International Journal of Pharmaceutics 474 (2014) 202–211
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Carboxymethyl-b-cyclodextrin conjugated nanoparticles facilitate therapy for folate receptor-positive tumor with the mediation of folic acid Chang Su a,1, Hongdan Li b,1, Yijie Shi c , Guan Wang b , Liwei Liu c, Liang Zhao c, *, Rongjian Su b, ** a b c
School of Veterinary Medicine, Liaoning Medical University, Jinzhou 121000, PR China Central Laboratory of Liaoning Medical University, Jinzhou 121000, PR China School of Pharmacy, Liaoning Medical University, Jinzhou 121000, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 June 2014 Received in revised form 7 August 2014 Accepted 15 August 2014 Available online 19 August 2014
Currently, clinical operation treatments, chemotherapy and radiotherapy just could eliminate local tumor cells. However, chemotherapy and radiotherapy also injury normal cells and lead to serious side effects and toxicities. So, it is necessary to find an effective target cancer carrier that delivers the anticancer agents into tumor cells and reduces normal cells’ injury. Folic acid (FA) is a classical targeting agent mediates internalization of chemical drugs into tumor cells which over-express folate receptor (FR) on their surface. We herein report that based on host–guest interaction, NPs decorated by novel folate enhance antitumor drug delivery. BSA-NPs were prepared by desolvation method and carboxymethyl-b-cyclodextrin (CM-b-CD) was conjugated to the surface of NPs by carbodiimide coupling to hold FA. From in vitro cytotoxicity assay, cell apoptosis study, intracellular ATP level assay and western blot, we can see that FA-CM-b-CD-BSA NPs as good monodispersity, negative charge, and homogenous particle size have a high encapsulation efficiency. The results showed that MTT and cell apoptosis demonstrated that FA-decorated NPs exhibit stronger inhibition rate and induce obvious apoptosis in FR positive Hela cells as compared to free drug and FA undecorated NPs. Moreover, 5-fluorouracil (5-Fu) loaded FA-CM-b-CD-BSA NPs down-regulate ATP levels and increase the expression of caspase-3. Taken together, FA-CM-b-CD-BSA NPs enhance FR receptor-mediated endocytosis and lead to more intracellular uptake of drug, inducing the higher apoptosis ratio of cells than free 5-Fu. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Folic acid Nanoparticles Carboxymethyl-b-cyclodextrin 5-Fu Apoptosis
1. Introduction Although cancer therapy has made some great progress nowadays, cancer is still a serious threat to human health. The main drawback of anti-cancer formulations is that besides inhibiting the growth of tumor cells, they can also significantly inhibit the proliferation of normal cells. Recently, the application of nanoparticles has become a very important research field, attracting more and more researchers. Nanopaticle is characterized by a small particle covering a range from 1 to 500 nm in size usually behaving
* Corresponding author. Tel.: +86 416 4673439; fax: +86 416 4673439. ** Corresponding author. E-mail addresses: [email protected] (L. Zhao), [email protected] (R. Su). 1 These two authors contributed equally to the manuscript. http://dx.doi.org/10.1016/j.ijpharm.2014.08.026 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
good transport and special biological properties. Compared with traditional carriers, nanoparticles have following advantages: (1) due to the small size of particles, NPs are suitable for administration through the blood vessels and they are also known to be able to even penetrate the blood–brain barrier into the brain tissue (Lin et al., 2012; Yim et al., 2012); (2) NPs are capable of encapsulating more drug molecules compared with traditional drug delivery systems and, they can also further control the drug release (Singh and Lillard, 2009; Aryal et al., 2013; Mattheolabakis et al., 2009; Hyung Park et al., 2006); (3) drug loaded NPs can be selectively retained in the lesion site by active or passive targeting known as EPR effects, thus reducing the total dose of drugs and lowering the toxicity (Huang et al., 2013; Verderio et al., 2013; Wu et al., 2014); (4) NPs can improve drug stability in vivo by protecting nucleic acids, peptide drugs from enzyme’s degradation. In order to improve the targeting ability of NPs on organs and tissues, it is necessary to conjugate specific targeting molecules at the surface of nanoparticles to achieve active
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targeting therapy by binding with specific cell surface receptors (Xu et al., 2005; Cho et al., 2001; Gref et al., 2003; Li et al., 2014) Folic acid plays important roles on cell division, proliferation, and synthesis of some biological macromolecules (Weinstein et al., 2003; Leamon, 2008). In proliferation process of tumor cells, a large amount of folic acids are required. Particularly, folate receptors (FRs) were highly expressed in tumor cells like uterus, breast, brain, lung, kidney cancer cells, while low expression of FRs was found in normal tissues (Sudimack and Lee, 2000; Weitman et al., 1992). As the folate receptors most expressed in cancer cells are a – FRs, free folic acid has a strong affinity for a – FR and it was reported to be covalently bound to macromolecules to facilitate the internalization of the macromolecules into cancer cells (Lu et al., 2004; Pinhassi et al., 2010). Therefore, nanoparticles can be coupled to folic acid to achieve the active targeting delivery of anti-cancer drugs (Hou et al., 2011; Zhang et al., 2012). In order to prepare FA decorated nanoparticles, traditionally, organic reagents and toxic coupling reagents were commonly used, leading to toxic reagents residue and cellular toxic effects (Yang et al., 2012; Shen et al., 2011). In addition, nanoparticles conjugated directly with FA tend to aggregate in aqueous media duo to the absence of the shielding action (Porta et al., 2013). Cyclodextrins are potential materials to be exploited in this case as they may form complex with FA through the host–guest interactions (Okamatsu et al., 2013a,b,b; Zhao et al., 2013). Cyclodextrins are a family of compounds made up of sugar molecules bound together in a ring (cyclic oligosaccharides). In a typical cyclodextrin molecule, 6–8 glucopyranoside units are interconnected to form toroids with the larger and the smaller openings. As the exterior of this toroid was polar and hydrophilic and the interior cavity was relatively nonpolar and hydrophobic, the inclusion complex between CD and small hydrophobic molecules was easily obtained by inserting guest molecules into the cavity of host molecules (Dorokhin et al., 2010; Loftsson and Duchêne, 2007; Xin et al., 2010; He et al., 2013). Based on the special characteristics of the structure of CD, we prepared biocompatible folate-decorated carboxymethyl-b-cyclodextrin conjugated bovine serum albumin nanoparticles (FA-CM-b-CD-BSA NPs) with FA–FR binding specificity to achieve targeted delivery of drugs into tumor cells. Instead of using chemical linkers, FA was inserted into the cavity of CM-b-CD which was covalently bonded on the surface of nanoparticles to prevent the clustering effects and precipitation in buffered media. The structure and properties of NPs were characterized and determined by Fourier transform infrared (FT-IR) spectra, dynamic light scattering (DLS) and transmission electron microscope (TEM) analyses. The drug loading and in vitro release studies were performed by using 5-Fu as a model anticancer drug. The cell uptake, in vitro cytotoxicity and cellular apoptosis were performed to confirm the FA mediated tumor targeting ability of FA-CM-b-CD-BSA NPs. 2. Materials and methods
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2.2. The preparation of BSA NPs 175 mg of BSA was added into an aqueous solution of acetic acid (5 mL, 0.5%, v/v) under vortex mixing. To this solution, 20 mL of ethanol was added at a rate of 1 mL/min at 37 C under continuous stirring (1000 rpm) and the resulting white suspension was further stirred for 24 h. Ethanol was removed with vacuum distillation and 8% glutaraldehyde in water (0.5 mL per mg of BSA) was added to induce particle cross-linking. The resulting nanoparticles were redispersed in 10 mL of water. The nanoparticles were collected, washed with deionized water three times and centrifuged at 16,000 rpm to remove the supernatant. To prepare the drug-loaded BSA NPs, 10 mL of stock solution containing 5-Fu at 0.1 mg/mL was added into BSA solution prior to the addition of ethanol and the rest of the procedure was the same as that described above for blank BSA NPs. Rhodamine B or FITC as the fluorescent marker was encapsulated for labeling NPs. To prepare rhodamine B or FITC-labeled BSA NPs, 5 mL stock solution (0.1 mg/mL) containing FITC or rhodamine B was added into BSA solution prior to the addition of ethanol and the rest of the procedure was the same as that described above for blank BSA NPs. 2.3. Method of preparation of CM-b-CD-BSA NPs 10.0 mg of CM-b-CD was dissolved in 10 mL of PBS buffer (pH 5.8). The resulting solution was added into a biochemical centrifugal tube and activated by addition of 40 mg of EDC and 16 mg NHS at constant vibration for 1 h. Nanoparticles were added and the resulting reaction mixture was rotated overnight. Finally, CM-b-CD-BSA NPs were collected and washed three times with deionized water and centrifuged at 16,000 rpm to remove uncoupled residues. 2.4. Preparation and characterization of FA-CM-b-CD-BSA NPs 5 mg FA was incubated with 20 mg of CM-b-CD-BSA NPs in 10.0 mL of phosphate buffer (pH 7.4) under oscillation for 4 h. Collected NPs were washed 3–4 times with deionized water and centrifuged at 16,000 rpm for 20 min. The characterization of FA-CM-b-CD-BSA NP was investigated by affinity-1 infrared spectroscopy (Shimadzu, Kyoto, Japan) and the morphology of particles was studied by JEM-1200EX (Jeol, Tokyo, Japan) transmission electron microscope (TEM). Drug loading efficiency (LE) was determined as the ratio of the amount of 5-Fu in the NPs to the weight of NPs. The difference between the initially added drug amount and drug in the supernatant was measured at detecting absorbance at 256 nm using a UV/vis spectrophotometer (model 1601, Shimadzu, Japan) to determine the encapsulation efficiency (EE) of 5-Fu in nanoparticles. The EE was calculated using the equation listed below Eq. (1): EEð%Þ ¼
Weight of initially added drug Weight of free drug in supernatant Weight of initially added drug
100
(1)
2.1. Chemicals
2.5. Assessment of drug release
BSA was purchased from Sigma (USA), 5-fluorouracil was purchased from Nantong Jinghua Pharmaceutical Co., Ltd. (China) and carboxymethyl-b-cyclodextrin sodium salt (CM-b-CD) was purchased from Zhiyuan Bio-Technology Co., Ltd. (China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), acetic acid and folic acid were obtained from Sigma Chemicals (St. Louis, US). All other chemicals were of reagent grade and were used as received.
5-Fu loaded FA-CM-b-CD-BSA NPs and free 5-Fu were redispersed in 2 mL of PBS solution (pH 7.4) respectively and placed into dialysis bags with molecular weight cutoff of 1000. The dialysis bag was immersed into 60 mL of PBS buffer solution (pH 7.4) and shaken horizontally at 60 rpm at 37 C. After certain time intervals (0.5, 1–4, 6, 8, 10, 12, 24, 48 h), 2 mL dialysate fluid was taken out from the media and 2 mL of fresh PBS solution was added to remain the equivalent volume. Content of 5-Fu in
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Fig. 1. The process of preparing 5-Fu loaded biocompatible folate-decorated carboxymethyl-b-cyclodextrin conjugated bovine serum albumin nanoparticles.
dialysate fluid was determined by UV spectrophotometry at 256 nm and the cumulative amount of released 5-Fu was plotted against time.
10% fetal bovine serum (Hyclone) and penicillin/streptomycin (100 units/mL and 100 mg/mL, Gibco). All cells were maintained at 37 C in a humidified atmosphere of 5% CO2, 95% air.
2.6. Cell culture
2.7. Receptor expression
Human hepatocellular carcinoma cell line SMMC-7721 (folate receptor negative tumor cells line) and human cervical carcinoma (Hela) cells (folate receptor positive tumor cells line) were purchased from the Institute of Biochemistry and Cell Biology of Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone) containing
The total protein from each sample was extracted using RIPA buffer. Equal amount of protein (50 mg each lane) were separated by 10% SDS-PAGE and transferred to PVDF membrane. The membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and incubated with primary antibody anti-FA (Rabbit,1:1000) at 4 C overnight. The membranes were washed and then incubated with
Fig. 2. FT-IR spectra results. A: FT-IR spectra of CM-b-CD (A), CM-b-CD-BSA NPs (B) and BSA NPs (C); B: FT-IR spectra of FA (D), mixture of FA and CM-b-CD-BSA NPs (E) and FA-CM-b-CD-BSA NPs (F).
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Table 1 Key parameters of NPs. Group
Diameter (nm)
Zeta potential (mV)
Polydispersity
Encapsulation efficiency (%)
Loading efficiency (%)
BSA NPs CM-b-CD-BSA NPs FA-CM-b-CD-BSA NPs
254 19 277 15 311 32
25.97 1.94 10.85 1.21 7.53 0.51
0.51 0.06 0.21 0.09 0.15 0.05
80.1 3.96 79.4 4.13 81.5 5.32
4.0 0.21 3.5 0.32 3.4 0.27
the HRP conjugated secondary antibodies for 1 h at room temperature. After washing, the bands were revealed by in ECL and photographed by gel imaging system (I-box, UVP). 2.8. In vitro cellular uptake The cellular distributions of rhodamine B-labeled CM-b-CD-BSA NPs and FA-CM-b-CD-BSA NPs were observed by confocal laser scanning microscopy and quantified by microplate reader (Synergy2, Biotek, USA). Briefly, Confluent SMMC-7721 cells and Hela cells were incubated to a density of 5 104/mL in 6-well plate filled with serum free medium at 37 C and 5% CO2. After 24 h, both rhodamine B-labeled NPs were added into the medium and incubated with cells, respectively. After 6 h, the supernatant was discarded after centrifugation and cells in well were washed with ice-cold PBS for 3 times. Finally the internalization of rhodamine B-labeled NPs into cells was observed by confocal laser scanning microscopy. FITC as the fluorescent marker was encapsulated in NPs to quantify the intensity change of fluorescence in cells by using a microplate reader. The medium was replaced by serum free medium in the presence of different amount of free FA. After 6 h, the supernatant was removed. In order to remove the remnant NPs on the cell surface, the cells in wells were washed with ice-cold PBS for 3 times. The difference between intensity from the initially added FITC labeled NPs and that from FITC labeled NPs internalized in cells was measured at detecting fluorescence from FITC which is excited at 485 nm and emitted at 528 nm. Relative fluorescent ratio (RFR, %) was calculated using equation below. RFR% ¼
FIinternalized 100 FItotal
(2)
FItotal is fluorescent intensity from the initially added FITC labeled NPs, FIinternalized is fluorescent intensity from FITC labeled NPs internalized in cells. 2.9. In vitro cell viability assay
exposed to suspension of NPs at different amounts. After 24 h, 20 mL MTT with concentration of 5 mg/mL was added into 96-well plate and incubated for 4 h at 37 C . Then, the supernatant was aspirated, 150 mL of DMSO was added to each well, and the absorbance was measured at 490 nm. 2.10. Cell apoptosis study The quantitative apoptosis of 5-Fu loaded NPs was determined by flow cytometry analysis-annexin V-FITC/PI double staining. Hela cells were seeded into the 6-well plate and incubated to reach a density of 5 104/mL for 24 h at 37 C under 5% CO2 and 95% O2. Then, the cells were washed with PBS and incubated either with free 5-Fu, 5-Fu loaded CM-b-CD-BSA NPs, 5-Fu loaded FA-CMb-CD-BSA NPs for 48 h. After treatment, cells were collected and suspended in Nicoletti buffer containing propidium iodide (PI) and annexin V-FITC(AV). DNA content was determined on a fluorescence-activated cell sorter (FACSCalibur, BD, USA). The ratio of AV-positive cells and PI-positive cells were presented by calculating the amount of cells undergoing apoptosis at different stage. 2.11. Intracellular ATP level assay Hela cells seeded into the 96-well plate (100 mL each well) were incubated to reach a density of 5 104/mL for 24 h at 37 C under 5% CO2 and 95% O2. They were treated with free 5-Fu, 5-Fu loaded CM-b-CD-BSA NPs, 5-Fu loaded FA-CM-b-CD-BSA NPs at the same drug concentration for 48 h. The change of intracellular ATP level was determined by the luciferin–luciferase-based ATP luminescence assay kit. We took ATP level of untreated Hela cells as the control group and the changing rates of intracellular ATP level were (CR, %) calculated using equation below. ATP level of Hela cells treated with free drug or NPs ATP level of untreated Hela cells 100
CRð%Þ ¼
(3)
A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to investigate cell viability status. SMMC-7721 cells and Hela cells seeded into the 96-well plate (100 mL each well) were incubated to reach a density of 5 104/mL for 24 h at 37 C under 5% CO2 and 95% O2. Cells were
2.12. Western blot assay After treated with free 5-Fu, 5-Fu loaded CM-b-CD-BSA NPs, 5-Fu loaded FA-CM-b-CD-BSA NPs, cells were harvested, washed
Fig. 3. TEM images of BSA NPs (A), CM-b-CD-BSA NPs (B) and FA-CM-b-CD-BSA NPs (C).
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C. Su et al. / International Journal of Pharmaceutics 474 (2014) 202–211 Table 2 Fitting results of drug-release model of FA-CM-b-CD-BSA NPs. Model
Equations
R2
Zero-order First-order Higuchi Ritger–Peppas
Qt = 0.0103t + 0.1949 ln(100 Qt) = 0.0168t 0.2119 Qt = 0.0837t1/2 + 0.0766 lnQt = 0.3512 ln t 1.7798
0.7477 0.8640 0.9729 0.9844
Note: “Qt” is the accumulative release rate of 5-Fu in FA-CM-b-CD-BSA NPs at time “t”.
Fig. 4. FRa expression of Hela cells and SMMC-7721 cells (actin was used as control)
twice with ice cold PBS, then lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 1% SDS, 1 mM PMSF, 10 ug/mL leupeptin, 1 mM aprotinin, 50 mM Tris–Cl, pH 7.4). The cell lysate was cleared by centrifugation at 12,000 rpm for 25 min. Cell lysate containing 50 mg protein was separated by 10% SDS-PAGE and the protein was transferred onto polyvinylidene fluoride (PVDF) membrane. After blocking with 1% BSA, the PVDF membrane was incubated with the primary antibodies (cleaved caspase-3, bax, tubulin) at 4 C, overnight. Subsequently, incubated with appropriate secondary antibody for 1 h and stained with ECL. The level of the targeted proteins were photographed and analyzed by UVP gel analysis system. 3. Results and discussion 3.1. Synthesis of FA-CM-b-CD-BSA NPs As shown schematically in Fig. 1, BSA NPs were prepared using desolvation method. Firstly, BSA was dispersed and surrounded by water through hydrogen bonds reaction. Then, with the addition of ethanol as the dehydrating agent, BSA aggregates were precipitated into particles after removal of water at the surface of BSA. Finally, BSA NPs were collected by centrifugation and washed with deionized water for three times to remove organic agents and residues. Conjugation of CM-b-CD to the surface of BSA NPs was obtained by carbodiimide coupling. Furthermore, FA was completely or partially embedded into CM-b-CD cavity to form inclusion complex. The conjugation of CM-b-CD with BSA NPs was investigated using infrared spectroscopy as shown in Fig. 2A. The characteristic peak of the new formed amide bond between CM-b-CD molecules
and BSA NPs appeared at 1650 and 1540 cm1. In the IR spectrum (Fig. 2B.), the characteristic absorption peaks of FA from hydroxyl group (OH, 3556 cm1) and the aromatic amine groups (NH2, 3421 cm1 and 3327 cm1) were visible in the spectrum of the mixture of FA and CM-b-CD-BSA NPs. Conversely, these peaks disappeared in the sample of FA-CM-b-CD-BSA NPs, indicating that FA was partially embedded into CD cavity to form inclusion complex. 3.2. Physicochemical properties of NPs The morphology and size distribution of the prepared nanoparticles were determined by means of TEM and DLS. The results were displayed in Table 1. Compared with BSA NPs, the particle sizes of CM-b-CD-BSA NPs and FA-CM-b-CD-BSA NPs were bigger. This is mainly because the conjugation CM-b-CD on the surface of BSA NPs increased the size of particles. When FA was inserted into the cavity of CM-b-CD, the positive charges of amide group of FA were neutralized by the negative charges from CMb-CD-BSA NPs, leading to the increase on the zeta potential of FACM-b-CD-BSA NPs. In addition, the polydispersity index of BSA NPs was above 0.5, indicating an aggregating population and poor stability in media. With CM-b-CD, the polydispersity indexes of NPs were decreased significantly, suggesting that CM-b-CD improved the steric hindrance of NPs, therefore preventing the aggregation in media (Porta et al., 2013). The encapsulation efficiency and loading efficiency of 5-Fu for three NPs remained the same range. The morphology of three NPs was observed as being spherical and monodisperse spheres as shown in Fig. 3. FA-CMb-CD-BSA NPs presented a narrow size distribution with an average diameter about 311 32 nm and negative zeta potential about 7.53 0.51 mV. The 5-Fu encapsulation efficiency and loading efficiency of the FA-CM-b-CD-BSA NPs were 81.5 5.3% and 3.4 0.27%. Entrapment efficiency of FA conjugated with CM-b-CD-CS NPs was determined as the ratio of actual FA loading amount to the initial added FA amount. Furthermore, the CM-b-CD-CS NPs could conjugate with more FA (entrapment efficiency of FA: 60.9 2.3%) through host–guest interactions. 3.3. Receptor expression. The expression of FRa was evaluated on SMMC-7721 and Hela cells (Fig. 4) by western blot. It demonstrated that FRa was expressed higher on Hela cells than SMMC-7721 cells, consistent with previous results. Therefore, Hela cells were widely used as model for angiogenesis targeting delivery and treatment (Song et al., 2012; Feng et al., 2013). And FRa was lower expressed on SMMC-7721 cells. In this paper, Hela cells and SMMC-7721 cells were chosen to evaluate the cellular viability and targeting efficiency of NPs under the mediation of FA. 3.4. In vitro drug release study
Fig. 5. In vitro 5-Fu release profiles from FA-CM-b-CD-BSA NPs in aqueous solution at 37 C (n = 3).
The in vitro drug release profiles of 5-Fu loaded FA-CM-b-CD-BSA NPs and free 5-Fu in the medium were compared and results are shown in Fig. 5. The results showed
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Fig. 6. The results of cell viability assays. (A) Viability of SMMC-7221 cells after incubation with different amounts of naked FA-CM-b-CD-BSA NPs for 72 h (n = 3). (B) Viability of Hela cells after incubation with different amounts of naked FA-CM-b-CD-BSA NPs for 72 h (n = 3). (C) SMMC-7721 cell viability cultured with 5-Fu loaded NPs at various concentrations of 5-Fu in folate-free medium after 24 h. (D) Hela cell viability of 5-Fu loaded NPs at various concentrations of 5-Fu in folate-free medium after 24 h. Data were presented as mean S.D. (n = 3).
that the free drug as the control group was dissolved faster and about 98.7% was released into the medium within the first 6 h. Compared with free drugs, drugs encapsulated in FA-CM-b-CDBSA NPs exhibited a prolonged and slow release pattern in medium. The in vitro release data were fitted into release kinetics models to determine the best-fit release mode. The release kinetic data analysis and correlation coefficient (R2) are shown in Table 2. The relationship between cumulative release rate and time was tested using Zero-order, First-order, Higuchi and Ritger–Peppas models. In vitro drug release was best fitted to Ritger–Peppas model which showed the highest correlation coefficient (R2). The diffusional exponent (n) in Ritger–Peppas model was 0.3512, lower than 0.45, suggesting that drug release from 5-Fu loaded FA-CMb-CD-BSA NPs followed Fickian diffusing theory and was influenced by drug diffusion.
both cells incubated with FA-decorated NPs in vitro was significantly higher than undecorated one after 24 h. However, the 5-Fu loaded FA-CM-b-CD-BSA NPs could inhibit both cells viability from Fig. 6, we could find Hela cells which expressed higher FR showed more susceptible to 5-Fu loaded FA-CM-b-CDBSA NPs than SMMC-7721 cells. Briefly, FA improved the drug delivery and encapsulation by tumor cells which folate receptor was expressed high. Compared with low expression of FR at the surface of SMMC-7721 cells, more FRs were expressed in Hela cells, which led to the more uptake of FA-CM-b-CD-BSA NPs via acting endocytosis mediated by FA–FR interaction. Therefore, 5-Fu loaded FA-CM-b-CD-BSA NPs exhibited stronger cell inhibition activity on Hela cells than on SMMC7721 cells (Fig. 6C and D). 3.6. Cellular uptake of NPs and competition assay
3.5. Cell viability assays It can be seen from Fig. 6 that the inhibiting rates of blank FACM-b-CD-BSA NPs for tumor cells were very low and different amount of blank FA-CM-b-CD-BSA NPs showed no obvious cell inhibition within 72 h (Fig. 6A and B). When Hela and SMMC7721 cells were treated with CM-b-CD-BSA NPs and FA-CM-b-CDBSA NPs encapsulating the same amount of 5-Fu, the viability of
The cellular uptake of rhodamine B-labeled NPs in SMMC7721 and Hela cells was performed by confocal laser scanning microscopy, as shown in Fig. 7. When the cells were treated with CM-b-CD-BSA NPs, the red fluorescence intensity of the cells was weak indicating that most of nanoparticles were not taken up by cells, only a small portion of NPs was internalized into cells by endocytic process. On the contrary, rhodamine B-labeled
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Fig. 7. Confocal images of cells after incubating for 6 h with FA-CM-b-CD-BSA NPs and CM-b-CD-BSA NPs. Nucleus was stained with hochest (blue) for 15 min at 37 C and all of NPs are labeled by rhodamine B (red); (A) incubated with hela cells, (B) incubated with SMMC-7721 cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
FA-CM-b-CD-BSA NPs showed significant red fluorescence throughout the cytoplasm, which is closely located around the nuclei (blue). This observation indicated that FA-CM-b-CD-BSA NPs could effectively enhance drug accumulation in cancer cells, which may be attributed to specific binding between FA and folate
receptor on the surface of cancer cells. Hereby, FA-CM-b-CD-BSA NPs as an anticancer drug carrier will accumulate inside cells, improving the curing effects of anti-tumor drugs. The cellular distribution of CM-b-CD-BSA NPs and FA-CMb-CD-BSA NPs was quantified by micro-plate reader as shown in
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Fig. 8. Fluorescence spectrum analysis of the uptake of FA-CM-b-CD-BSA NPs labeled by FITC. (A) The uptake of FA-CM-b-CD-BSA NPs labeled by FITC incubated with SMMC7721 cells for 6 h in media containing different amount of free FA, n = 3, *P < 0.05, vs. the FA-CM-b-CD-BSA NPs group in media without folate, (B) incubated with Hela cells, n = 3, *P < 0.05, vs. the FA-CM-b-CD-BSA NPs group in media without folate.
Fig. 8. When FA-CM-b-CD-BSA NPs were incubated with two tumor cells for 6 h, The RFR observed in cells was increased gradually with the decreased amount of free FA in medium,
suggesting that free FA existing in the medium competed with FA-CM-b-CD-BSA NPs to bind the folate receptor on the cell surface, thus resulting in the saturation of receptors and
Fig. 9. Cell apoptosis determined by annexin V-FITC staining. Flow cytometer analysis of the apoptosis and necrosis cells after 48 h incubation with the free 5-Fu, 5-Fu loaded CM-b-CD-BSA NPs and 5-Fu loaded FA-CM-b-CD-BSA NPs respectively. Results were expressed as means S.D. (n = 3).
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FA-CM-b-CD-BSA NPs treated cells was markedly higher than other treatments. It proved that 5-Fu loaded NPs with the mediation of FA significantly enhanced 5-Fu-induced apoptosis. 3.8. Intracellular ATP level assay and western blot assay
Fig. 10. Intracellular ATP level assay after 48 h incubation with the free 5-Fu, 5-Fu loaded CM-b-CD-BSA NPs and 5-Fu loaded FA-CM-b-CD-BSA NPs respectively. Results were expressed as means S.D. (n = 3).
preventing the transportation of FA-CM-b-CD-BSA NPs into cells. In contrast, the addition of FA took no obvious effects on the internalization of CM-b-CD-BSA NPs. This suggested that FA-CM-b-CD-BSA NPs may be internalized via FA mediated uptake and the FA unconjugated NPs were probably internalized by different mechanisms. Taken together, more FA-CM-b-CD-BSA NPs were internalized in FR high expressed Hela cells than SMMC-7721 cells, which expressed lower FR and, the targeting effect is effective and the side effect will be decreased correspondently. 3.7. Cell apoptosis and necrosis In order to verify the cell apoptosis rate induced by the 5-Fu loaded NPs, annexin V-FITC/PI staining assay was performed and the apoptotic and necrotic cells were quantified by flow cytometry. The percentages of early apoptotic (Q4), late apoptotic (Q2), necrotic (Q1) and live cells (Q3) are shown in Fig. 9. Flow cytometry analysis revealed that after 48 h incubation with free 5-Fu, 5-Fu loaded CM-b-CD-BSA NPs and 5-Fu loaded FA-CM-b-CD-BSA NPs, the ratio of AV-positive and PI-positive cells was increased to 27.48%, 49.90% and 67.68%, respectively. It was worth to note that the ratio of AV-positive and PI-positive fractions in 5-Fu loaded
Apoptosis of cells usually depended on the supply of energy and required the participation of ATP (adenosine triphosphate). Decreased ATP level can not maintain basic metabolic functions of cells, and lead to cells’ apoptosis. A typical apoptotic cell was presented by significant decline of ATP level. Therefore, the change of intracellular ATP level was highly related with apoptosis, necrosis or toxicity in cells. We took ATP level of untreated Hela cells as the control group and the changing rates of intracellular ATP level were shown in Fig. 10. Compared with ATP level of the control group, after 48 h incubation with free 5-Fu, 5-Fu loaded CM-b-CD-BSA NPs and 5-Fu loaded FA-CM-b-CD-BSA NPs, The changing rates of intracellular ATP level were decreased to 88.28%, 74.58% and 55.12%, respectively. It was also suggested that more drug loaded FA-CM-b-CD-BSA NPs were internalized into cells and increased the intracellular drug concentration with the mediation of FA, thus leading to the significant decrease of intracellular ATP and accelerating the apoptosis of cells by accumulation of drugs in cells. In order to further explore drug loaded NPs-induced apoptosis of Hela cells, the expression of two apoptosis relevant proteins (Bax and cleaved caspase-3) was evaluated in Fig. 11. It is well known that caspase family plays a very important role in mediating apoptosis process of cells, wherein the caspase-3 as a key executing molecule mainly functions on the regulation of the apoptosis signal transduction pathway. In the early stages of apoptosis, caspase-3 was activated and corresponding cytoplasmic and nuclear substrates began to cleave, then resulting in apoptosis. It was observed that the expressions of cleaved caspase-3 protein in cells treated with the three 5-Fu containing formulations were obviously elevated. Especially, cleaved caspase-3 protein in cells treated with 5-Fu loaded FA-CM-b-CD-BSA NPs was expressed at the highest level, suggesting that the targeted delivery of 5-Fu loaded FA-CM-b-CD-BSA NPs significantly increased the intracellular drug concentration with the mediation of FA, thus accelerating the apoptosis of cells by accumulation of drugs in cells. On the contrast, the regulation of Bax in cells treated with three drug groups was not significantly different from control group. It was demonstrated that involvement of drug had little direct effect on the expression of Bax acting as a well-known pro-apoptotic protein. From all the above, to elucidate effect of FA-CM-b-CD-BSA NPs, we use 5-Fu to exam the drug delivery, and the cell viability and cell apoptosis analysis show the 5-Fu loaded FA-CM-b-CD-BSA NPs increased the cytotoxicity of folate receptor positive Hela cells. Furthermore, we detect the expression of ATP and cleaved capase3 to explain the apoptosis mechanism of FA-CM-b-CD-BSA NPs, and find FA-CM-b-CD-BSA NPs could inhibit energy production (ATP) and promote cleaved caspase-3 expression. So, FA-CM-b-CDBSA NPs provide a more effective way to assist drugs to target folate receptor positive tumor cells. Through this article, we can see 5-Fu loaded FA-CM-b-CD-BSA NPs could be uptaked by FR positive Hela cells, and facilitate 5-Fu chemotherapy and decrease chemoresistance with the interaction between FA and FR. 4. Conclusion
Fig. 11. Apoptotic effects of various 5-Fu formulations on Hela cells. Western blot analyses of the expression levels of Bax and cleaved caspase-3 proteins in Hela cells after treatments.
FA as a targeting ligand was successfully inserted into CM-b-CD-BSA NPs to fabricate folate decorated target nanoparticles based on the interaction between FA and FR for
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enhancing antitumor drug delivery. FA-CM-b-CD-BSA NPs obtained in our experiments were characterized of good monodispersity, negative charge and homogenous particle size, high encapsulating efficiency and 5-Fu could be released in a prolonged and slow pattern in medium in vitro. Fluorescent images and the free folic acid competition study proved that FA-CM-b-CD-BSA NPs could obviously enhance cellular uptake through folate mediated internalization in Hela cells, which FR is higher expressed in cell surface. MTT and cell apoptosis demonstrated that FA-decorated NPs exhibited stronger inhibition and induced obvious apoptosis in FR positive Hela cells as compared to free 5-Fu and FA undecorated NPs. This can be attributed to the possibility that FA-CM-b-CD-BSA NPs enhanced FR receptor-mediated endocytosis which led to more intracellular uptake of drugs, then inducing the apoptosis of cells by down-regulation of ATP level and over-expression of caspase-3. These properties suggest us that folate decorated targeted CM-b-CD-BSA NPs may become a promising active FR positive tumor-targeting carrier candidate and it may potentially find clinical usage after further modifications in future. Disclosure The authors report no conflicts of interest in this work. Acknowledgements This work is supported by Natural Science Foundation of Liaoning Province (No. 2013022035), National Natural Science Foundation of China (No. 81172048), and Grant of Liaoning Medical University (No. XZJJ20130104-05). We thank Prof. Yunhong Jia and Dr. Bo Zhang for polishing manuscript. References Aryal, S., Hu, C.M., Fang, R.H., Dehaini, D., Carpenter, C., Zhang, D.E., Zhang, L., 2013. Erythrocyte membrane-cloaked polymeric nanoparticles for controlled drug loading and release. Nanomedicine 8, 1271–1280. Cho, C.S., Kobayashi, A., Takei, R., Ishihara, T., Maruyama, A., Akaike, T., 2001. Receptor-mediated cell modulator delivery to hepatocyte using nanoparticles coated with carbohydrate-carrying polymers. Biomaterials 22, 45–51. Dorokhin, D., Hsu, S.H., Tomczak, N., Reinhoudt, D.N., Huskens, J., Velders, A.H., Vancso, G.J., 2010. Fabrication and luminescence of designer surface patterns with beta-cyclodextrin functionalized quantum dots via multivalent supramolecular coupling. ACS Nano 4, 137–142. Feng, D., Song, Y., Shi, W., Li, X., Ma, H., 2013. Distinguishing folate-receptor-positive cells from folate-receptor-negative cells using a fluorescence off–on nanoprobe. Anal. Chem. 85, 6530–6535. Gref, R., Couvreur, P., Barratt, G., Mysiakine, E., 2003. Surface-engineered nanoparticles for multiple ligand coupling. Biomaterials 24, 4529–4537. He, Q., Wu, W., Xiu, K., Zhang, Q., Xu, F., Li, J., 2013. Controlled drug release system based on cyclodextrin-conjugated poly(lactic acid)-b-poly(ethylene glycol) micelles. Int. J. Pharm. 443, 110–119. Hou, Z., Zhan, C., Jiang, Q., Hu, Q., Li, L., Chang, D., Yang, X., Wang, Y., Li, Y., Ye, S., Xie, L., Yi, Y., Zhang, Q., 2011. Both FA-and mPEG-conjugated chitosan nanoparticles for targeted cellular uptake and enhanced tumor tissue distribution. Nanoscale Res. Lett. 6, 563. Huang, S., Shao, K., Kuang, Y., Liu, Y., Li, J., An, S., Guo, Y., Ma, H., He, X., Jiang, C., 2013. Tumor targeting and microenvironment-responsive nanoparticles for gene delivery. Biomaterials 34, 5294–5302. Hyung Park, J., Kwon, S., Lee, M., Chung, H., Kim, J.H., Kim, Y.S., Park, R.W., Kim, I.S., Bong Seo, S., Kwon, I.C., Young Jeong, S., 2006. Self-assembled nanoparticles based on glycol chitosan bearing hydrophobic moieties as carriers for doxorubicin: in vivo biodistribution and anti-tumor activity. Biomaterials 27, 119–126.
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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Carboxymethyl-b-cyclodextrin conjugated nanoparticles facilitate therapy for folate receptor-positive tumor with the mediation of folic acid Chang Su a,1, Hongdan Li b,1, Yijie Shi c , Guan Wang b , Liwei Liu c, Liang Zhao c, *, Rongjian Su b, ** a b c
School of Veterinary Medicine, Liaoning Medical University, Jinzhou 121000, PR China Central Laboratory of Liaoning Medical University, Jinzhou 121000, PR China School of Pharmacy, Liaoning Medical University, Jinzhou 121000, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 June 2014 Received in revised form 7 August 2014 Accepted 15 August 2014 Available online 19 August 2014
Currently, clinical operation treatments, chemotherapy and radiotherapy just could eliminate local tumor cells. However, chemotherapy and radiotherapy also injury normal cells and lead to serious side effects and toxicities. So, it is necessary to find an effective target cancer carrier that delivers the anticancer agents into tumor cells and reduces normal cells’ injury. Folic acid (FA) is a classical targeting agent mediates internalization of chemical drugs into tumor cells which over-express folate receptor (FR) on their surface. We herein report that based on host–guest interaction, NPs decorated by novel folate enhance antitumor drug delivery. BSA-NPs were prepared by desolvation method and carboxymethyl-b-cyclodextrin (CM-b-CD) was conjugated to the surface of NPs by carbodiimide coupling to hold FA. From in vitro cytotoxicity assay, cell apoptosis study, intracellular ATP level assay and western blot, we can see that FA-CM-b-CD-BSA NPs as good monodispersity, negative charge, and homogenous particle size have a high encapsulation efficiency. The results showed that MTT and cell apoptosis demonstrated that FA-decorated NPs exhibit stronger inhibition rate and induce obvious apoptosis in FR positive Hela cells as compared to free drug and FA undecorated NPs. Moreover, 5-fluorouracil (5-Fu) loaded FA-CM-b-CD-BSA NPs down-regulate ATP levels and increase the expression of caspase-3. Taken together, FA-CM-b-CD-BSA NPs enhance FR receptor-mediated endocytosis and lead to more intracellular uptake of drug, inducing the higher apoptosis ratio of cells than free 5-Fu. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Folic acid Nanoparticles Carboxymethyl-b-cyclodextrin 5-Fu Apoptosis
1. Introduction Although cancer therapy has made some great progress nowadays, cancer is still a serious threat to human health. The main drawback of anti-cancer formulations is that besides inhibiting the growth of tumor cells, they can also significantly inhibit the proliferation of normal cells. Recently, the application of nanoparticles has become a very important research field, attracting more and more researchers. Nanopaticle is characterized by a small particle covering a range from 1 to 500 nm in size usually behaving
* Corresponding author. Tel.: +86 416 4673439; fax: +86 416 4673439. ** Corresponding author. E-mail addresses: [email protected] (L. Zhao), [email protected] (R. Su). 1 These two authors contributed equally to the manuscript. http://dx.doi.org/10.1016/j.ijpharm.2014.08.026 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
good transport and special biological properties. Compared with traditional carriers, nanoparticles have following advantages: (1) due to the small size of particles, NPs are suitable for administration through the blood vessels and they are also known to be able to even penetrate the blood–brain barrier into the brain tissue (Lin et al., 2012; Yim et al., 2012); (2) NPs are capable of encapsulating more drug molecules compared with traditional drug delivery systems and, they can also further control the drug release (Singh and Lillard, 2009; Aryal et al., 2013; Mattheolabakis et al., 2009; Hyung Park et al., 2006); (3) drug loaded NPs can be selectively retained in the lesion site by active or passive targeting known as EPR effects, thus reducing the total dose of drugs and lowering the toxicity (Huang et al., 2013; Verderio et al., 2013; Wu et al., 2014); (4) NPs can improve drug stability in vivo by protecting nucleic acids, peptide drugs from enzyme’s degradation. In order to improve the targeting ability of NPs on organs and tissues, it is necessary to conjugate specific targeting molecules at the surface of nanoparticles to achieve active
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targeting therapy by binding with specific cell surface receptors (Xu et al., 2005; Cho et al., 2001; Gref et al., 2003; Li et al., 2014) Folic acid plays important roles on cell division, proliferation, and synthesis of some biological macromolecules (Weinstein et al., 2003; Leamon, 2008). In proliferation process of tumor cells, a large amount of folic acids are required. Particularly, folate receptors (FRs) were highly expressed in tumor cells like uterus, breast, brain, lung, kidney cancer cells, while low expression of FRs was found in normal tissues (Sudimack and Lee, 2000; Weitman et al., 1992). As the folate receptors most expressed in cancer cells are a – FRs, free folic acid has a strong affinity for a – FR and it was reported to be covalently bound to macromolecules to facilitate the internalization of the macromolecules into cancer cells (Lu et al., 2004; Pinhassi et al., 2010). Therefore, nanoparticles can be coupled to folic acid to achieve the active targeting delivery of anti-cancer drugs (Hou et al., 2011; Zhang et al., 2012). In order to prepare FA decorated nanoparticles, traditionally, organic reagents and toxic coupling reagents were commonly used, leading to toxic reagents residue and cellular toxic effects (Yang et al., 2012; Shen et al., 2011). In addition, nanoparticles conjugated directly with FA tend to aggregate in aqueous media duo to the absence of the shielding action (Porta et al., 2013). Cyclodextrins are potential materials to be exploited in this case as they may form complex with FA through the host–guest interactions (Okamatsu et al., 2013a,b,b; Zhao et al., 2013). Cyclodextrins are a family of compounds made up of sugar molecules bound together in a ring (cyclic oligosaccharides). In a typical cyclodextrin molecule, 6–8 glucopyranoside units are interconnected to form toroids with the larger and the smaller openings. As the exterior of this toroid was polar and hydrophilic and the interior cavity was relatively nonpolar and hydrophobic, the inclusion complex between CD and small hydrophobic molecules was easily obtained by inserting guest molecules into the cavity of host molecules (Dorokhin et al., 2010; Loftsson and Duchêne, 2007; Xin et al., 2010; He et al., 2013). Based on the special characteristics of the structure of CD, we prepared biocompatible folate-decorated carboxymethyl-b-cyclodextrin conjugated bovine serum albumin nanoparticles (FA-CM-b-CD-BSA NPs) with FA–FR binding specificity to achieve targeted delivery of drugs into tumor cells. Instead of using chemical linkers, FA was inserted into the cavity of CM-b-CD which was covalently bonded on the surface of nanoparticles to prevent the clustering effects and precipitation in buffered media. The structure and properties of NPs were characterized and determined by Fourier transform infrared (FT-IR) spectra, dynamic light scattering (DLS) and transmission electron microscope (TEM) analyses. The drug loading and in vitro release studies were performed by using 5-Fu as a model anticancer drug. The cell uptake, in vitro cytotoxicity and cellular apoptosis were performed to confirm the FA mediated tumor targeting ability of FA-CM-b-CD-BSA NPs. 2. Materials and methods
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2.2. The preparation of BSA NPs 175 mg of BSA was added into an aqueous solution of acetic acid (5 mL, 0.5%, v/v) under vortex mixing. To this solution, 20 mL of ethanol was added at a rate of 1 mL/min at 37 C under continuous stirring (1000 rpm) and the resulting white suspension was further stirred for 24 h. Ethanol was removed with vacuum distillation and 8% glutaraldehyde in water (0.5 mL per mg of BSA) was added to induce particle cross-linking. The resulting nanoparticles were redispersed in 10 mL of water. The nanoparticles were collected, washed with deionized water three times and centrifuged at 16,000 rpm to remove the supernatant. To prepare the drug-loaded BSA NPs, 10 mL of stock solution containing 5-Fu at 0.1 mg/mL was added into BSA solution prior to the addition of ethanol and the rest of the procedure was the same as that described above for blank BSA NPs. Rhodamine B or FITC as the fluorescent marker was encapsulated for labeling NPs. To prepare rhodamine B or FITC-labeled BSA NPs, 5 mL stock solution (0.1 mg/mL) containing FITC or rhodamine B was added into BSA solution prior to the addition of ethanol and the rest of the procedure was the same as that described above for blank BSA NPs. 2.3. Method of preparation of CM-b-CD-BSA NPs 10.0 mg of CM-b-CD was dissolved in 10 mL of PBS buffer (pH 5.8). The resulting solution was added into a biochemical centrifugal tube and activated by addition of 40 mg of EDC and 16 mg NHS at constant vibration for 1 h. Nanoparticles were added and the resulting reaction mixture was rotated overnight. Finally, CM-b-CD-BSA NPs were collected and washed three times with deionized water and centrifuged at 16,000 rpm to remove uncoupled residues. 2.4. Preparation and characterization of FA-CM-b-CD-BSA NPs 5 mg FA was incubated with 20 mg of CM-b-CD-BSA NPs in 10.0 mL of phosphate buffer (pH 7.4) under oscillation for 4 h. Collected NPs were washed 3–4 times with deionized water and centrifuged at 16,000 rpm for 20 min. The characterization of FA-CM-b-CD-BSA NP was investigated by affinity-1 infrared spectroscopy (Shimadzu, Kyoto, Japan) and the morphology of particles was studied by JEM-1200EX (Jeol, Tokyo, Japan) transmission electron microscope (TEM). Drug loading efficiency (LE) was determined as the ratio of the amount of 5-Fu in the NPs to the weight of NPs. The difference between the initially added drug amount and drug in the supernatant was measured at detecting absorbance at 256 nm using a UV/vis spectrophotometer (model 1601, Shimadzu, Japan) to determine the encapsulation efficiency (EE) of 5-Fu in nanoparticles. The EE was calculated using the equation listed below Eq. (1): EEð%Þ ¼
Weight of initially added drug Weight of free drug in supernatant Weight of initially added drug
100
(1)
2.1. Chemicals
2.5. Assessment of drug release
BSA was purchased from Sigma (USA), 5-fluorouracil was purchased from Nantong Jinghua Pharmaceutical Co., Ltd. (China) and carboxymethyl-b-cyclodextrin sodium salt (CM-b-CD) was purchased from Zhiyuan Bio-Technology Co., Ltd. (China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), acetic acid and folic acid were obtained from Sigma Chemicals (St. Louis, US). All other chemicals were of reagent grade and were used as received.
5-Fu loaded FA-CM-b-CD-BSA NPs and free 5-Fu were redispersed in 2 mL of PBS solution (pH 7.4) respectively and placed into dialysis bags with molecular weight cutoff of 1000. The dialysis bag was immersed into 60 mL of PBS buffer solution (pH 7.4) and shaken horizontally at 60 rpm at 37 C. After certain time intervals (0.5, 1–4, 6, 8, 10, 12, 24, 48 h), 2 mL dialysate fluid was taken out from the media and 2 mL of fresh PBS solution was added to remain the equivalent volume. Content of 5-Fu in
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Fig. 1. The process of preparing 5-Fu loaded biocompatible folate-decorated carboxymethyl-b-cyclodextrin conjugated bovine serum albumin nanoparticles.
dialysate fluid was determined by UV spectrophotometry at 256 nm and the cumulative amount of released 5-Fu was plotted against time.
10% fetal bovine serum (Hyclone) and penicillin/streptomycin (100 units/mL and 100 mg/mL, Gibco). All cells were maintained at 37 C in a humidified atmosphere of 5% CO2, 95% air.
2.6. Cell culture
2.7. Receptor expression
Human hepatocellular carcinoma cell line SMMC-7721 (folate receptor negative tumor cells line) and human cervical carcinoma (Hela) cells (folate receptor positive tumor cells line) were purchased from the Institute of Biochemistry and Cell Biology of Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone) containing
The total protein from each sample was extracted using RIPA buffer. Equal amount of protein (50 mg each lane) were separated by 10% SDS-PAGE and transferred to PVDF membrane. The membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and incubated with primary antibody anti-FA (Rabbit,1:1000) at 4 C overnight. The membranes were washed and then incubated with
Fig. 2. FT-IR spectra results. A: FT-IR spectra of CM-b-CD (A), CM-b-CD-BSA NPs (B) and BSA NPs (C); B: FT-IR spectra of FA (D), mixture of FA and CM-b-CD-BSA NPs (E) and FA-CM-b-CD-BSA NPs (F).
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Table 1 Key parameters of NPs. Group
Diameter (nm)
Zeta potential (mV)
Polydispersity
Encapsulation efficiency (%)
Loading efficiency (%)
BSA NPs CM-b-CD-BSA NPs FA-CM-b-CD-BSA NPs
254 19 277 15 311 32
25.97 1.94 10.85 1.21 7.53 0.51
0.51 0.06 0.21 0.09 0.15 0.05
80.1 3.96 79.4 4.13 81.5 5.32
4.0 0.21 3.5 0.32 3.4 0.27
the HRP conjugated secondary antibodies for 1 h at room temperature. After washing, the bands were revealed by in ECL and photographed by gel imaging system (I-box, UVP). 2.8. In vitro cellular uptake The cellular distributions of rhodamine B-labeled CM-b-CD-BSA NPs and FA-CM-b-CD-BSA NPs were observed by confocal laser scanning microscopy and quantified by microplate reader (Synergy2, Biotek, USA). Briefly, Confluent SMMC-7721 cells and Hela cells were incubated to a density of 5 104/mL in 6-well plate filled with serum free medium at 37 C and 5% CO2. After 24 h, both rhodamine B-labeled NPs were added into the medium and incubated with cells, respectively. After 6 h, the supernatant was discarded after centrifugation and cells in well were washed with ice-cold PBS for 3 times. Finally the internalization of rhodamine B-labeled NPs into cells was observed by confocal laser scanning microscopy. FITC as the fluorescent marker was encapsulated in NPs to quantify the intensity change of fluorescence in cells by using a microplate reader. The medium was replaced by serum free medium in the presence of different amount of free FA. After 6 h, the supernatant was removed. In order to remove the remnant NPs on the cell surface, the cells in wells were washed with ice-cold PBS for 3 times. The difference between intensity from the initially added FITC labeled NPs and that from FITC labeled NPs internalized in cells was measured at detecting fluorescence from FITC which is excited at 485 nm and emitted at 528 nm. Relative fluorescent ratio (RFR, %) was calculated using equation below. RFR% ¼
FIinternalized 100 FItotal
(2)
FItotal is fluorescent intensity from the initially added FITC labeled NPs, FIinternalized is fluorescent intensity from FITC labeled NPs internalized in cells. 2.9. In vitro cell viability assay
exposed to suspension of NPs at different amounts. After 24 h, 20 mL MTT with concentration of 5 mg/mL was added into 96-well plate and incubated for 4 h at 37 C . Then, the supernatant was aspirated, 150 mL of DMSO was added to each well, and the absorbance was measured at 490 nm. 2.10. Cell apoptosis study The quantitative apoptosis of 5-Fu loaded NPs was determined by flow cytometry analysis-annexin V-FITC/PI double staining. Hela cells were seeded into the 6-well plate and incubated to reach a density of 5 104/mL for 24 h at 37 C under 5% CO2 and 95% O2. Then, the cells were washed with PBS and incubated either with free 5-Fu, 5-Fu loaded CM-b-CD-BSA NPs, 5-Fu loaded FA-CMb-CD-BSA NPs for 48 h. After treatment, cells were collected and suspended in Nicoletti buffer containing propidium iodide (PI) and annexin V-FITC(AV). DNA content was determined on a fluorescence-activated cell sorter (FACSCalibur, BD, USA). The ratio of AV-positive cells and PI-positive cells were presented by calculating the amount of cells undergoing apoptosis at different stage. 2.11. Intracellular ATP level assay Hela cells seeded into the 96-well plate (100 mL each well) were incubated to reach a density of 5 104/mL for 24 h at 37 C under 5% CO2 and 95% O2. They were treated with free 5-Fu, 5-Fu loaded CM-b-CD-BSA NPs, 5-Fu loaded FA-CM-b-CD-BSA NPs at the same drug concentration for 48 h. The change of intracellular ATP level was determined by the luciferin–luciferase-based ATP luminescence assay kit. We took ATP level of untreated Hela cells as the control group and the changing rates of intracellular ATP level were (CR, %) calculated using equation below. ATP level of Hela cells treated with free drug or NPs ATP level of untreated Hela cells 100
CRð%Þ ¼
(3)
A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to investigate cell viability status. SMMC-7721 cells and Hela cells seeded into the 96-well plate (100 mL each well) were incubated to reach a density of 5 104/mL for 24 h at 37 C under 5% CO2 and 95% O2. Cells were
2.12. Western blot assay After treated with free 5-Fu, 5-Fu loaded CM-b-CD-BSA NPs, 5-Fu loaded FA-CM-b-CD-BSA NPs, cells were harvested, washed
Fig. 3. TEM images of BSA NPs (A), CM-b-CD-BSA NPs (B) and FA-CM-b-CD-BSA NPs (C).
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C. Su et al. / International Journal of Pharmaceutics 474 (2014) 202–211 Table 2 Fitting results of drug-release model of FA-CM-b-CD-BSA NPs. Model
Equations
R2
Zero-order First-order Higuchi Ritger–Peppas
Qt = 0.0103t + 0.1949 ln(100 Qt) = 0.0168t 0.2119 Qt = 0.0837t1/2 + 0.0766 lnQt = 0.3512 ln t 1.7798
0.7477 0.8640 0.9729 0.9844
Note: “Qt” is the accumulative release rate of 5-Fu in FA-CM-b-CD-BSA NPs at time “t”.
Fig. 4. FRa expression of Hela cells and SMMC-7721 cells (actin was used as control)
twice with ice cold PBS, then lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 1% SDS, 1 mM PMSF, 10 ug/mL leupeptin, 1 mM aprotinin, 50 mM Tris–Cl, pH 7.4). The cell lysate was cleared by centrifugation at 12,000 rpm for 25 min. Cell lysate containing 50 mg protein was separated by 10% SDS-PAGE and the protein was transferred onto polyvinylidene fluoride (PVDF) membrane. After blocking with 1% BSA, the PVDF membrane was incubated with the primary antibodies (cleaved caspase-3, bax, tubulin) at 4 C, overnight. Subsequently, incubated with appropriate secondary antibody for 1 h and stained with ECL. The level of the targeted proteins were photographed and analyzed by UVP gel analysis system. 3. Results and discussion 3.1. Synthesis of FA-CM-b-CD-BSA NPs As shown schematically in Fig. 1, BSA NPs were prepared using desolvation method. Firstly, BSA was dispersed and surrounded by water through hydrogen bonds reaction. Then, with the addition of ethanol as the dehydrating agent, BSA aggregates were precipitated into particles after removal of water at the surface of BSA. Finally, BSA NPs were collected by centrifugation and washed with deionized water for three times to remove organic agents and residues. Conjugation of CM-b-CD to the surface of BSA NPs was obtained by carbodiimide coupling. Furthermore, FA was completely or partially embedded into CM-b-CD cavity to form inclusion complex. The conjugation of CM-b-CD with BSA NPs was investigated using infrared spectroscopy as shown in Fig. 2A. The characteristic peak of the new formed amide bond between CM-b-CD molecules
and BSA NPs appeared at 1650 and 1540 cm1. In the IR spectrum (Fig. 2B.), the characteristic absorption peaks of FA from hydroxyl group (OH, 3556 cm1) and the aromatic amine groups (NH2, 3421 cm1 and 3327 cm1) were visible in the spectrum of the mixture of FA and CM-b-CD-BSA NPs. Conversely, these peaks disappeared in the sample of FA-CM-b-CD-BSA NPs, indicating that FA was partially embedded into CD cavity to form inclusion complex. 3.2. Physicochemical properties of NPs The morphology and size distribution of the prepared nanoparticles were determined by means of TEM and DLS. The results were displayed in Table 1. Compared with BSA NPs, the particle sizes of CM-b-CD-BSA NPs and FA-CM-b-CD-BSA NPs were bigger. This is mainly because the conjugation CM-b-CD on the surface of BSA NPs increased the size of particles. When FA was inserted into the cavity of CM-b-CD, the positive charges of amide group of FA were neutralized by the negative charges from CMb-CD-BSA NPs, leading to the increase on the zeta potential of FACM-b-CD-BSA NPs. In addition, the polydispersity index of BSA NPs was above 0.5, indicating an aggregating population and poor stability in media. With CM-b-CD, the polydispersity indexes of NPs were decreased significantly, suggesting that CM-b-CD improved the steric hindrance of NPs, therefore preventing the aggregation in media (Porta et al., 2013). The encapsulation efficiency and loading efficiency of 5-Fu for three NPs remained the same range. The morphology of three NPs was observed as being spherical and monodisperse spheres as shown in Fig. 3. FA-CMb-CD-BSA NPs presented a narrow size distribution with an average diameter about 311 32 nm and negative zeta potential about 7.53 0.51 mV. The 5-Fu encapsulation efficiency and loading efficiency of the FA-CM-b-CD-BSA NPs were 81.5 5.3% and 3.4 0.27%. Entrapment efficiency of FA conjugated with CM-b-CD-CS NPs was determined as the ratio of actual FA loading amount to the initial added FA amount. Furthermore, the CM-b-CD-CS NPs could conjugate with more FA (entrapment efficiency of FA: 60.9 2.3%) through host–guest interactions. 3.3. Receptor expression. The expression of FRa was evaluated on SMMC-7721 and Hela cells (Fig. 4) by western blot. It demonstrated that FRa was expressed higher on Hela cells than SMMC-7721 cells, consistent with previous results. Therefore, Hela cells were widely used as model for angiogenesis targeting delivery and treatment (Song et al., 2012; Feng et al., 2013). And FRa was lower expressed on SMMC-7721 cells. In this paper, Hela cells and SMMC-7721 cells were chosen to evaluate the cellular viability and targeting efficiency of NPs under the mediation of FA. 3.4. In vitro drug release study
Fig. 5. In vitro 5-Fu release profiles from FA-CM-b-CD-BSA NPs in aqueous solution at 37 C (n = 3).
The in vitro drug release profiles of 5-Fu loaded FA-CM-b-CD-BSA NPs and free 5-Fu in the medium were compared and results are shown in Fig. 5. The results showed
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Fig. 6. The results of cell viability assays. (A) Viability of SMMC-7221 cells after incubation with different amounts of naked FA-CM-b-CD-BSA NPs for 72 h (n = 3). (B) Viability of Hela cells after incubation with different amounts of naked FA-CM-b-CD-BSA NPs for 72 h (n = 3). (C) SMMC-7721 cell viability cultured with 5-Fu loaded NPs at various concentrations of 5-Fu in folate-free medium after 24 h. (D) Hela cell viability of 5-Fu loaded NPs at various concentrations of 5-Fu in folate-free medium after 24 h. Data were presented as mean S.D. (n = 3).
that the free drug as the control group was dissolved faster and about 98.7% was released into the medium within the first 6 h. Compared with free drugs, drugs encapsulated in FA-CM-b-CDBSA NPs exhibited a prolonged and slow release pattern in medium. The in vitro release data were fitted into release kinetics models to determine the best-fit release mode. The release kinetic data analysis and correlation coefficient (R2) are shown in Table 2. The relationship between cumulative release rate and time was tested using Zero-order, First-order, Higuchi and Ritger–Peppas models. In vitro drug release was best fitted to Ritger–Peppas model which showed the highest correlation coefficient (R2). The diffusional exponent (n) in Ritger–Peppas model was 0.3512, lower than 0.45, suggesting that drug release from 5-Fu loaded FA-CMb-CD-BSA NPs followed Fickian diffusing theory and was influenced by drug diffusion.
both cells incubated with FA-decorated NPs in vitro was significantly higher than undecorated one after 24 h. However, the 5-Fu loaded FA-CM-b-CD-BSA NPs could inhibit both cells viability from Fig. 6, we could find Hela cells which expressed higher FR showed more susceptible to 5-Fu loaded FA-CM-b-CDBSA NPs than SMMC-7721 cells. Briefly, FA improved the drug delivery and encapsulation by tumor cells which folate receptor was expressed high. Compared with low expression of FR at the surface of SMMC-7721 cells, more FRs were expressed in Hela cells, which led to the more uptake of FA-CM-b-CD-BSA NPs via acting endocytosis mediated by FA–FR interaction. Therefore, 5-Fu loaded FA-CM-b-CD-BSA NPs exhibited stronger cell inhibition activity on Hela cells than on SMMC7721 cells (Fig. 6C and D). 3.6. Cellular uptake of NPs and competition assay
3.5. Cell viability assays It can be seen from Fig. 6 that the inhibiting rates of blank FACM-b-CD-BSA NPs for tumor cells were very low and different amount of blank FA-CM-b-CD-BSA NPs showed no obvious cell inhibition within 72 h (Fig. 6A and B). When Hela and SMMC7721 cells were treated with CM-b-CD-BSA NPs and FA-CM-b-CDBSA NPs encapsulating the same amount of 5-Fu, the viability of
The cellular uptake of rhodamine B-labeled NPs in SMMC7721 and Hela cells was performed by confocal laser scanning microscopy, as shown in Fig. 7. When the cells were treated with CM-b-CD-BSA NPs, the red fluorescence intensity of the cells was weak indicating that most of nanoparticles were not taken up by cells, only a small portion of NPs was internalized into cells by endocytic process. On the contrary, rhodamine B-labeled
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Fig. 7. Confocal images of cells after incubating for 6 h with FA-CM-b-CD-BSA NPs and CM-b-CD-BSA NPs. Nucleus was stained with hochest (blue) for 15 min at 37 C and all of NPs are labeled by rhodamine B (red); (A) incubated with hela cells, (B) incubated with SMMC-7721 cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
FA-CM-b-CD-BSA NPs showed significant red fluorescence throughout the cytoplasm, which is closely located around the nuclei (blue). This observation indicated that FA-CM-b-CD-BSA NPs could effectively enhance drug accumulation in cancer cells, which may be attributed to specific binding between FA and folate
receptor on the surface of cancer cells. Hereby, FA-CM-b-CD-BSA NPs as an anticancer drug carrier will accumulate inside cells, improving the curing effects of anti-tumor drugs. The cellular distribution of CM-b-CD-BSA NPs and FA-CMb-CD-BSA NPs was quantified by micro-plate reader as shown in
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Fig. 8. Fluorescence spectrum analysis of the uptake of FA-CM-b-CD-BSA NPs labeled by FITC. (A) The uptake of FA-CM-b-CD-BSA NPs labeled by FITC incubated with SMMC7721 cells for 6 h in media containing different amount of free FA, n = 3, *P < 0.05, vs. the FA-CM-b-CD-BSA NPs group in media without folate, (B) incubated with Hela cells, n = 3, *P < 0.05, vs. the FA-CM-b-CD-BSA NPs group in media without folate.
Fig. 8. When FA-CM-b-CD-BSA NPs were incubated with two tumor cells for 6 h, The RFR observed in cells was increased gradually with the decreased amount of free FA in medium,
suggesting that free FA existing in the medium competed with FA-CM-b-CD-BSA NPs to bind the folate receptor on the cell surface, thus resulting in the saturation of receptors and
Fig. 9. Cell apoptosis determined by annexin V-FITC staining. Flow cytometer analysis of the apoptosis and necrosis cells after 48 h incubation with the free 5-Fu, 5-Fu loaded CM-b-CD-BSA NPs and 5-Fu loaded FA-CM-b-CD-BSA NPs respectively. Results were expressed as means S.D. (n = 3).
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FA-CM-b-CD-BSA NPs treated cells was markedly higher than other treatments. It proved that 5-Fu loaded NPs with the mediation of FA significantly enhanced 5-Fu-induced apoptosis. 3.8. Intracellular ATP level assay and western blot assay
Fig. 10. Intracellular ATP level assay after 48 h incubation with the free 5-Fu, 5-Fu loaded CM-b-CD-BSA NPs and 5-Fu loaded FA-CM-b-CD-BSA NPs respectively. Results were expressed as means S.D. (n = 3).
preventing the transportation of FA-CM-b-CD-BSA NPs into cells. In contrast, the addition of FA took no obvious effects on the internalization of CM-b-CD-BSA NPs. This suggested that FA-CM-b-CD-BSA NPs may be internalized via FA mediated uptake and the FA unconjugated NPs were probably internalized by different mechanisms. Taken together, more FA-CM-b-CD-BSA NPs were internalized in FR high expressed Hela cells than SMMC-7721 cells, which expressed lower FR and, the targeting effect is effective and the side effect will be decreased correspondently. 3.7. Cell apoptosis and necrosis In order to verify the cell apoptosis rate induced by the 5-Fu loaded NPs, annexin V-FITC/PI staining assay was performed and the apoptotic and necrotic cells were quantified by flow cytometry. The percentages of early apoptotic (Q4), late apoptotic (Q2), necrotic (Q1) and live cells (Q3) are shown in Fig. 9. Flow cytometry analysis revealed that after 48 h incubation with free 5-Fu, 5-Fu loaded CM-b-CD-BSA NPs and 5-Fu loaded FA-CM-b-CD-BSA NPs, the ratio of AV-positive and PI-positive cells was increased to 27.48%, 49.90% and 67.68%, respectively. It was worth to note that the ratio of AV-positive and PI-positive fractions in 5-Fu loaded
Apoptosis of cells usually depended on the supply of energy and required the participation of ATP (adenosine triphosphate). Decreased ATP level can not maintain basic metabolic functions of cells, and lead to cells’ apoptosis. A typical apoptotic cell was presented by significant decline of ATP level. Therefore, the change of intracellular ATP level was highly related with apoptosis, necrosis or toxicity in cells. We took ATP level of untreated Hela cells as the control group and the changing rates of intracellular ATP level were shown in Fig. 10. Compared with ATP level of the control group, after 48 h incubation with free 5-Fu, 5-Fu loaded CM-b-CD-BSA NPs and 5-Fu loaded FA-CM-b-CD-BSA NPs, The changing rates of intracellular ATP level were decreased to 88.28%, 74.58% and 55.12%, respectively. It was also suggested that more drug loaded FA-CM-b-CD-BSA NPs were internalized into cells and increased the intracellular drug concentration with the mediation of FA, thus leading to the significant decrease of intracellular ATP and accelerating the apoptosis of cells by accumulation of drugs in cells. In order to further explore drug loaded NPs-induced apoptosis of Hela cells, the expression of two apoptosis relevant proteins (Bax and cleaved caspase-3) was evaluated in Fig. 11. It is well known that caspase family plays a very important role in mediating apoptosis process of cells, wherein the caspase-3 as a key executing molecule mainly functions on the regulation of the apoptosis signal transduction pathway. In the early stages of apoptosis, caspase-3 was activated and corresponding cytoplasmic and nuclear substrates began to cleave, then resulting in apoptosis. It was observed that the expressions of cleaved caspase-3 protein in cells treated with the three 5-Fu containing formulations were obviously elevated. Especially, cleaved caspase-3 protein in cells treated with 5-Fu loaded FA-CM-b-CD-BSA NPs was expressed at the highest level, suggesting that the targeted delivery of 5-Fu loaded FA-CM-b-CD-BSA NPs significantly increased the intracellular drug concentration with the mediation of FA, thus accelerating the apoptosis of cells by accumulation of drugs in cells. On the contrast, the regulation of Bax in cells treated with three drug groups was not significantly different from control group. It was demonstrated that involvement of drug had little direct effect on the expression of Bax acting as a well-known pro-apoptotic protein. From all the above, to elucidate effect of FA-CM-b-CD-BSA NPs, we use 5-Fu to exam the drug delivery, and the cell viability and cell apoptosis analysis show the 5-Fu loaded FA-CM-b-CD-BSA NPs increased the cytotoxicity of folate receptor positive Hela cells. Furthermore, we detect the expression of ATP and cleaved capase3 to explain the apoptosis mechanism of FA-CM-b-CD-BSA NPs, and find FA-CM-b-CD-BSA NPs could inhibit energy production (ATP) and promote cleaved caspase-3 expression. So, FA-CM-b-CDBSA NPs provide a more effective way to assist drugs to target folate receptor positive tumor cells. Through this article, we can see 5-Fu loaded FA-CM-b-CD-BSA NPs could be uptaked by FR positive Hela cells, and facilitate 5-Fu chemotherapy and decrease chemoresistance with the interaction between FA and FR. 4. Conclusion
Fig. 11. Apoptotic effects of various 5-Fu formulations on Hela cells. Western blot analyses of the expression levels of Bax and cleaved caspase-3 proteins in Hela cells after treatments.
FA as a targeting ligand was successfully inserted into CM-b-CD-BSA NPs to fabricate folate decorated target nanoparticles based on the interaction between FA and FR for
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enhancing antitumor drug delivery. FA-CM-b-CD-BSA NPs obtained in our experiments were characterized of good monodispersity, negative charge and homogenous particle size, high encapsulating efficiency and 5-Fu could be released in a prolonged and slow pattern in medium in vitro. Fluorescent images and the free folic acid competition study proved that FA-CM-b-CD-BSA NPs could obviously enhance cellular uptake through folate mediated internalization in Hela cells, which FR is higher expressed in cell surface. MTT and cell apoptosis demonstrated that FA-decorated NPs exhibited stronger inhibition and induced obvious apoptosis in FR positive Hela cells as compared to free 5-Fu and FA undecorated NPs. This can be attributed to the possibility that FA-CM-b-CD-BSA NPs enhanced FR receptor-mediated endocytosis which led to more intracellular uptake of drugs, then inducing the apoptosis of cells by down-regulation of ATP level and over-expression of caspase-3. These properties suggest us that folate decorated targeted CM-b-CD-BSA NPs may become a promising active FR positive tumor-targeting carrier candidate and it may potentially find clinical usage after further modifications in future. Disclosure The authors report no conflicts of interest in this work. Acknowledgements This work is supported by Natural Science Foundation of Liaoning Province (No. 2013022035), National Natural Science Foundation of China (No. 81172048), and Grant of Liaoning Medical University (No. XZJJ20130104-05). We thank Prof. Yunhong Jia and Dr. Bo Zhang for polishing manuscript. References Aryal, S., Hu, C.M., Fang, R.H., Dehaini, D., Carpenter, C., Zhang, D.E., Zhang, L., 2013. Erythrocyte membrane-cloaked polymeric nanoparticles for controlled drug loading and release. Nanomedicine 8, 1271–1280. Cho, C.S., Kobayashi, A., Takei, R., Ishihara, T., Maruyama, A., Akaike, T., 2001. Receptor-mediated cell modulator delivery to hepatocyte using nanoparticles coated with carbohydrate-carrying polymers. Biomaterials 22, 45–51. Dorokhin, D., Hsu, S.H., Tomczak, N., Reinhoudt, D.N., Huskens, J., Velders, A.H., Vancso, G.J., 2010. Fabrication and luminescence of designer surface patterns with beta-cyclodextrin functionalized quantum dots via multivalent supramolecular coupling. ACS Nano 4, 137–142. Feng, D., Song, Y., Shi, W., Li, X., Ma, H., 2013. Distinguishing folate-receptor-positive cells from folate-receptor-negative cells using a fluorescence off–on nanoprobe. Anal. Chem. 85, 6530–6535. Gref, R., Couvreur, P., Barratt, G., Mysiakine, E., 2003. Surface-engineered nanoparticles for multiple ligand coupling. Biomaterials 24, 4529–4537. He, Q., Wu, W., Xiu, K., Zhang, Q., Xu, F., Li, J., 2013. Controlled drug release system based on cyclodextrin-conjugated poly(lactic acid)-b-poly(ethylene glycol) micelles. Int. J. Pharm. 443, 110–119. Hou, Z., Zhan, C., Jiang, Q., Hu, Q., Li, L., Chang, D., Yang, X., Wang, Y., Li, Y., Ye, S., Xie, L., Yi, Y., Zhang, Q., 2011. Both FA-and mPEG-conjugated chitosan nanoparticles for targeted cellular uptake and enhanced tumor tissue distribution. Nanoscale Res. Lett. 6, 563. Huang, S., Shao, K., Kuang, Y., Liu, Y., Li, J., An, S., Guo, Y., Ma, H., He, X., Jiang, C., 2013. Tumor targeting and microenvironment-responsive nanoparticles for gene delivery. Biomaterials 34, 5294–5302. Hyung Park, J., Kwon, S., Lee, M., Chung, H., Kim, J.H., Kim, Y.S., Park, R.W., Kim, I.S., Bong Seo, S., Kwon, I.C., Young Jeong, S., 2006. Self-assembled nanoparticles based on glycol chitosan bearing hydrophobic moieties as carriers for doxorubicin: in vivo biodistribution and anti-tumor activity. Biomaterials 27, 119–126.
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