solution was dropped into the CS solution under magnetic stirring at different reaction temperature. After scheduled reaction time the resultant products were coagulated by adding 300 mL acetone. The coagulation was purified by dialysis against DMSO for two days and then by distilled water for another two days. Finally, yellow colored FA–CS products were collected and freeze-dried at 50 °C for 24 h.
phate (STPP) was dissolved in distilled water with a concentration of 2 mg/mL. After that, 4 mL STPP solution was added dropwise into 10 mL CS or FA–CS solution under magnetic stirring at room temperature. The nanoparticles were collected by centrifugation at 1200 rpm for 20 min and dried by lyophilization at 40 °C for
Table 2 Orthogonal experimental design and conjugational efficiency results No.
Factors
1 2 3 4 5 6 7 8 9 X1 X2 X3 R
A
B
C
Coupling ratio
1 1 1 2 2 2 3 3 3 0.2250 0.3890 0.4763 0.2513
1 2 3 1 2 3 1 2 3 0.3081 0.3674 0.4148 0.1067
1 2 3 3 1 2 2 3 1 0.3481 0.3822 0.3600 0.0341
0.0614 0.0819 0.0817 0.1094 0.1166 0.1630 0.1373 0.1689 0.1701
a
2.3. Preparation of nanoparticles 1485 1694
3419
T (%)
The preparation of CS or FA–CS nanoparticles was based on ionic gelation technology.24 Briefly, CS or FA–CS was dissolved in 1% acetic acid at a concentration of 3 mg/mL, and sodium tripolyphos-
1603
1652
b 1592 1087
3456
Table 1 The levels of experimental factors
c
Factors Levels
A FA:CS (w/w)
B Reaction time (h)
C Reaction temperature (°C)
1 2 3
1:5 1:3 1:1
2 4 6
40 50 60
1633
3435
4000
3500
3000
2500
2000 cm -1
1017
1500
1000
500
Figure 2. FTIR spectra of (a) FA, (b) CS and (c) FA–CS conjugates.
803
P. Li et al. / Carbohydrate Research 346 (2011) 801–806
48 h. The fluorescence marker loaded nanoparticles were prepared by dissolving calcein into STPP solution before the formation of nanoparticles.
2.5. Characterization of FA–CS conjugates FTIR spectrum was obtained by scanning samples from 4000 to 400 cm1 on a FTIR (GX-1, PerkinElmer, USA). 1H NMR spectrum was measured on a spectrometer (Mercury Plus 400, Varian, Inc., USA) using deuterated DMSO as solvent for FA and deuterated water (containing 1% deuterated trifluoroacetic acid) for FA–CS conjugates. XRD was conducted using X-ray diffractometer (D8 Advance, Bruck, Germany) with Cu as a target at a voltage of 40 kV. Samples were analyzed in 2h angle range of 3–60° at a scanning rate of 3°/2h/min. Morphology of the nanoparticles was observed at 10 kV on a scanning electronic microscopy (SEM, S-4800, Hitachi, Japan). Particle size distribution and zeta potential of nanoparticles were analyzed on a Nano-ZS (Malvern, UK).
2.4. Quantitative analysis of FA to CS coupling ratio In order to quantitatively analyze the content of FA in FA–CS conjugates, FA–CS was dissolved in 2% acetic acid solution at a concentration of 2 mg/mL. The content of FA in FA–CS conjugate was quantitatively analyzed by measuring the UV absorption at 363 nm on a UV–vis spectrophotometer (Lambda 35, PerkinElmer, USA) and its weight was calculated using a standard calibration curve. The coupling ratio (CR) was obtained:
CR ¼
W FA W FA—CS W FA
ð1Þ
2.6. Cell uptake of CS and FA–CS nanoparticles
where WFA–CS is the total amount of FA–CS conjugates, WFA is the amount of FA in the FA–CS conjugate. 4 OH
HT-29 cells were cultured into FA free RPMI1640 culture medium containing 10% fetal bovine serum (FBS) at 37 °C, 5% CO2 and 12
5
N 3 N
CH 2
H2N
N
N
2
1
8
20
13
O
10
9
6
NH
11
7
COOH
C
NH
17
18
14
CH 19 CH 2 21 CH 2 22 COOH
15
16
23
7
13/15
12/16 22
19 2
18
(a)
12
21
9
20/23
10
8
6
4
2
0
ppm 6' CH2 OH 5' 4'
OH
O
O
O
1'
3'
2'
n
NH 23 C 22 CH 2 HO 20
O
O
21 CH 2 C
CH 19
O
NH 18
13
C 17
12 10 NH
11 15
6
CH2
OH 4
5 N
9
N 3
7
16
N 8
22 21
(b)
12
2'
13/15 2 12/16 10
8
6
2 NH2
3'
4'
5' 20
N 1
4
ppm Figure 3. 1H NMR spectrum of (a) FA and (b) FA–CS conjugate.
2
0
P. Li et al. / Carbohydrate Research 346 (2011) 801–806
95% relative humidity in an incubator. After 24 h incubation, cell lines in exponential growth phase were digested with 0.25% trypsin and the cell concentration was adjusted to 1 105 cells/mL by using RPMI1640 culture medium. The cells were seeded into a 6well plastic plate with 18 mm 18 mm cover slips. After incubation for 24 h, HT-29 cells were exposed to calcein-loaded CS or FA–CS nanoparticles with or without the presence of 1 mM free FA. After 4 h incubation at 37 °C, the culture medium was removed and the wells were washed six times with PBS. Finally, the distribution of calcein-loaded nanoparticles in the cells was viewed under fluorescent microscope (IX71, Olympus, Japan). 3. Results and discussion 3.1. The preparation of FA–CS conjugates FA is a yellow-colored crystal powder which shows poor solubility in cold water. Increasing the water temperature or changing the pH of the solution (in basic or acidic condition) can improve its solubility, but will damage its stability. Thus DMSO was chosen as the solute and EDC was used to activate it. All the reactions were carried out in a dark environment to prohibit the FA from decomposition, as the UV absorption at 363 nm decreases with time when the FA DMSO solution is exposed to natural light. The schematic representation of the formulation process of FA–CS conjugates is presented in Figure 1. Firstly, EDC reacted with the carboxyl of FA to form an amine reactive O-acylisourea intermediate; then, this intermediate reacted with the amine of CS to form a conjugate of the two molecules which are joined by a stable amide bond. The conjugation of FA to CS molecules was affected by several parameters. Therefore, the key preparation parameters, FA to CS weight ratio, reaction time and reaction temperature, were chosen as the most influential factors (Table 1). By choosing the FA to CS coupling ratio as an index, three factors of three levels were investigated in order to obtain optimum preparation parameters. Table 2 shows the L9(33) orthogonal experimental design and the corresponding experimental data. The experimental data were evaluated with statistic analysis software (SAS version 6.0) and the results were summarized in Table 2. The ranking of the three factors is A > B > C, and the ranking of levels within each factor is: A: 3 > 2 > 1; B: 3 > 2 > 1; C: 1 > 2 > 3. Therefore, the optimized formulation for the conjugation of FA to CS is A3B3C1. The increase of FA to CS weight ratio and the increase of reaction time lead to a high degree of substitution, while increasing reaction temperature results in a decreased CR. This is due to the fact that high temperature leads to the degradation of CS and decomposition of FA. Therefore, the optimized formulation for preparing FA–CS conjugates is A3B3C1.
and two new peaks appear in 1633 and 1017 cm1, which belong to the vibration of C–N. The absorption peak of amide at 1652 cm1 of CS shifts to 1633 cm1, which is overlapped with the absorption peak of the newly formed C–N bond. 3.3. 1H NMR analysis The appearance of characteristic peaks in the 1H NMR spectrum of FA–CS conjugates confirms that FA is successfully incorporated with CS. As shown in the 1H NMR spectrum of FA (Fig. 3a), the signals at 8.67, 8.16, 7.64, 6.94, 6.64, 4.5, 4.34, 2.51, and 2.04 ppm were corresponding to the FA protons of H-18, H-13/15, H-10, H12/16, H-19, H-19, H-22, and H-21, respectively. The coupling of FA with CS leads to the overlapping of some sympathetic vibration peaks due to the influence of solvent and the interaction between FA and CS. The characteristic peaks at 11.0 ppm (H-20, FA), 2.30 ppm (H-21, FA), 2.14 ppm (H-22, FA) and the characteristic peaks at 3.31 ppm (H-2/H-5/H-6’), 1.83 ppm (H-4’, CS) can still be observed. 3.4. XRD analysis FA–CS conjugates are more likely to form amorphous rather than crystal structure as observed from XRD scattering pattern (Fig. 4). Intense peaks can be observed at 2h = 15.4°, 18.3°, 21.8°, 23.8°, 28.5°, 30.9°, 33.3°, 34.4°, 37° in the DSC scattering pattern of CS (Fig. 4a), indicating the crystal nature of CS. Intense peaks are also observed in the scattering pattern of FA (Fig. 4b) at
a Intensity
804
b
c 10
20
30 40 2 theta ( θ)
50
Figure 4. X-ray diffraction pattern of (a) CS; (b) FA; (c) FA–CS conjugate (FA/CS = 1/ 5).
3.2. FTIR analysis of FA–CS conjugates The significant change of FTIR peaks of FA–CS from those of CS confirms the effective formulation of FA–CS conjugates (Fig. 2). Strong absorption peaks in 3419, 1694, 1603 and 1485 cm1 can be observed in the FTIR spectrum of FA (Fig. 2a), which are corresponding to the vibration of N–H, C@O, amino group in the pteridine ring and C@C or C@N of FA. In the case of IR spectrum of CS (Fig. 2b), the absorption peaks in 1652 and 1592 cm1 belong to the vibration of amide I and amide II groups, and the wide absorption band in 3456 cm1 is the vibration of OH; the strong absorption peaks in 1087 cm1 belongs to the vibration of C–O– C. Significant difference is observed between the IR spectra of CS and FA–CS. It can be seen that the absorption peak in 3435 cm1 becomes stronger due to the overlapping of the vibration of OH and N–H functional group. The absorption in 1592 cm1 disappears
60
Figure 5. SEM image of FA–CS nanoparticles.
P. Li et al. / Carbohydrate Research 346 (2011) 801–806
2h = 5.6°, 11.1°, 13.4°, 16.6°, 17.3°, 19.5°, 22.0°, 26.9°, 28.07°, 29.8° due to its crystal property. Although intense peaks are observed at either CS or FA, only a hump peak is observed at 2h = 20.6° for FA– CS conjugates (Fig. 4c), which indicates the partly loss of crystalline
805
character for their resultant products. The coupling of FA with CS can hamper the movement of the molecular chain of CS in solid state due to the more complex structure of FA as compared to amino group. 3.5. Characterization of FA–CS nanoparticles
Figure 6. Particle size distribution of FA–CS nanoparticles.
The surface morphology and particle size distribution of FA–CS nanoparticles were investigated by SEM (Fig. 5) and laser light scattering (DLS) technology (Fig. 6). The FA–CS nanoparticles are spherical in shape with monodispersity and the average particle size is 100 nm (Fig. 5). The average particle size of FA–CS nanoparticles obtained by DLS method is 112.7 nm (Fig. 6), which is in good agreement with the measurement from SEM. The minor difference could be derived from the different sample states of the nanoparticles as SEM only visualizes the core of nanoparticles, while DLS measures the hydrodynamic radius of nanoparticles. The zeta potential of FA–CS nanoparticles is narrowly distributed with an average zeta potential of 12.5 mV (Fig. 7). In general, the average zeta potential of the CS nanoparticles is about 30– 50 mV. It is obvious that the modification of CS with FA leads to a sharp decrease in the zeta potential. This might be ascribed to the partial substitution of –NH2 with FA in the coupling process. Another possible reason might be the presence of side chain of FA, which destroys the entanglement of CS molecular chains in 1% acetic acid solution. Thus, more amino groups are exposed, which subsequently leads to more intense crosslinking between FA–CS and STPP. 3.6. Cellular uptake by HT-29
Figure 7. Zeta potential of FA–CS NPs (12.5 mV).
To investigate the cellular uptake of FA–CS nanoparticles, calcein was encapsulated into FA–CS and CS nanoparticles. The cellular uptake of ligand incorporated nanoparticles was qualitatively assayed by fluorescence microscopy in colorectal cancer cells HT-29. For the measurement of fluorescence intensity, HT-29 cells were incubated with calcein-loaded FA–CS or CS nanoparticles for 24 h with or without the presence of 1 mM free FA. The cellular uptake of FA–CS or CS nanoparticles was viewed by fluorescence microscopy. As shown in Figure 8a, strong fluorescence intensity is observed in the case of FA incorporated FA–CS nanoparticles, while very weak intensity is observed in the CS nanoparticles
Figure 8. Fluorescent microscopic images of HT-29 cells after incubation with calcein-loaded CS or FA–CS NPs for 4 h (200 magnification): (a) FA–CS NPs; (b) CS NPs; (c) FA– CS NPs + free FA; (d) CS NPs + free FA; (e and f) are their corresponding microscopic images without fluorescence.
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P. Li et al. / Carbohydrate Research 346 (2011) 801–806
(Fig. 8b). The presence of free FA in the culture medium leads to the decrease of fluorescence intensity of FA–CS nanoparticles (Fig. 8c). However, a slight increase in fluorescence intensity is observed for CS nanoparticles incubated with free FA. Due to the strong binding affinity between FA and FA-receptors, FA–CS nanoparticles could bind to FA receptors overexpressed in cancerous cells, which are subsequently internalized by HT-29 cells via the receptormediated endocytosis. The presence of free FA in the culture medium leads to the combination of FA with the receptors of HT-29 cells, which hamper the binding of FA–CS nanoparticles with receptors of HT-29. The results of the cellular uptake indicate that the coupling of FA into CS could significantly improve the cellular uptake of FA–CS nanoparticles by HT-29. It is obvious from the cellular uptake results that the FA–CS nanoparticles are a promising vehicle for the targeting anticancer drug to cancer tumor cells which are overexpressed with FA receptors. 4. Conclusions The chemical modification of CS with FA leads to the successful formulation of FA–CS conjugates which combine the original properties of CS and the targeting ligand of FA. The preparation parameters were optimized with orthogonal experimental design by using coupling ratio as an index. Carboxyl group of FA was successfully conjugated with the amino group of CS as confirmed by FTIR and 1H NMR. FA–CS displayed less crystal structure when compared with CS. FA–CS nanoparticles are spherical in shape and display positive charge properties. The specific binding affinity between the FA incorporated FA–CS nanoparticles and the FAreceptor was studied in colorectal cancer cell (HT-29). It was found that the FA incorporated FA–CS nanoparticles exhibit improved cellular uptake in HT-29 cells. These novel properties of FA–CS conjugates demonstrated that it is a promising biomaterial for the colon targeted drug delivery. Acknowledgments Mr. Puwang Li gratefully acknowledges the financial support from Australian Federal Government’s International Postgraduate
Research Scholarship through Deakin University. The work is also partially supported by Chinese Central Government 948 research program (2010-S8) via the Agricultural Product Processing Research Institute at Chinese Academy of Tropical Agricultural Sciences. References 1. Koning, G. A.; Kamps, J.; Scherphof, G. L. Cancer Detect. Prev. 2002, 26, 299– 307. 2. Castillo-Fernández, O.; Santibáñez, M.; Bauza, A.; Calderillo, G.; Castro, C.; Herrera, R.; Serrano, A.; Arrieta, O.; Herrera, L. A. Arch. Med. Res. 2010, 41, 430– 435. 3. Kornmann, M.; Formentini, A.; Ette, C.; Henne-Bruns, D.; Kron, M.; Sander, S.; Baumann, W.; Kreuser, E. D.; Staib, L.; Link, K. H. Eur. J. Surg. Oncol. 2008, 34, 1316–1321. 4. Chien, Y. W. Med. Res. Rev. 1990, 10, 477–504. 5. Hu, Y.; Jiang, X.; Ding, Y.; Ge, H. X.; Yuan, Y. Y.; Yang, C. Z. Biomaterials 2002, 23, 3193–3201. 6. Zhao, X. L.; Li, K. X.; Zhao, X. F.; Pang, D. H.; Chen, D. W. Chem. Pharm. Bull. 2008, 56, 963–968. 7. Byrne, J. D.; Betancourt, T.; Brannon-Peppas, L. Adv. Drug Delivery Rev. 2008, 60, 1615–1626. 8. Farokhzad, O. C.; Jon, S. Y.; Khadelmhosseini, A.; Tran, T. N. T.; LaVan, D. A.; Langer, R. Cancer Res. 2004, 64, 7668–7672. 9. Jain, A.; Jain, S. K. Eur. J. Pharm. Sci. 2008, 35, 404–416. 10. Ravi Kumar, M. N. V. React. Funct. Polym. 2000, 46, 1–27. 11. Jayakumar, R.; Menon, D.; Manzoor, K.; Nair, S. V.; Tamura, H. Carbohydr. Polym. 2010, 82, 227–232. 12. Harish Prashanth, K. V.; Tharanathan, R. N. Trends Food Sci. Technol. 2007, 18, 117–131. 13. Sajomsang, W.; Tantayanon, S.; Tangpasuthadol, V.; Daly, W. H. Carbohydr. Res. 2009, 344, 2502–2511. 14. Wang, T. W.; Xu, Q.; Wu, Y.; Zeng, A. J.; Li, M.; Gao, H. Carbohydr. Res. 2009, 344, 908–914. 15. Mourya, V. K.; Inamdar, N. N. React. Funct. Polym. 2008, 68, 1013–1051. 16. Subramanian, A.; Rau, A. V.; Kaligotla, H. Carbohydr. Polym. 2006, 66, 321–332. 17. Jain, A.; Jain, S. K.; Ganesh, N.; Barve, J.; Beg, A. M. Nanomed. Nanotechnol. Biol. Med. 2010, 6, 179–190. 18. Mamot, C.; Ritschard, R.; Kung, W.; Park, J. W.; Herrmann, R.; Rochlitz, C. F. J. Drug Targeting 2006, 14, 215–223. 19. Low, P. S.; Antony, A. C. Adv. Drug Delivery Rev. 2004, 56, 1055–1058. 20. Wang, S.; Low, P. S. J. Controlled Release 1998, 53, 39–48. 21. Wu, J.; Liu, Q.; Lee, R. J. Int. J. Pharm. 2006, 316, 148–153. 22. Mathew, M. E.; Mohan, J. C.; Manzoor, K.; Nair, S. V.; Tamura, H.; Jayakumar, R. Carbohydr. Polym. 2010, 80, 442–448. 23. Wan, A. J.; Sun, Y.; Li, H. L. Int. J. Biol. Macromol. 2008, 43, 415–421. 24. Calvo, P.; RemunanLopez, C.; VilaJato, J. L.; Alonso, M. J. J. Appl. Polym. Sci. 1997, 63, 125–132.
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Synthesis and characterization of folate conjugated chitosan and cellular uptake of its nanoparticles in HT-29 cells Puwang Li a,b, Yichao Wang a, Fanbo Zeng c, Lijue Chen c, Zheng Peng b, Ling Xue Kong a,⇑ a
Centre for Material and Fiber Innovation, Deakin University, Geelong Vic 3217, Australia Agricultural Product Processing Research Institute, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang 524001, PR China c Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, PR China b
a r t i c l e
i n f o
Article history: Received 15 September 2010 Received in revised form 21 January 2011 Accepted 27 January 2011
Keywords: Folate Chitosan Chemical modification Cell uptake HT-29 cells
a b s t r a c t Folate–chitosan (FA–CS) conjugates synthesized by coupling FA with CS render new and improved functions because the original properties of CS are maintained and the targeting ligand of FA is incorporated. In this work, FA–CS conjugates were synthesized based on chemical linking of carboxylic group of FA with amino group of CS as confirmed by Fourier transform spectroscopy (FTIR) and nuclear magnetic resonance (1H NMR). FA–CS conjugates displayed less crystal nature when compared to CS. The FA–CS nanoparticles (NPs) were prepared by crosslinking FA–CS conjugates with sodium tripolyphosphate (STPP). Positively charged FA–CS nanoparticles were spherical in shape with a particle size of about 100 nm. Cellular uptake of CS or FA–CS nanoparticles was assayed by fluorescent microscopy using calcein as fluorescent marker in colon cancer cells (HT-29). The FA–CS nanoparticles exhibited improved uptake of HT-29 and could become a potential targeted drug delivery system for colorectal cancer. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Colon cancer is one of the leading causes of deaths in the world, which accounts for 655,000 deaths worldwide every year.1 Various drugs or drug combinations2,3 have been employed for the chemotherapeutic treatment of colon cancer. To achieve efficient chemotherapy, anticancer drug concentration in the blood should be maintained between the minimum effective therapeutic level and the maximum tolerable level for prolonged time interval.4 However, in many cases, the drug concentration exceeds the maximum tolerable level immediately after being administrated, which leads to serious side effects, and then declines quickly below the minimum effective therapeutic level, which makes therapy inefficient. Frequent dosing is required to maintain the therapeutic level, which cause serious side effect due to the presence of anticancer drugs in normal cells.5,6 Therefore, an ideal drug delivery system should be able to control the release rate of drugs.7 Nanoparticle drug delivery systems8 are believed to provide sustained and controlled release of drugs which leads to improved therapeutic index. However, those systems were still limited by the lack of selectivity9 toward tumor cells. Chitosan is attracting more and more attention in the applications as biomaterials due to its inherent biocompatibility and biodegradability.10,11 The abundant functional groups existing in CS monomers12 make CS amenable to various chemical ⇑ Corresponding author. Tel.: +61 3 5227 2087, fax: +61 3 5227 1103. E-mail address: [email protected] (L.X. Kong). 0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2011.01.027
modifications.13,14 The incorporation of targeting ligand with CS can not only keep the original physicochemical and biochemical properties without changing the original fundamental structure of CS but also introduce targeting ligand.15,16 Tumor-targeted drug delivery using CS nanoparticles could be achieved by surface decoration of CS nanoparticles with targeting ligand such as antibodies, hormones, peptides, and small compounds like FA and hyaluronic acid (HA).9,17,18 When compared to other targeting ligands, FA is less expensive, more easily conjugated to drug delivery carriers, and more stable in processing, storage and application.7 Therefore, it is widely used for targeting cell membrane and enhancing endocytosis of nanoparticles. FA has been covalently incorporated to CS nanoparticles as targeting ligand to selectively deliver anticancer drugs to cancer cells.19–21 However, there were only a few reports22 about targeting colon cancer cells by using FA incorporated CS. In addition, the incorporation of FA with CS is affected by many factors such as FA to CS ratio, reaction temperature and time. Nevertheless, up to now, no systemic investigation has been conducted in literature to optimize the preparation parameters and systematically investigate the properties of FA incorporated CS. Working on this rationale, the parameters for preparing FA–CS conjugates was optimized using orthogonal experimental design. FA–CS nanoparticles were prepared by ionic gelation technology, the resultant FA–CS conjugates were characterized with FTIR, 1H NMR and X-ray diffraction (XRD), and their physicochemical properties were examined in terms of surface morphology, particle size and zeta potential. Moreover, their cellular uptake was assayed in HT-29 cancerous cells by fluorescence microscope technology.
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P. Li et al. / Carbohydrate Research 346 (2011) 801–806
Figure 1. The preparation of FA–CS conjugates.
2. Experimental 2.1. Materials Chitosan (medium viscosity, deacetylation degree >84%) was purchased from Zhanjiang Xinmao Chemical & Glass Company (Zhanjiang, China). FA (cell culture tested), and 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Sigma Chemicals (Sydney, Australia). All other reagents were of analytical grade and obtained from the chemical store of Deakin University (Waurn Ponds Campus, Australia). 2.2. Synthesis of FA–CS conjugates The synthesis of FA–CS conjugates was based on the mechanism of carbodiimide chemistry.23 CS (1 g) was dissolved in 100 mL acetic acid solution (1%). A mixture solution of FA and EDC was prepared by dissolving FA and EDC simultaneously in anhydrous dimethyl sulfoxide (DMSO). Then, the mixture solution was dropped into the CS solution under magnetic stirring at different reaction temperature. After scheduled reaction time the resultant products were coagulated by adding 300 mL acetone. The coagulation was purified by dialysis against DMSO for two days and then by distilled water for another two days. Finally, yellow colored FA–CS products were collected and freeze-dried at 50 °C for 24 h.
phate (STPP) was dissolved in distilled water with a concentration of 2 mg/mL. After that, 4 mL STPP solution was added dropwise into 10 mL CS or FA–CS solution under magnetic stirring at room temperature. The nanoparticles were collected by centrifugation at 1200 rpm for 20 min and dried by lyophilization at 40 °C for
Table 2 Orthogonal experimental design and conjugational efficiency results No.
Factors
1 2 3 4 5 6 7 8 9 X1 X2 X3 R
A
B
C
Coupling ratio
1 1 1 2 2 2 3 3 3 0.2250 0.3890 0.4763 0.2513
1 2 3 1 2 3 1 2 3 0.3081 0.3674 0.4148 0.1067
1 2 3 3 1 2 2 3 1 0.3481 0.3822 0.3600 0.0341
0.0614 0.0819 0.0817 0.1094 0.1166 0.1630 0.1373 0.1689 0.1701
a
2.3. Preparation of nanoparticles 1485 1694
3419
T (%)
The preparation of CS or FA–CS nanoparticles was based on ionic gelation technology.24 Briefly, CS or FA–CS was dissolved in 1% acetic acid at a concentration of 3 mg/mL, and sodium tripolyphos-
1603
1652
b 1592 1087
3456
Table 1 The levels of experimental factors
c
Factors Levels
A FA:CS (w/w)
B Reaction time (h)
C Reaction temperature (°C)
1 2 3
1:5 1:3 1:1
2 4 6
40 50 60
1633
3435
4000
3500
3000
2500
2000 cm -1
1017
1500
1000
500
Figure 2. FTIR spectra of (a) FA, (b) CS and (c) FA–CS conjugates.
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48 h. The fluorescence marker loaded nanoparticles were prepared by dissolving calcein into STPP solution before the formation of nanoparticles.
2.5. Characterization of FA–CS conjugates FTIR spectrum was obtained by scanning samples from 4000 to 400 cm1 on a FTIR (GX-1, PerkinElmer, USA). 1H NMR spectrum was measured on a spectrometer (Mercury Plus 400, Varian, Inc., USA) using deuterated DMSO as solvent for FA and deuterated water (containing 1% deuterated trifluoroacetic acid) for FA–CS conjugates. XRD was conducted using X-ray diffractometer (D8 Advance, Bruck, Germany) with Cu as a target at a voltage of 40 kV. Samples were analyzed in 2h angle range of 3–60° at a scanning rate of 3°/2h/min. Morphology of the nanoparticles was observed at 10 kV on a scanning electronic microscopy (SEM, S-4800, Hitachi, Japan). Particle size distribution and zeta potential of nanoparticles were analyzed on a Nano-ZS (Malvern, UK).
2.4. Quantitative analysis of FA to CS coupling ratio In order to quantitatively analyze the content of FA in FA–CS conjugates, FA–CS was dissolved in 2% acetic acid solution at a concentration of 2 mg/mL. The content of FA in FA–CS conjugate was quantitatively analyzed by measuring the UV absorption at 363 nm on a UV–vis spectrophotometer (Lambda 35, PerkinElmer, USA) and its weight was calculated using a standard calibration curve. The coupling ratio (CR) was obtained:
CR ¼
W FA W FA—CS W FA
ð1Þ
2.6. Cell uptake of CS and FA–CS nanoparticles
where WFA–CS is the total amount of FA–CS conjugates, WFA is the amount of FA in the FA–CS conjugate. 4 OH
HT-29 cells were cultured into FA free RPMI1640 culture medium containing 10% fetal bovine serum (FBS) at 37 °C, 5% CO2 and 12
5
N 3 N
CH 2
H2N
N
N
2
1
8
20
13
O
10
9
6
NH
11
7
COOH
C
NH
17
18
14
CH 19 CH 2 21 CH 2 22 COOH
15
16
23
7
13/15
12/16 22
19 2
18
(a)
12
21
9
20/23
10
8
6
4
2
0
ppm 6' CH2 OH 5' 4'
OH
O
O
O
1'
3'
2'
n
NH 23 C 22 CH 2 HO 20
O
O
21 CH 2 C
CH 19
O
NH 18
13
C 17
12 10 NH
11 15
6
CH2
OH 4
5 N
9
N 3
7
16
N 8
22 21
(b)
12
2'
13/15 2 12/16 10
8
6
2 NH2
3'
4'
5' 20
N 1
4
ppm Figure 3. 1H NMR spectrum of (a) FA and (b) FA–CS conjugate.
2
0
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95% relative humidity in an incubator. After 24 h incubation, cell lines in exponential growth phase were digested with 0.25% trypsin and the cell concentration was adjusted to 1 105 cells/mL by using RPMI1640 culture medium. The cells were seeded into a 6well plastic plate with 18 mm 18 mm cover slips. After incubation for 24 h, HT-29 cells were exposed to calcein-loaded CS or FA–CS nanoparticles with or without the presence of 1 mM free FA. After 4 h incubation at 37 °C, the culture medium was removed and the wells were washed six times with PBS. Finally, the distribution of calcein-loaded nanoparticles in the cells was viewed under fluorescent microscope (IX71, Olympus, Japan). 3. Results and discussion 3.1. The preparation of FA–CS conjugates FA is a yellow-colored crystal powder which shows poor solubility in cold water. Increasing the water temperature or changing the pH of the solution (in basic or acidic condition) can improve its solubility, but will damage its stability. Thus DMSO was chosen as the solute and EDC was used to activate it. All the reactions were carried out in a dark environment to prohibit the FA from decomposition, as the UV absorption at 363 nm decreases with time when the FA DMSO solution is exposed to natural light. The schematic representation of the formulation process of FA–CS conjugates is presented in Figure 1. Firstly, EDC reacted with the carboxyl of FA to form an amine reactive O-acylisourea intermediate; then, this intermediate reacted with the amine of CS to form a conjugate of the two molecules which are joined by a stable amide bond. The conjugation of FA to CS molecules was affected by several parameters. Therefore, the key preparation parameters, FA to CS weight ratio, reaction time and reaction temperature, were chosen as the most influential factors (Table 1). By choosing the FA to CS coupling ratio as an index, three factors of three levels were investigated in order to obtain optimum preparation parameters. Table 2 shows the L9(33) orthogonal experimental design and the corresponding experimental data. The experimental data were evaluated with statistic analysis software (SAS version 6.0) and the results were summarized in Table 2. The ranking of the three factors is A > B > C, and the ranking of levels within each factor is: A: 3 > 2 > 1; B: 3 > 2 > 1; C: 1 > 2 > 3. Therefore, the optimized formulation for the conjugation of FA to CS is A3B3C1. The increase of FA to CS weight ratio and the increase of reaction time lead to a high degree of substitution, while increasing reaction temperature results in a decreased CR. This is due to the fact that high temperature leads to the degradation of CS and decomposition of FA. Therefore, the optimized formulation for preparing FA–CS conjugates is A3B3C1.
and two new peaks appear in 1633 and 1017 cm1, which belong to the vibration of C–N. The absorption peak of amide at 1652 cm1 of CS shifts to 1633 cm1, which is overlapped with the absorption peak of the newly formed C–N bond. 3.3. 1H NMR analysis The appearance of characteristic peaks in the 1H NMR spectrum of FA–CS conjugates confirms that FA is successfully incorporated with CS. As shown in the 1H NMR spectrum of FA (Fig. 3a), the signals at 8.67, 8.16, 7.64, 6.94, 6.64, 4.5, 4.34, 2.51, and 2.04 ppm were corresponding to the FA protons of H-18, H-13/15, H-10, H12/16, H-19, H-19, H-22, and H-21, respectively. The coupling of FA with CS leads to the overlapping of some sympathetic vibration peaks due to the influence of solvent and the interaction between FA and CS. The characteristic peaks at 11.0 ppm (H-20, FA), 2.30 ppm (H-21, FA), 2.14 ppm (H-22, FA) and the characteristic peaks at 3.31 ppm (H-2/H-5/H-6’), 1.83 ppm (H-4’, CS) can still be observed. 3.4. XRD analysis FA–CS conjugates are more likely to form amorphous rather than crystal structure as observed from XRD scattering pattern (Fig. 4). Intense peaks can be observed at 2h = 15.4°, 18.3°, 21.8°, 23.8°, 28.5°, 30.9°, 33.3°, 34.4°, 37° in the DSC scattering pattern of CS (Fig. 4a), indicating the crystal nature of CS. Intense peaks are also observed in the scattering pattern of FA (Fig. 4b) at
a Intensity
804
b
c 10
20
30 40 2 theta ( θ)
50
Figure 4. X-ray diffraction pattern of (a) CS; (b) FA; (c) FA–CS conjugate (FA/CS = 1/ 5).
3.2. FTIR analysis of FA–CS conjugates The significant change of FTIR peaks of FA–CS from those of CS confirms the effective formulation of FA–CS conjugates (Fig. 2). Strong absorption peaks in 3419, 1694, 1603 and 1485 cm1 can be observed in the FTIR spectrum of FA (Fig. 2a), which are corresponding to the vibration of N–H, C@O, amino group in the pteridine ring and C@C or C@N of FA. In the case of IR spectrum of CS (Fig. 2b), the absorption peaks in 1652 and 1592 cm1 belong to the vibration of amide I and amide II groups, and the wide absorption band in 3456 cm1 is the vibration of OH; the strong absorption peaks in 1087 cm1 belongs to the vibration of C–O– C. Significant difference is observed between the IR spectra of CS and FA–CS. It can be seen that the absorption peak in 3435 cm1 becomes stronger due to the overlapping of the vibration of OH and N–H functional group. The absorption in 1592 cm1 disappears
60
Figure 5. SEM image of FA–CS nanoparticles.
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2h = 5.6°, 11.1°, 13.4°, 16.6°, 17.3°, 19.5°, 22.0°, 26.9°, 28.07°, 29.8° due to its crystal property. Although intense peaks are observed at either CS or FA, only a hump peak is observed at 2h = 20.6° for FA– CS conjugates (Fig. 4c), which indicates the partly loss of crystalline
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character for their resultant products. The coupling of FA with CS can hamper the movement of the molecular chain of CS in solid state due to the more complex structure of FA as compared to amino group. 3.5. Characterization of FA–CS nanoparticles
Figure 6. Particle size distribution of FA–CS nanoparticles.
The surface morphology and particle size distribution of FA–CS nanoparticles were investigated by SEM (Fig. 5) and laser light scattering (DLS) technology (Fig. 6). The FA–CS nanoparticles are spherical in shape with monodispersity and the average particle size is 100 nm (Fig. 5). The average particle size of FA–CS nanoparticles obtained by DLS method is 112.7 nm (Fig. 6), which is in good agreement with the measurement from SEM. The minor difference could be derived from the different sample states of the nanoparticles as SEM only visualizes the core of nanoparticles, while DLS measures the hydrodynamic radius of nanoparticles. The zeta potential of FA–CS nanoparticles is narrowly distributed with an average zeta potential of 12.5 mV (Fig. 7). In general, the average zeta potential of the CS nanoparticles is about 30– 50 mV. It is obvious that the modification of CS with FA leads to a sharp decrease in the zeta potential. This might be ascribed to the partial substitution of –NH2 with FA in the coupling process. Another possible reason might be the presence of side chain of FA, which destroys the entanglement of CS molecular chains in 1% acetic acid solution. Thus, more amino groups are exposed, which subsequently leads to more intense crosslinking between FA–CS and STPP. 3.6. Cellular uptake by HT-29
Figure 7. Zeta potential of FA–CS NPs (12.5 mV).
To investigate the cellular uptake of FA–CS nanoparticles, calcein was encapsulated into FA–CS and CS nanoparticles. The cellular uptake of ligand incorporated nanoparticles was qualitatively assayed by fluorescence microscopy in colorectal cancer cells HT-29. For the measurement of fluorescence intensity, HT-29 cells were incubated with calcein-loaded FA–CS or CS nanoparticles for 24 h with or without the presence of 1 mM free FA. The cellular uptake of FA–CS or CS nanoparticles was viewed by fluorescence microscopy. As shown in Figure 8a, strong fluorescence intensity is observed in the case of FA incorporated FA–CS nanoparticles, while very weak intensity is observed in the CS nanoparticles
Figure 8. Fluorescent microscopic images of HT-29 cells after incubation with calcein-loaded CS or FA–CS NPs for 4 h (200 magnification): (a) FA–CS NPs; (b) CS NPs; (c) FA– CS NPs + free FA; (d) CS NPs + free FA; (e and f) are their corresponding microscopic images without fluorescence.
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(Fig. 8b). The presence of free FA in the culture medium leads to the decrease of fluorescence intensity of FA–CS nanoparticles (Fig. 8c). However, a slight increase in fluorescence intensity is observed for CS nanoparticles incubated with free FA. Due to the strong binding affinity between FA and FA-receptors, FA–CS nanoparticles could bind to FA receptors overexpressed in cancerous cells, which are subsequently internalized by HT-29 cells via the receptormediated endocytosis. The presence of free FA in the culture medium leads to the combination of FA with the receptors of HT-29 cells, which hamper the binding of FA–CS nanoparticles with receptors of HT-29. The results of the cellular uptake indicate that the coupling of FA into CS could significantly improve the cellular uptake of FA–CS nanoparticles by HT-29. It is obvious from the cellular uptake results that the FA–CS nanoparticles are a promising vehicle for the targeting anticancer drug to cancer tumor cells which are overexpressed with FA receptors. 4. Conclusions The chemical modification of CS with FA leads to the successful formulation of FA–CS conjugates which combine the original properties of CS and the targeting ligand of FA. The preparation parameters were optimized with orthogonal experimental design by using coupling ratio as an index. Carboxyl group of FA was successfully conjugated with the amino group of CS as confirmed by FTIR and 1H NMR. FA–CS displayed less crystal structure when compared with CS. FA–CS nanoparticles are spherical in shape and display positive charge properties. The specific binding affinity between the FA incorporated FA–CS nanoparticles and the FAreceptor was studied in colorectal cancer cell (HT-29). It was found that the FA incorporated FA–CS nanoparticles exhibit improved cellular uptake in HT-29 cells. These novel properties of FA–CS conjugates demonstrated that it is a promising biomaterial for the colon targeted drug delivery. Acknowledgments Mr. Puwang Li gratefully acknowledges the financial support from Australian Federal Government’s International Postgraduate
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