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carboxymethyl chitosan hybrid nanoparticles with improved drug delivery efficiency
International Journal of Pharmaceutics 446 (2013) 205–210
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Peptide decorated calcium phosphate/carboxymethyl chitosan hybrid nanoparticles with improved drug delivery efficiency Jun Wang a , Bin Chen a , Dong Zhao a , Yan Peng b , Ren-Xi Zhuo a , Si-Xue Cheng a,∗ a b
Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, People’s Republic of China Department of Pharmacy, The Renmin Hospital of Wuhan University, Wuhan 430060, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 5 October 2012 Received in revised form 16 January 2013 Accepted 9 February 2013 Available online 16 February 2013 Keywords: Nanoparticles Drug delivery Calcium phosphate Peptide Self-assembly
a b s t r a c t In this study, a facile strategy to effectively improve the delivery efficiency of nano-sized drug delivery systems was developed. Calcium phosphate/carboxymethyl chitosan (Ca–P/CMC) hybrid nanoparticles were prepared by the precipitation of calcium phosphate in the aqueous solution containing CMC. The obtained Ca–P/CMC nanoparticles were characterized by SEM, XPS and TGA. Doxorubicin hydrochloride (DOX), a water-soluble anticancer drug, was loaded in the nanoparticles with high encapsulation efficiency. The in vitro drug release showed that the release of DOX from the nanoparticles could be effectively sustained. After drug loading, the nanoparticles were decorated by peptide KALA by self-assembly through the electrostatic interaction between the positively charged KALA and the negatively charged CMC chains to obtain drug loaded Ca–P/CMC/KALA nanoparticles. The size and size distribution of the nanoparticles were measured by a particle size analyzer. The KALA decorated nanoparticles exhibited a larger size and an increased zeta potential. The effect of KALA content on the HeLa cell inhibition was studied. The in vitro study showed that the cell inhibition effect could be significantly enhanced by the presence of KALA. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Compared with conventional formulations, drug delivery systems offer numerous advantages such as improved efficacy, reduced toxicity, reduced frequency of doses, and convenience. Of the different drug delivery systems reported, drug loaded nanoparticles have attracted great interest because of the convenience in administration through different routes, the possibility to achieve passive targeting when their sizes are in particular ranges (Kim et al., 2009a,b; Talelli et al., 2010; Chawla and Amiji, 2002). Among different nano-sized drug carriers, nanoparticles based on bioresorbable inorganic materials such as calcium phosphate (Ca–P) and calcium carbonate (CaCO3 ) have unique advantages, including ideal biocompatibility and biodegradability, mild and simple preparation which does not involve any organic solvent and surfactant, capability to load various therapeutics including proteins, genes, and chemotherapeutic agents with different hydrophilicity, and pH-dependent dissolution which favors intracellular drug delivery (Adair et al., 2010; Dasgupta et al., 2009; Andreeva et al., 2007; Morgan et al., 2008; Kester et al., 2008; Wei et al., 2008; Ueno et al., 2005). Despite of these well known
∗ Corresponding author. Tel.: +86 27 68754061; fax: +86 27 68754509. E-mail addresses: [email protected], [email protected] (S.-X. Cheng). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.02.028
advantages, the major limitation of these delivery systems is the difficulty in controlling the preparation conditions since the successful delivery strongly depends on the size of the delivery systems containing inorganic compounds. To avoid the growth of co-precipitates into ineffective microcrystals, the experimental parameters such as ion concentrations, pH, and precipitation time should be precisely controlled (Olton et al., 2007; Kumta et al., 2005). To the end of controlling the particle size and crystallization, improving thermodynamic stability of particles, and enhancing the delivery efficiency, various modification strategies have been developed. For example, poly(ethylene glycol)-bpoly(aspartic acid) was used to control the crystallization of Ca–P and form a hybrid vector with improved gene transfection efficiency (Kakizawa et al., 2004). Tri-block and di-block copolymers containing poly(ethylene glycol) and polylactide segments could form hybrid nanoparticles with Ca–P with various sizes (Wang et al., 2010a,b). Carboxylmethyl cellulose was utilized to form hybrid particles with CaCO3 with controllable sizes (Peng et al., 2010; Zhao et al., 2007). As far as we know, most studies carried out in this field were mainly focused on preparation of the hybrid nanoparticles with controllable size and morphology (Ethirajan and Landfester, 2010; Sugawara et al., 2006; Sokolova et al., 2006). For nano-sized drug delivery systems, cellular internalization is a critical issue because the plasma membranes are nearly impermeable barriers. To solve this issue, cell-penetrating peptides (CPPs) have been investigated for delivery of various therapeutic agents
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(Gupta et al., 2005; Fonseca et al., 2009). Among these CPPs, KALA, a cationic endosomolytic and fusogenic peptide, has been extensively investigated as a gene vector to condense pDNA (Rittner et al., 2002) and siRNA (Kim et al., 2009a,b), and an additive to be incorporated into vector/DNA complexes to achieve enhanced transfection efficiency. Cationic KALA and KALA derivatives such as PEG-KALA could be incorporated into polymer/DNA complexes, including polyethylenimine/DNA (Min et al., 2006; Lee et al., 2001), PEG-g-polylysine (Lee et al., 2002) and poly(DMAEMA-NVP)-bPEG-galactose (Lim et al., 2000), to increase the transfection efficiency. In our previous work, it was found that the addition of KALA to Ca–P/DNA co-precipitates resulted in enhanced gene delivery efficiency (Chen et al., 2010). In our study on the KALA decorated gene delivery system, KALA was added to the system by mixing the positively charged KALA and negatively charged plasmid DNA first and then the nanoparticle gene delivery system was prepared by co-precipitation. As a result, KALA was mainly encapsulated in the nanoparticles. In this study, to improve the drug delivery efficiency, we prepared by KALA decorated calcium carbonate/carboxymethyl chitosan (Ca–P/CMC) hybrid nanoparticles through the electrostatic interaction between the positively charged peptide and the Ca–P/CMC nanoparticles with negative surface charges. CMC used in the current study not only played an important role to control the particle size but also provided the negatively charged groups for the self-assembly to incorporate KALA onto the surfaces of the nanoparticles. The components of current delivery systems are exceedingly suitable for the applications in biomedical fields since Ca–P, CMC and the peptide have good biocompatibility and biodegradability property. The whole preparation procedure was carried out in aqueous solutions under mild conditions, and did not involve any organic solvent and surfactant. Doxorubicin hydrochloride (DOX), a water-soluble anticancer drug, was encapsulated in the nanoparticles. The cell inhibition evaluation showed the drug delivery efficiency could be obviously enhanced after peptide decoration on the surface of the nanoparticles. In addition, the hydrophilic out-layer of the nanoparticles containing CMC and KALA could endow the particles with good dispersion stability in water. The current study provides a new strategy for peptide decoration, i.e. the peptide is introduced to the surface of the delivery system by self-assembly conveniently. Our results demonstrate that the facile method for the peptide decoration can modify inorganic/polymer hybrid drug delivery systems easily and the peptide decorated on the particle surface can effectively improve the drug delivery efficiency. 2. Materials and methods 2.1. Materials Chitosan (Mw = 50 kDa) was purchased from Jinan Haidebei Marine Bioengineering Co., Ltd. (China). Doxorubicin hydrochloride was provided by Zhejiang Hisun Pharmaceutical Co., Ltd. (China). Monochloroacetic acid was supplied by Sinopharm Chemical Reagent Co., Ltd. (China). All other reagents were of analytical grade and used as received. 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) was from Amresco. Dimethylsulfoxide (DMSO) was from Sigma. KALA peptide (WEAKLAKALAKALAKHLAKALAKALKACEA) was from GL Biochem (Shanghai, China). HeLa cells were obtained from China Center for Typical Culture Collection (Wuhan, China) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco), supplemented with 10% fetal bovine serum (FBS), 2 mg/mL NaHCO3 , and 100 unit/mL penicillin/streptomycin. Cells were incubated at 37 ◦ C in humidified air/5% CO2 .
2.2. Synthesis of carboxymethyl chitosan Carboxymethyl chitosan (CMC) was synthesized according to a literature procedure (Liu et al., 2001). 6.8 g of sodium hydroxide was dissolved in 10 mL of water, and 40 mL of isopropanol was added into the solution. Then 5 g of chitosan was dispersed into the solution and stirred at 50 ◦ C for 1 h. After that, 7.5 g of monochloroacetic acid in 10 mL of isopropanol was added into the mixture dropwise and the reaction was carried out at 50 ◦ C for 4 h. The resulted solution was filtered, washed with 80% alcohol until the filtrate was neutral, and then dried in an oven to obtain CMC. The substitution degree of CMC was determined to be 89% by potentiometric titration (Ge and Luo, 2005). 2.3. Preparation and characterizations of Ca–P/CMC hybrid nanoparticles 0.1 g of CMC was dissolved in 10 mL of de-ionized water at room temperature, then 15 mL of Na3 PO4 (0.02 M) was added and stirred to form an uniform solution. After that, 4.5 mL of CaCl2 (0.1 M) was added into the mixtures dropwise and stirred for 10 min, and then the mixture was put in a dialysis bag and dialyzed against de-ionized water to obtain Ca–P/CMC hybrid nanoparticles. The surface morphology of the Ca–P/CMC hybrid nanoparticles was observed by a Hitachi X650 scanning electron microscope (SEM) operating at an accelerating voltage of 15 kV. Before SEM observation, the sample was sputter-coated with gold. The relative amounts of C, O, Ca, P and N on the surfaces of the Ca–P/CMC hybrid nanoparticles were measured by XPS (Kratos XSAM 800) using a magnesium anode (1253.6 eV) as the exciting source. The Ca–P/CMC hybrid nanoparticles were characterized by thermogravimetric analyis (TGA) (Netzsch STA 449 C) in the temperature range of 20–1200 ◦ C with a heating rate of 10 ◦ C/min in N2 . 2.4. Drug loading and characterizations of drug loaded Ca–P/CMC nanoparticles 1.5 mg of DOX was dissolved in 3 mL of de-ionized water. Then the drug solution was added into 12 mL of solution containing 30 mg of nanoparticles and stirred for 12 h. The mixture was then put in a dialysis bag and dialyzed for 24 h to remove the free drug and to obtain drug loaded Ca–P/CMC nanoparticles. The drug loading content and encapsulation efficiency were calculated as follows. Drug loading content = (WT − WF )/WNP × 100%, where WT is the total weight of drug fed, WF is the weight of non-encapsulated free drug, and WNP is the weight of nanoparticles. Encapsulation efficiency = (WT − WF )/WT × 100%, where WT is the total weight of drug fed, and WF is the weight of nonencapsulated free drug. To evaluate the in vitro drug release property, the drug loaded Ca–P/CMC nanoparticles (10 mg) were put in a dialysis bag and immersed in 30 mL of PBS solution (pH 7.4) in a centrifuge tube and shaken in a shaking water bath at 37 ◦ C. At predetermined intervals, 10 mL of solution outside the dialysis bag was taken out and replaced by 10 mL of fresh PBS solution. The drug concentration was determined by the absorbance at 485 nm in a UV–vis spectrophotometer (Perkin–Elmer Lambda Bio 40). The data are given as mean ± standard deviation (SD) based on the measurements of the samples from 3 batches. 2.5. Peptide decoration of drug loaded Ca–P/CMC nanoparticles 60 L of KALA solution with a particular concentration (1.5 mg/mL and 3 mg/mL, respectively) was added to 240 L of
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solution containing 900 g of drug loaded Ca–P/CMC nanoparticles under stirring and then the mixture was co-incubated for 30 min. Due to the electrostatic interaction between the positively charged KALA and DOX loaded Ca–P/CMC nanoaprticles with negatively charged surfaces, KALA was coated on the surfaces of the nanoaprticles and drug loaded Ca–P/CMC/KALA nanoparticles were obtained. The solution containing nanoparticles was centrifuged. After centrifugation, the amount of free KALA remaining in the supernatant of solution was determined. The encapsulation efficiency of KALA was calculated as follows. Encapsulation efficiency = (WT − WF )/WT × 100%, where WT is the total weight of KALA fed, and WF is the weight of free KALA. 2.6. Size and zeta potential measurements The size and zeta potential of nanoparticles were measured using a Nano-ZS ZEN3600 particle sizer (Malvern Instruments). The data are given as mean ± standard deviation (SD) based on 3 independent measurements. 2.7. In vitro cell inhibition evaluation The cytotoxicities of different nanoparticles and free drug were examined by MTT assay. The HeLa cells were seeded in the 24-well plates at a density of 5 × 104 cells/well in 1 mL of DMEM containing 10% FBS. After incubation for 24 h, the medium was replaced by 1 mL of fresh DMEM containing a particular amount of nanoparticles or free drug. After co-incubation at 37 ◦ C for 48 h, 800 L fresh DMEM and 80 L of MTT (5 mg/mL) solution was added to each well and further incubated for 4 h. Thereafter, the medium was removed carefully and 800 L DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 570 nm by a microplate reader (Bio-rad 550). The cells without any treatment were used as the control. The data are given as mean ± standard deviation (SD) based on 3 measurements. 3. Results and discussion 3.1. Preparation and characterizations of Ca–P/CMC hybird nanoparticles According to previous studies, polysaccharides could act as an efficient stabilizer and prevent the precipitation of inorganic compounds (Peng et al., 2010; Zhao et al., 2007; Wang et al., 2010a,b). In this study, calcium phosphate/carboxymethyl chitosan (Ca–P/CMC) hybrid nanoparticles were prepared coprecipitation of Ca–P in the presence of CMC. Due to the existence of CMC, the precipitation of Ca–P could be effectively retarded and the size of Ca–P particles could be well controlled. During the preparation, CMC was co-dissolved with Na3 PO4 , and then CaCl2 solution was added. As we know, CMC has two types of protonable groups. The COOH groups have a protonation constant (pKa) around 2.2–3.2 and NH3 groups have a pKa at 6.2–7.8 depending on the deacetylation degree, substitution degree and molecular weight of CMC (Wang et al., 2008). Two-step dissociation of the groups occurs with an increase in pH, i.e. deprotonation of COOH groups and then deprotonation of NH3 groups. In the basic and neutral solutions, the CMC chains exist in the form of stretching conformation due to the repulsion between the deprotonated carboxyl groups, and CMC chains have the ability to bind Ca2+ cations. In the current study, with the addition of CaCl2 solution into the CMC solution, Ca2+ ions interact with CMC chains. The presence of bound Ca2+ ions reduces the electrostatic repulsion between COO− groups in the CMC chains. So the CMC chains in the Ca2+ -rich domains become more condense, while the chains
Fig. 1. SEM image of Ca–P/CMC nanoparticles.
in the Ca2+ deficient domains have the stronger electrostatic repulsion between the COO− groups and thus have a higher affinity with water molecules. As a result, Ca–P/CMC hybrid particles with high CMC content in the surface layer are formed. The negatively charged CMC chains on the particle surface not only ensure the good dispersibility and high colloidal stability of the nanoparticles in water but also facilitate the further decoration by positively charged peptide KALA. As shown in Fig. 1, SEM image shows that the Ca–P/CMC hybrid nanoparticles exhibit a regular spherical shape with a mean size about 50 nm. The particle size of nanoparticles in aqueous solutions measured by a particle size analyzer (Fig. 2) exhibits a unimodal size distribution with a relatively narrow distribution. Compared with the size of water-soaked nanoparticles measured by the particle size analyzer, the size of dried nanoparticles from SEM observation is smaller. This is due to that fact that the CMC-rich outer layers of the nanoparticles are highly hydrolyzed in water, leading to a larger size measured by the particle size analyzer based on laser light scattering technique. In addition, from the SEM photograph, there exists some extent of nanoparticles aggregation, which may also lead to the larger size determined by laser light scattering technique.
Fig. 2. Size distribution of Ca–P/CMC nanoparticles.
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Fig. 3. XPS spectrum of Ca–P/CMC nanoparticles with relative atomic concentrations of C: 39.2%, O: 47.4%, Ca: 7.3%, P: 4.9% and N: 1.2%.
The Ca–P/CMC hybrid nanoparticles were characterized by XPS and the relative atomic concentrations of different elements on the surface layers of the particles were determined (Fig. 3). In the XPS measurement, the Ca concentration is an overall contribution from Ca–P and the Ca2+ ions coordinated with CMC, the C and N concentrations are the only contribution from CMC, and the P concentration is the only contribution from Ca–P. The overall CMC content in the Ca–P/CMC hybrid nanoparticles could be determined by TGA since the weight loss at the temperature below 800 ◦ C in TGA characterization is caused by the decomposition of CMC. According to TGA (Fig. 4), the overall CMC content in the hybrid nanoparticles is about 32 wt.%. 3.2. Properties of drug loaded Ca–P/CMC hybrid nanoparticles In our study, doxorubicin hydrochloride (DOX) was chosen as a model drug. According to previous studies, the nanopores in the inorganic/polymer hybrid particles provide a strong capability to load drugs regardless of their surface charge and hydrophilicity (Peng et al., 2010; Wang et al., 2010a,b). In the current study, the negatively charged CMC in the hybrid nanosperes could provide
Fig. 5. In vitro drug release from drug loaded Ca–P/CMC nanoparticles.
additional attractive forces for the positively charged drug. The drug could be readily loaded in the Ca–P/CMC hybrid nanoparticles with encapsulation efficiency of 70.2% and drug loading content of 3.3 wt.%. The in vitro drug release profile of the Ca–P/CMC hybrid nanoparticles in Fig. 5 clearly shows the Ca–P/CMC particles could effectively sustain the drug release. As we know, polysaccharides are highly hydrolyzed in water. Since the drugs with low molecular weights encapsulated in the polysaccharides can be diffused out quickly and easily, the polysaccharide based drug delivery systems most commonly have low encapsulation efficiency and a fast drug release rate for water soluble drugs with low molecular weights. In our hybrid system, the content of inorganic compound Ca–P is relatively high (higher than 60 wt.%). As a result, the encapsulation efficiency of the current system is much higher than the previously reported systems with polysaccharide as the main component (Yu et al., 2008). 3.3. Peptide decoration of drug loaded Ca–P/CMC hybrid nanoparticles In the current study, KALA, a cationic endosomolytic and fusogenic peptide, was used to decorate the drug loaded Ca–P/CMC hybrid nanoparticles by self-assembly through the electrostatic interaction between the natively charged drug loaded Ca–P/CMC nanoparticles with the positively charged KALA. The KALA encapsulation efficiencies for drug loaded Ca–P/CMC/KALA 1 and drug loaded Ca–P/CMC/KALA 2 nanoparticles were 87.5% and 90.0%, respectively, indicating this facile method for peptide decoration could effectively introduce the peptide to the delivery system by self-assembly. As listed in Table 1, the zeta potential of drug loaded Ca–P/CMC nanoparticles is −23.7 mV. After self-assembly with KALA with a positive charge, the resultant drug loaded Ca–P/CMC/KALA nanoparticles exhibit an increased zeta potential Table 1 Size and zeta potential of different nanoparticles.
Fig. 4. TGA curve of Ca–P/CMC nanoparticles.
Sample
Drug loaded Ca–P/CMC:KALA feed ratio (wt:wt)
Size (nm)
Zeta potential (mV)
Ca–P/CMC Drug loaded Ca–P/CMC Drug loaded Ca–P/CMC/KALA 1 Drug loaded Ca–P/CMC/KALA 2
– – 10:1 5:1
208.3 234.2 237.7 278.1
−26.1 −23.7 −19.5 −16.9
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nanoparticles could enter the cell easily and thus have a higher inhibition activity. For the cells treated with free DOX or DOX loaded nanoparticles with an equivalent DOX concentration, the DOX loaded nanoparticles exhibit a weaker cell inhibition effect as compared with free DOX. Those results could be attributed to the different cellular uptake mechanisms of free DOX and DOX loaded nanoparticles. The free DOX transports into cells via a passive diffusion mechanism and thus can suppress the cancer cells immediately, while nanosized drug loaded particles enter the cells by endocytosis-mediated cellular uptake, and the release of DOX from the nanoparticles is a prerequisite requirement for inducing cell apoptosis. 4. Conclusions Ca–P/CMC/KALA nanoparticles were prepared through selfassembly via electrostatic interaction between positively charged KALA and negatively charged Ca–P/CMC nanoparticles in an aqueous solution. The in vitro study shows that the cell inhibition effect of DOX loaded nanoparticles could be significantly enhanced by the presence of KALA and the cell inhibition increases with increasing KALA content due to the enhanced cell uptake, indicating the Ca–P/CMC/KALA nanoparticles are suitable for the delivery of antitumor drugs. This self-assembly method provides a new strategy for modifying inorganic/polymer hybrid drug delivery systems easily and effectively. Acknowledgments Financial supports from National Natural Science Foundation of China (21074099), Ministry of Science and Technology of China (National Basic Research Program of China 2009CB930300), and Ministry of Education of China (Program for Changjiang Scholars and Innovative Research Team in University IRT1030) are gratefully acknowledged. References
Fig. 6. Cell viability after being treated by different agents: (A) blank nanoparticles, and (B) drug loaded nanoparticles and free drug.
with a larger size. As expected, the zeta potential and the particle size of the nanoparticles increase with increasing KALA concentration for self-assembly, i.e. the Ca–P/CMC/KALA 2 nanoparticles with a higher KALA content exhibit a higher zeta potential and a larger size. To evaluate the cell inhibition effects of different nanaospheres, we determined the in vitro cytotoxicity by MTT assay. As shown in Fig. 6A, all blank nanaoparticles do not exhibit apparent cytotoxicity. The cytotoxicity values of drug loaded nanaoparticles are shown in Fig. 6B. The presence of KALA in the nanosphere surface results in a significant improvement in the cell inhibition. Compared with drug loaded Ca–P/CMC/KALA 1 nanoparticles, drug loaded Ca–P/CMC/KALA 2 nanoparticles exhibit a much stronger inhibition effect due to the higher KALA content. According to previous research, KALA could interact with and destabilize lipid membranes and facilitate the cellular entry by its ability to form an amphipathic ␣-helical structure and thus to induce enhanced cell uptake (Min et al., 2006; Lee et al., 2001; Lim et al., 2000). In our study, the KALA decorated drug loaded
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Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Peptide decorated calcium phosphate/carboxymethyl chitosan hybrid nanoparticles with improved drug delivery efficiency Jun Wang a , Bin Chen a , Dong Zhao a , Yan Peng b , Ren-Xi Zhuo a , Si-Xue Cheng a,∗ a b
Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, People’s Republic of China Department of Pharmacy, The Renmin Hospital of Wuhan University, Wuhan 430060, People’s Republic of China
a r t i c l e
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Article history: Received 5 October 2012 Received in revised form 16 January 2013 Accepted 9 February 2013 Available online 16 February 2013 Keywords: Nanoparticles Drug delivery Calcium phosphate Peptide Self-assembly
a b s t r a c t In this study, a facile strategy to effectively improve the delivery efficiency of nano-sized drug delivery systems was developed. Calcium phosphate/carboxymethyl chitosan (Ca–P/CMC) hybrid nanoparticles were prepared by the precipitation of calcium phosphate in the aqueous solution containing CMC. The obtained Ca–P/CMC nanoparticles were characterized by SEM, XPS and TGA. Doxorubicin hydrochloride (DOX), a water-soluble anticancer drug, was loaded in the nanoparticles with high encapsulation efficiency. The in vitro drug release showed that the release of DOX from the nanoparticles could be effectively sustained. After drug loading, the nanoparticles were decorated by peptide KALA by self-assembly through the electrostatic interaction between the positively charged KALA and the negatively charged CMC chains to obtain drug loaded Ca–P/CMC/KALA nanoparticles. The size and size distribution of the nanoparticles were measured by a particle size analyzer. The KALA decorated nanoparticles exhibited a larger size and an increased zeta potential. The effect of KALA content on the HeLa cell inhibition was studied. The in vitro study showed that the cell inhibition effect could be significantly enhanced by the presence of KALA. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Compared with conventional formulations, drug delivery systems offer numerous advantages such as improved efficacy, reduced toxicity, reduced frequency of doses, and convenience. Of the different drug delivery systems reported, drug loaded nanoparticles have attracted great interest because of the convenience in administration through different routes, the possibility to achieve passive targeting when their sizes are in particular ranges (Kim et al., 2009a,b; Talelli et al., 2010; Chawla and Amiji, 2002). Among different nano-sized drug carriers, nanoparticles based on bioresorbable inorganic materials such as calcium phosphate (Ca–P) and calcium carbonate (CaCO3 ) have unique advantages, including ideal biocompatibility and biodegradability, mild and simple preparation which does not involve any organic solvent and surfactant, capability to load various therapeutics including proteins, genes, and chemotherapeutic agents with different hydrophilicity, and pH-dependent dissolution which favors intracellular drug delivery (Adair et al., 2010; Dasgupta et al., 2009; Andreeva et al., 2007; Morgan et al., 2008; Kester et al., 2008; Wei et al., 2008; Ueno et al., 2005). Despite of these well known
∗ Corresponding author. Tel.: +86 27 68754061; fax: +86 27 68754509. E-mail addresses: [email protected], [email protected] (S.-X. Cheng). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.02.028
advantages, the major limitation of these delivery systems is the difficulty in controlling the preparation conditions since the successful delivery strongly depends on the size of the delivery systems containing inorganic compounds. To avoid the growth of co-precipitates into ineffective microcrystals, the experimental parameters such as ion concentrations, pH, and precipitation time should be precisely controlled (Olton et al., 2007; Kumta et al., 2005). To the end of controlling the particle size and crystallization, improving thermodynamic stability of particles, and enhancing the delivery efficiency, various modification strategies have been developed. For example, poly(ethylene glycol)-bpoly(aspartic acid) was used to control the crystallization of Ca–P and form a hybrid vector with improved gene transfection efficiency (Kakizawa et al., 2004). Tri-block and di-block copolymers containing poly(ethylene glycol) and polylactide segments could form hybrid nanoparticles with Ca–P with various sizes (Wang et al., 2010a,b). Carboxylmethyl cellulose was utilized to form hybrid particles with CaCO3 with controllable sizes (Peng et al., 2010; Zhao et al., 2007). As far as we know, most studies carried out in this field were mainly focused on preparation of the hybrid nanoparticles with controllable size and morphology (Ethirajan and Landfester, 2010; Sugawara et al., 2006; Sokolova et al., 2006). For nano-sized drug delivery systems, cellular internalization is a critical issue because the plasma membranes are nearly impermeable barriers. To solve this issue, cell-penetrating peptides (CPPs) have been investigated for delivery of various therapeutic agents
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(Gupta et al., 2005; Fonseca et al., 2009). Among these CPPs, KALA, a cationic endosomolytic and fusogenic peptide, has been extensively investigated as a gene vector to condense pDNA (Rittner et al., 2002) and siRNA (Kim et al., 2009a,b), and an additive to be incorporated into vector/DNA complexes to achieve enhanced transfection efficiency. Cationic KALA and KALA derivatives such as PEG-KALA could be incorporated into polymer/DNA complexes, including polyethylenimine/DNA (Min et al., 2006; Lee et al., 2001), PEG-g-polylysine (Lee et al., 2002) and poly(DMAEMA-NVP)-bPEG-galactose (Lim et al., 2000), to increase the transfection efficiency. In our previous work, it was found that the addition of KALA to Ca–P/DNA co-precipitates resulted in enhanced gene delivery efficiency (Chen et al., 2010). In our study on the KALA decorated gene delivery system, KALA was added to the system by mixing the positively charged KALA and negatively charged plasmid DNA first and then the nanoparticle gene delivery system was prepared by co-precipitation. As a result, KALA was mainly encapsulated in the nanoparticles. In this study, to improve the drug delivery efficiency, we prepared by KALA decorated calcium carbonate/carboxymethyl chitosan (Ca–P/CMC) hybrid nanoparticles through the electrostatic interaction between the positively charged peptide and the Ca–P/CMC nanoparticles with negative surface charges. CMC used in the current study not only played an important role to control the particle size but also provided the negatively charged groups for the self-assembly to incorporate KALA onto the surfaces of the nanoparticles. The components of current delivery systems are exceedingly suitable for the applications in biomedical fields since Ca–P, CMC and the peptide have good biocompatibility and biodegradability property. The whole preparation procedure was carried out in aqueous solutions under mild conditions, and did not involve any organic solvent and surfactant. Doxorubicin hydrochloride (DOX), a water-soluble anticancer drug, was encapsulated in the nanoparticles. The cell inhibition evaluation showed the drug delivery efficiency could be obviously enhanced after peptide decoration on the surface of the nanoparticles. In addition, the hydrophilic out-layer of the nanoparticles containing CMC and KALA could endow the particles with good dispersion stability in water. The current study provides a new strategy for peptide decoration, i.e. the peptide is introduced to the surface of the delivery system by self-assembly conveniently. Our results demonstrate that the facile method for the peptide decoration can modify inorganic/polymer hybrid drug delivery systems easily and the peptide decorated on the particle surface can effectively improve the drug delivery efficiency. 2. Materials and methods 2.1. Materials Chitosan (Mw = 50 kDa) was purchased from Jinan Haidebei Marine Bioengineering Co., Ltd. (China). Doxorubicin hydrochloride was provided by Zhejiang Hisun Pharmaceutical Co., Ltd. (China). Monochloroacetic acid was supplied by Sinopharm Chemical Reagent Co., Ltd. (China). All other reagents were of analytical grade and used as received. 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) was from Amresco. Dimethylsulfoxide (DMSO) was from Sigma. KALA peptide (WEAKLAKALAKALAKHLAKALAKALKACEA) was from GL Biochem (Shanghai, China). HeLa cells were obtained from China Center for Typical Culture Collection (Wuhan, China) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco), supplemented with 10% fetal bovine serum (FBS), 2 mg/mL NaHCO3 , and 100 unit/mL penicillin/streptomycin. Cells were incubated at 37 ◦ C in humidified air/5% CO2 .
2.2. Synthesis of carboxymethyl chitosan Carboxymethyl chitosan (CMC) was synthesized according to a literature procedure (Liu et al., 2001). 6.8 g of sodium hydroxide was dissolved in 10 mL of water, and 40 mL of isopropanol was added into the solution. Then 5 g of chitosan was dispersed into the solution and stirred at 50 ◦ C for 1 h. After that, 7.5 g of monochloroacetic acid in 10 mL of isopropanol was added into the mixture dropwise and the reaction was carried out at 50 ◦ C for 4 h. The resulted solution was filtered, washed with 80% alcohol until the filtrate was neutral, and then dried in an oven to obtain CMC. The substitution degree of CMC was determined to be 89% by potentiometric titration (Ge and Luo, 2005). 2.3. Preparation and characterizations of Ca–P/CMC hybrid nanoparticles 0.1 g of CMC was dissolved in 10 mL of de-ionized water at room temperature, then 15 mL of Na3 PO4 (0.02 M) was added and stirred to form an uniform solution. After that, 4.5 mL of CaCl2 (0.1 M) was added into the mixtures dropwise and stirred for 10 min, and then the mixture was put in a dialysis bag and dialyzed against de-ionized water to obtain Ca–P/CMC hybrid nanoparticles. The surface morphology of the Ca–P/CMC hybrid nanoparticles was observed by a Hitachi X650 scanning electron microscope (SEM) operating at an accelerating voltage of 15 kV. Before SEM observation, the sample was sputter-coated with gold. The relative amounts of C, O, Ca, P and N on the surfaces of the Ca–P/CMC hybrid nanoparticles were measured by XPS (Kratos XSAM 800) using a magnesium anode (1253.6 eV) as the exciting source. The Ca–P/CMC hybrid nanoparticles were characterized by thermogravimetric analyis (TGA) (Netzsch STA 449 C) in the temperature range of 20–1200 ◦ C with a heating rate of 10 ◦ C/min in N2 . 2.4. Drug loading and characterizations of drug loaded Ca–P/CMC nanoparticles 1.5 mg of DOX was dissolved in 3 mL of de-ionized water. Then the drug solution was added into 12 mL of solution containing 30 mg of nanoparticles and stirred for 12 h. The mixture was then put in a dialysis bag and dialyzed for 24 h to remove the free drug and to obtain drug loaded Ca–P/CMC nanoparticles. The drug loading content and encapsulation efficiency were calculated as follows. Drug loading content = (WT − WF )/WNP × 100%, where WT is the total weight of drug fed, WF is the weight of non-encapsulated free drug, and WNP is the weight of nanoparticles. Encapsulation efficiency = (WT − WF )/WT × 100%, where WT is the total weight of drug fed, and WF is the weight of nonencapsulated free drug. To evaluate the in vitro drug release property, the drug loaded Ca–P/CMC nanoparticles (10 mg) were put in a dialysis bag and immersed in 30 mL of PBS solution (pH 7.4) in a centrifuge tube and shaken in a shaking water bath at 37 ◦ C. At predetermined intervals, 10 mL of solution outside the dialysis bag was taken out and replaced by 10 mL of fresh PBS solution. The drug concentration was determined by the absorbance at 485 nm in a UV–vis spectrophotometer (Perkin–Elmer Lambda Bio 40). The data are given as mean ± standard deviation (SD) based on the measurements of the samples from 3 batches. 2.5. Peptide decoration of drug loaded Ca–P/CMC nanoparticles 60 L of KALA solution with a particular concentration (1.5 mg/mL and 3 mg/mL, respectively) was added to 240 L of
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solution containing 900 g of drug loaded Ca–P/CMC nanoparticles under stirring and then the mixture was co-incubated for 30 min. Due to the electrostatic interaction between the positively charged KALA and DOX loaded Ca–P/CMC nanoaprticles with negatively charged surfaces, KALA was coated on the surfaces of the nanoaprticles and drug loaded Ca–P/CMC/KALA nanoparticles were obtained. The solution containing nanoparticles was centrifuged. After centrifugation, the amount of free KALA remaining in the supernatant of solution was determined. The encapsulation efficiency of KALA was calculated as follows. Encapsulation efficiency = (WT − WF )/WT × 100%, where WT is the total weight of KALA fed, and WF is the weight of free KALA. 2.6. Size and zeta potential measurements The size and zeta potential of nanoparticles were measured using a Nano-ZS ZEN3600 particle sizer (Malvern Instruments). The data are given as mean ± standard deviation (SD) based on 3 independent measurements. 2.7. In vitro cell inhibition evaluation The cytotoxicities of different nanoparticles and free drug were examined by MTT assay. The HeLa cells were seeded in the 24-well plates at a density of 5 × 104 cells/well in 1 mL of DMEM containing 10% FBS. After incubation for 24 h, the medium was replaced by 1 mL of fresh DMEM containing a particular amount of nanoparticles or free drug. After co-incubation at 37 ◦ C for 48 h, 800 L fresh DMEM and 80 L of MTT (5 mg/mL) solution was added to each well and further incubated for 4 h. Thereafter, the medium was removed carefully and 800 L DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 570 nm by a microplate reader (Bio-rad 550). The cells without any treatment were used as the control. The data are given as mean ± standard deviation (SD) based on 3 measurements. 3. Results and discussion 3.1. Preparation and characterizations of Ca–P/CMC hybird nanoparticles According to previous studies, polysaccharides could act as an efficient stabilizer and prevent the precipitation of inorganic compounds (Peng et al., 2010; Zhao et al., 2007; Wang et al., 2010a,b). In this study, calcium phosphate/carboxymethyl chitosan (Ca–P/CMC) hybrid nanoparticles were prepared coprecipitation of Ca–P in the presence of CMC. Due to the existence of CMC, the precipitation of Ca–P could be effectively retarded and the size of Ca–P particles could be well controlled. During the preparation, CMC was co-dissolved with Na3 PO4 , and then CaCl2 solution was added. As we know, CMC has two types of protonable groups. The COOH groups have a protonation constant (pKa) around 2.2–3.2 and NH3 groups have a pKa at 6.2–7.8 depending on the deacetylation degree, substitution degree and molecular weight of CMC (Wang et al., 2008). Two-step dissociation of the groups occurs with an increase in pH, i.e. deprotonation of COOH groups and then deprotonation of NH3 groups. In the basic and neutral solutions, the CMC chains exist in the form of stretching conformation due to the repulsion between the deprotonated carboxyl groups, and CMC chains have the ability to bind Ca2+ cations. In the current study, with the addition of CaCl2 solution into the CMC solution, Ca2+ ions interact with CMC chains. The presence of bound Ca2+ ions reduces the electrostatic repulsion between COO− groups in the CMC chains. So the CMC chains in the Ca2+ -rich domains become more condense, while the chains
Fig. 1. SEM image of Ca–P/CMC nanoparticles.
in the Ca2+ deficient domains have the stronger electrostatic repulsion between the COO− groups and thus have a higher affinity with water molecules. As a result, Ca–P/CMC hybrid particles with high CMC content in the surface layer are formed. The negatively charged CMC chains on the particle surface not only ensure the good dispersibility and high colloidal stability of the nanoparticles in water but also facilitate the further decoration by positively charged peptide KALA. As shown in Fig. 1, SEM image shows that the Ca–P/CMC hybrid nanoparticles exhibit a regular spherical shape with a mean size about 50 nm. The particle size of nanoparticles in aqueous solutions measured by a particle size analyzer (Fig. 2) exhibits a unimodal size distribution with a relatively narrow distribution. Compared with the size of water-soaked nanoparticles measured by the particle size analyzer, the size of dried nanoparticles from SEM observation is smaller. This is due to that fact that the CMC-rich outer layers of the nanoparticles are highly hydrolyzed in water, leading to a larger size measured by the particle size analyzer based on laser light scattering technique. In addition, from the SEM photograph, there exists some extent of nanoparticles aggregation, which may also lead to the larger size determined by laser light scattering technique.
Fig. 2. Size distribution of Ca–P/CMC nanoparticles.
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Fig. 3. XPS spectrum of Ca–P/CMC nanoparticles with relative atomic concentrations of C: 39.2%, O: 47.4%, Ca: 7.3%, P: 4.9% and N: 1.2%.
The Ca–P/CMC hybrid nanoparticles were characterized by XPS and the relative atomic concentrations of different elements on the surface layers of the particles were determined (Fig. 3). In the XPS measurement, the Ca concentration is an overall contribution from Ca–P and the Ca2+ ions coordinated with CMC, the C and N concentrations are the only contribution from CMC, and the P concentration is the only contribution from Ca–P. The overall CMC content in the Ca–P/CMC hybrid nanoparticles could be determined by TGA since the weight loss at the temperature below 800 ◦ C in TGA characterization is caused by the decomposition of CMC. According to TGA (Fig. 4), the overall CMC content in the hybrid nanoparticles is about 32 wt.%. 3.2. Properties of drug loaded Ca–P/CMC hybrid nanoparticles In our study, doxorubicin hydrochloride (DOX) was chosen as a model drug. According to previous studies, the nanopores in the inorganic/polymer hybrid particles provide a strong capability to load drugs regardless of their surface charge and hydrophilicity (Peng et al., 2010; Wang et al., 2010a,b). In the current study, the negatively charged CMC in the hybrid nanosperes could provide
Fig. 5. In vitro drug release from drug loaded Ca–P/CMC nanoparticles.
additional attractive forces for the positively charged drug. The drug could be readily loaded in the Ca–P/CMC hybrid nanoparticles with encapsulation efficiency of 70.2% and drug loading content of 3.3 wt.%. The in vitro drug release profile of the Ca–P/CMC hybrid nanoparticles in Fig. 5 clearly shows the Ca–P/CMC particles could effectively sustain the drug release. As we know, polysaccharides are highly hydrolyzed in water. Since the drugs with low molecular weights encapsulated in the polysaccharides can be diffused out quickly and easily, the polysaccharide based drug delivery systems most commonly have low encapsulation efficiency and a fast drug release rate for water soluble drugs with low molecular weights. In our hybrid system, the content of inorganic compound Ca–P is relatively high (higher than 60 wt.%). As a result, the encapsulation efficiency of the current system is much higher than the previously reported systems with polysaccharide as the main component (Yu et al., 2008). 3.3. Peptide decoration of drug loaded Ca–P/CMC hybrid nanoparticles In the current study, KALA, a cationic endosomolytic and fusogenic peptide, was used to decorate the drug loaded Ca–P/CMC hybrid nanoparticles by self-assembly through the electrostatic interaction between the natively charged drug loaded Ca–P/CMC nanoparticles with the positively charged KALA. The KALA encapsulation efficiencies for drug loaded Ca–P/CMC/KALA 1 and drug loaded Ca–P/CMC/KALA 2 nanoparticles were 87.5% and 90.0%, respectively, indicating this facile method for peptide decoration could effectively introduce the peptide to the delivery system by self-assembly. As listed in Table 1, the zeta potential of drug loaded Ca–P/CMC nanoparticles is −23.7 mV. After self-assembly with KALA with a positive charge, the resultant drug loaded Ca–P/CMC/KALA nanoparticles exhibit an increased zeta potential Table 1 Size and zeta potential of different nanoparticles.
Fig. 4. TGA curve of Ca–P/CMC nanoparticles.
Sample
Drug loaded Ca–P/CMC:KALA feed ratio (wt:wt)
Size (nm)
Zeta potential (mV)
Ca–P/CMC Drug loaded Ca–P/CMC Drug loaded Ca–P/CMC/KALA 1 Drug loaded Ca–P/CMC/KALA 2
– – 10:1 5:1
208.3 234.2 237.7 278.1
−26.1 −23.7 −19.5 −16.9
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nanoparticles could enter the cell easily and thus have a higher inhibition activity. For the cells treated with free DOX or DOX loaded nanoparticles with an equivalent DOX concentration, the DOX loaded nanoparticles exhibit a weaker cell inhibition effect as compared with free DOX. Those results could be attributed to the different cellular uptake mechanisms of free DOX and DOX loaded nanoparticles. The free DOX transports into cells via a passive diffusion mechanism and thus can suppress the cancer cells immediately, while nanosized drug loaded particles enter the cells by endocytosis-mediated cellular uptake, and the release of DOX from the nanoparticles is a prerequisite requirement for inducing cell apoptosis. 4. Conclusions Ca–P/CMC/KALA nanoparticles were prepared through selfassembly via electrostatic interaction between positively charged KALA and negatively charged Ca–P/CMC nanoparticles in an aqueous solution. The in vitro study shows that the cell inhibition effect of DOX loaded nanoparticles could be significantly enhanced by the presence of KALA and the cell inhibition increases with increasing KALA content due to the enhanced cell uptake, indicating the Ca–P/CMC/KALA nanoparticles are suitable for the delivery of antitumor drugs. This self-assembly method provides a new strategy for modifying inorganic/polymer hybrid drug delivery systems easily and effectively. Acknowledgments Financial supports from National Natural Science Foundation of China (21074099), Ministry of Science and Technology of China (National Basic Research Program of China 2009CB930300), and Ministry of Education of China (Program for Changjiang Scholars and Innovative Research Team in University IRT1030) are gratefully acknowledged. References
Fig. 6. Cell viability after being treated by different agents: (A) blank nanoparticles, and (B) drug loaded nanoparticles and free drug.
with a larger size. As expected, the zeta potential and the particle size of the nanoparticles increase with increasing KALA concentration for self-assembly, i.e. the Ca–P/CMC/KALA 2 nanoparticles with a higher KALA content exhibit a higher zeta potential and a larger size. To evaluate the cell inhibition effects of different nanaospheres, we determined the in vitro cytotoxicity by MTT assay. As shown in Fig. 6A, all blank nanaoparticles do not exhibit apparent cytotoxicity. The cytotoxicity values of drug loaded nanaoparticles are shown in Fig. 6B. The presence of KALA in the nanosphere surface results in a significant improvement in the cell inhibition. Compared with drug loaded Ca–P/CMC/KALA 1 nanoparticles, drug loaded Ca–P/CMC/KALA 2 nanoparticles exhibit a much stronger inhibition effect due to the higher KALA content. According to previous research, KALA could interact with and destabilize lipid membranes and facilitate the cellular entry by its ability to form an amphipathic ␣-helical structure and thus to induce enhanced cell uptake (Min et al., 2006; Lee et al., 2001; Lim et al., 2000). In our study, the KALA decorated drug loaded
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