http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(8): 978–982 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.874533
ORIGINAL ARTICLE
Folate-modified doxorubicin-loaded nanoparticles for tumor-targeted therapy Guicun Wu, Zuozhi Wang, Xiaoshan Bian, Xiaojie Du, and Changhong Wei
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Department of Oncology, Shandong Provincial Crops Hospital, Chinese People’s Armed Forces, Jinan, China
Abstract
Keywords
Context: Polymeric nanoparticles (NPs) have been used frequently as drug delivery vehicles. Surface modification of polymeric NPs with specific ligands defines a new biological identity, which assists in targeting of the nanocarriers to specific cancers cells. Objective: The aim of this study is to develop a kind of modified vector which could target the cancer cells through receptor-mediated pathways to increase the uptake of doxorubicin (DOX). Methods: Folate (FA)-conjugated PEG–PE (FA–PEG–PE) ligands were used to modify the polymeric NPs. The modification rate was optimized and the physical–chemical characteristics, in vitro release, and cytotoxicity of the vehicle were evaluated. The in vivo therapeutic effect of the vectors was evaluated in human nasopharyngeal carcinoma KB cells baring mice by giving each mouse 100 ml of 10 mg/kg different solutions. Results: FA–PEG–PE-modified NPs/DOX (FA-NPs/DOX) have a particle size of 229 nm, and 86% of drug loading quantity. FA-NPs/DOX displayed remarkably higher cytotoxicity (812 mm3 tumor volume after 13 d of injection) than non-modified NPs/DOX (1290 mm3) and free DOX solution (1832 mm3) in vivo. Conclusion: The results demonstrate that the modified drug delivery system (DDS) could function comprehensively to improve the efficacy of cancer therapy. Consequently, the system was shown to be a promising carrier for delivery of DOX, leading to the efficiency of antitumor therapy.
Active targeted, cancer therapy, drug delivery system, folate–PEG–PE
Introduction The tumor-targeting ability of anticancer drugs is relying on the efficient targeted drug delivery systems (DDSs) (Watanabe et al., 2012). Non-viral DDS such as polymeric nanoparticles (Fields et al., 2012), liposomes (Kong et al., 2012), and other carriers (Brannon-Peppas & Blanchette, 2004; Danhier et al., 2010) have been widely developed as they have advantages such as less toxic, low immunogenic, and easily to be modified. Folate (FA) is used as a tumor-targeting ligand, which leads nanoparticles into cancer cells as a result of FA-receptor (FR)-mediated endocytosis, as FRs are over expressed in many human cancer cells, while they show limited expression in normal cells (Elnakat & Ratnam, 2004; Parker et al., 2005; Wu et al., 1999). FA-targeted drug delivery vectors showed enhanced accumulation in folate receptor expressing tumor model (Morris et al., 2010). FA-mediated transfection has been shown to facilitate particles internalization into cells through membrane receptors in vivo. This could improve the
History Received 14 September 2013 Revised 18 November 2013 Accepted 9 December 2013 Published online 9 July 2014
internalization of the vectors hence improving transfection efficiency (Yamada et al., 2008). PEG–phosphatidylethanolamine (PEG–PE) conjugates with various PEG lengths and terminal-targeted moieties can provide extremely stable, long-circulating, and actively targeted nanocarriers which spontaneously accumulate at specific sites (Lukyanov et al., 2002; Torchilin, 2005). These kinds of ligands were also used previously by our groups for surface modification of vehicles to achieve the targeted drug delivery (Jiang et al., 2012; Wang et al., 2012b; Wu et al., 2012). In this study, FA was linked to PEG–PE to form FA–PEG–PE as ligands for the surface modification of nanocarriers. In the present study, a FA-conjugated lipid, FA–PEG–PE, was synthesized and modified on the surface of DOX-loaded bioadhesive PLGA nanoparticles to form NPs/DOX. The in vivo effects were observed on mice-bearing KB human carcinoma cells (KB cells) model. Unmodified NPs (blank NPs), modified NPs not containing DOX (FA-NPs), and unmodified NPs/DOX are applied as controls.
Materials and methods Correspondence: Changhong Wei, Department of Oncology, Shandong Provincial Crops Hospital, Chinese People’s Armed Forces, 12-8 Jiang Shui Quan Road, Jinan 250031, People’s Republic of China. Tel: +86 531 81180828. E-mail: [email protected]
Materials Doxorubicin hydrochloride (DOXHCl), folic acid, and (3-[4,5-dimehyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium
Modified nanoparticles for cancer therapy
DOI: 10.3109/13880209.2013.874533
bromide (MTT) were purchased from Sigma-Aldrich Co., Ltd (St Louis, MO). Poly(D,L-lactic-co-glycolic) (PLGA, 50:50, avg. MW 25 000) was obtained from Shandong Institute of Medical Instrument (Shandong, China). Maleimide–PEG2000–COOH was purchased from Shanghai Yare Biotech Inc. (Shanghai, China). KB cells were obtained from the American Type Culture Collection (Manassas, VA). All other chemicals were of analytical grade or higher. Animals
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BALB/c mice (6-week-old, 20–25 g weight) were housed under standard laboratory conditions. All animal experiments complied with the requirements of the National Act on the Use of Experimental Animals (People’s Republic of China). Synthesis of FA–PEG–PE Maleimide–PEG2000–COOH (100 mg) was dissolved with dimethyl sulfoxide (DMSO) and stirred with PE (50 mg) as a mixture. 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide (EDCHCl) (80 mg) and triethylamine (TEA, 1 equivalent of EDCHCl) were dissolved in DMSO and added dropwise into the mixture in an ice bath, stirred for 12 h to produce maleimide–PEG–CO–NH–PE. The FA was then added to the maleimide–PEG2000–COOH solution and the whole solution was incubated for 1 h at room temperature with gentle stirring to get FA–PEG–PE ligands. The product was dialyzed against Milli-Q water (Millipore, Billerica, MA) for 18 h to form FA–PEG–PE solution. The mixture was centrifuged at 2000 g for 10 min at 4 C, and then resuspended in a phosphatebuffered saline (PBS) (pH 7.4). Preparation of NPs/DOX NPs/DOX complexes were prepared following the methods described previously by our group (solvent displacement technique) (Wu et al., 2012) (Figure 1). Briefly, Carbopol 940 (CP) (Guangzhou Yiming Chemical Materials Co., Ltd., Guangdong, China) was dispersed in distilled water at room temperature and left overnight to swell. The required amount of 1 M NaOH was added to neutralize the dispersion until pH 7.0 was reached and diluted with distilled water to afford a 0.02% (w/v) CP solution. PLGA polymer (50 mg) was accurately weighted and dissolved in 3 ml acetone. DOXHCl (200 mg) was weighted and dissolved in PBS. The organic phase and DOX containing PBS solution were added at the same time, dropwise into the 0.02% CP solution being stirred at 600 rpm at room temperature. When complete evaporation of the organic solvent had occurred, the redundant stabilizers and the nanoparticles were separated by
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ultracentrifugation at 100 g, 4 C for 10 min. The pellet was resuspended in Milli-Q water, washed three times, and filtered through a 0.45 mm membrane. Modification of NPs/DOX with FA–PEG–PE FA–PEG–PE ligands were dissolved in 5 ml of PBS (pH 7.4). Then the solution was added dropwise into 20 ml of NPs/DOX complexes that was stirred at 600 rpm at RT leading to the immediate modification. The obtained complexes was resuspended in Milli-Q water, washed three times, and filtered through a membrane with 0.45 mm pore size to obtain FA-NPs/ DOX (Figure 1). During this procedure, FA–PEG–PE ligands were coated onto the surface of the NPs/DOX continuously which would cause the decrease of zeta potential. To optimize the modification ratio, FA–PEG–PE ligands dissolved in PBS were designed at different weight ratios to the NPs/DOX (w/w) and the zeta potential of complexes was determined. NPs not containing DOX were also modified as the above mentioned method to form modified blank NPs (FA-NPs). Characterization of FA-NPs/DOX Physical–chemical characteristic The mean particle size, polydispersity index (PDI), and zeta potential of NPs, NPs/DOX, and FA-NPs/DOX were analyzed by photon correlation spectroscopy (PCS) with a Zetasizer 3000 (Malvern Instruments, Malvern, England, UK). The average particle size was expressed as volume mean diameter. Drug loading ability encapsulation efficiency assay The drug loading (DL) and encapsulation efficiency (EE) of the FA-NPs/DOX were determined by a subtraction method. Briefly, 0.2 ml of NPs/DOX complexes solution was centrifuged through a filter (EMD Millipore, Billerica, MA) with molecular weight cutoff of 3 kDa. Free DOX could pass through the filter, but FA-NPs/DOX could not pass through the filter. Unincorporated DOX in the solution was quantified by determining the absorbance at 485 nm using a spectrophotometer (Gao et al., 2013). DL and EE were calculated using the following equations: DL ¼ ðconcentration of ½total DOX free DOXÞ ðconcentration of½polymer þ total DOX 1
free DOXÞ
100%
EE ¼ concentration of ðtotal DOX free DOXÞ concentration of total DOX1 100%
Figure 1. Preparation and modification of NPs/DOX complexes.
ð1Þ
ð2Þ
G. Wu et al.
Pharm Biol, 2014; 52(8): 978–982
In vitro release of FA-NPs/DOX
Statistical analysis
The in vitro release study of FA-NPs/DOX was performed in a modified dialysis method (Men et al., 2012; Wang et al., 2012a). Briefly, 0.5 ml FA-NPs/DOX solution was transferred into a dialysis membrane (MWCO 3000), and 0.5 ml of free DOX solution in water (0.25 mg/ml) was used as the control. Then solutions were dialyzed against 20 ml acetic acid sodium buffer at pH 5.5 and PBS, both containing Tween 20 (0.5%), at 37 C with gentle shaking. A total of 20 ml of the surrounding dialysis medium was removed at predetermined time points for analysis, and 20 ml of fresh buffer at the relevant pH was added to the dialysis medium. The released DOX from FA-NPs/DOX was able to infiltrate through the dialysis bag because the molecular weight of the DOX was less than 3000. The released DOX was quantified by determining absorbance at 485 nm using the spectrophotometer.
All studies were repeated three times and all measurements were carried out in triplicate. Results were reported as means ± SD (SD ¼ standard deviation). Statistical significance was analyzed using Student’s t-test. Differences between experimental groups were considered significant when the p value was less than 0.05 (p50.05).
In vitro cytotoxicity evaluation To examine the cytotoxicity, KB cells were seeded in 48 well plates at 1 104 cells/well and incubated for 24 h to allow cell attachment. The cells were then exposed to a series of FA-NPs and NPs solutions at different concentrations for 48 h. The cell viability was then assessed by MTT assay. About 5 mg/ml of MTT in PBS was then added to each well and the plate was incubated for an additional 4 h at 37 C under the aforementioned 5% CO2 atmosphere. Then the MTT containing medium was removed, and the crystals formed by living cells were dissolved in 100 ml DMSO. Cells without treatment were used as a control. The absorbance at 570 nm of the solution in each well was recorded using a Microplate Reader (Model 680, BIO-RAD, Hercules, CA). The relative cell viability (%) was calculated as (AbssampleAbsblank)/ (AbscontrolAbsblank) 100. In vivo antitumor effect Tumor-bearing mice were prepared by inoculating (s.c.) a suspension of KB cells (1106 cells) into the right armpit of BALB/c mice (Li et al., 2012; Liu et al., 2011). Briefly, the mice were acclimatized at a temperature of 25 ± 2 C and a relative humidity of 70 ± 5% under natural light/dark conditions for 1 week before dosing. Then, the mice were injected subcutaneously in the right armpit with KB cells suspended in PBS. Tumors were permitted to reach 4–5 mm diameter before initiation of the studies. In vivo anticancer activity of FA-NPs/DOX was evaluated against KB solid tumors in mice. Five groups of tumorbearing mice (six per group) were used. The mice were injected with 10 mg/kg of (1) FA-NPs/DOX, (2) NPs/DOX, (3) NPs, (4) free DOX solution, and (5) 0.9% sodium chloride solution (blank control). All drugs were diluted with 0.9% sodium chloride (100 ml), and all were administered through direct intratumoral injection. After drug administration, the mortality was monitored daily and the tumor growth was determined by caliper measurement every 3 d. Tumor volume was calculated as follows (Jia et al., 2012) Tumor volume mm3 ¼ length width2 =2 ð3Þ
Results Optimization of the modification ratio FA–PEG–PE ligands were continuously coated onto the surface of the NPs/DOX. This procedure would mask the cationic surface charge NPs/DOX complexes and cause the decrease of zeta potential. The best ratio of the ligands to the carriers was optimized by measuring the change of zeta potential. As illustrated in Figure 2, the optimized ratio of the FA–PEG–PE to NPs/DOX was 35%. This ratio was determined and used for further experiments. Characterization of FA-NPs/DOX Size, zeta potential, DL, and EE Mean particle size, polydispersity index (PDI), zeta potential, DL, and EE of NPs, NPs/DOX, and FA-NPs/DOX were characterized and summarized in Table 1. In vitro release study The in vitro release profiles of FA-NPs/DOX and unmodified NPs/DOX are illustrated in Figure 3. Both the FA-NPs/DOX and unmodified NPs/DOX reached over 80% drug release at the time point of 72 h. 45 Zeta potential (mV)
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40 35 30 25 20 15 0%
5%
10% 15% 20% 25% 30% 35% 40% 45% 50% 55% Ratios of FA-PEG-PE to NPs/DOX
Figure 2. Optimization of the modification ratios of FA–PEG–PE to NPs/DOX.
Table 1. Particle size, zeta potential, and gene loading quantity of different vectors. Characteristics
Samples
Mean particle size (nm)
NPs 125.6 ± 3.2 NPs/DOX 159.3 ± 4.8 FA-NPs/ 229.2 ± 3.1 DOX
Zeta Polydispersity potential (mV) index (PDI) 0.12 ± 0.02 0.15 ± 0.03 0.16 ± 0.02
DL (%)
EE (%)
41.8 ± 2.8 – – 32.1 ± 2.3 9.8 ± 0.9 86.2 ± 1.3 21.8 ± 3.1 9.5 ± 0.8 85.6 ± 1.6
Modified nanoparticles for cancer therapy
DOI: 10.3109/13880209.2013.874533
90
were between 80% and 100% compared with controls (Figure 4).
NPs/DOX 80
FA-NPs/DOX
In vivo drug delivery and antitumor therapy
Cumulative released drug (%)
70
In vivo anticancer activity of FA-NPs/DOX was evaluated against KB solid tumors in mice. The in vivo antitumor efficiency of FA-NPs/DOX, NPs/DOX, NPs, and free DOX was observed. The tumor growth curves of each group are presented in Figure 5. Results indicate that NPs/DOX treatment resulted in smaller tumor volume compared with free DOX; FA-NPs/DOX treatment resulted in smaller tumor volume compared with all the others. The results illustrate that (1) encapsulation of DOX in NPs enhanced the anticancer activity of DOX in vivo; and (2) FA-modified NPs/DOX gain better antitumor therapeutic effect than unmodified ones.
60 50 40 30 20 10 0 0
12
24
36
48
60
72
84
96
108 120
Time (h)
Discussion
Figure 3. The in vitro release profiles of FA-NPs/DOX and NPs/DOX.
NPs FA-NPs
Relative cell viability (%)
100 80 60 40 20 0 10
20
50
100
200
400
600
800
Concentrations (µg/ml)
Figure 4. In vitro cytotoxicity evaluation of FA-NPs and NPs. 3000
FA-NPs/DOX NPs/DOX NPs DOX CONTROL
2500 Mean tumor volume (mm3)
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2000
1500
1000
500
0 0
2
4
6
8
10
12
14
Time (days)
Figure 5. The tumor growth curves of FA-NPs/DOX, NPs/DOX, NPs, and free DOX.
In vitro cytotoxicity evaluation In vitro cytotoxicity of FA-NPs and NPs was evaluated in KB cells at different concentrations. The cell viabilities of the vectors over the studied concentration range (10–800 mg/ml)
The intratumoral administration of anticancer drugs represents a growing trend for maximizing local tumor control with minimal systemic toxicity. However, it requires suitable DDS for treatment efficacy (Jia et al., 2012). In the present study, a biodegradable nanocarrier has been developed to reduce or minimize undesired interactions or undesired uptake into normal sites (Akbarzadeh et al., 2012). Moreover, an active targeting ligand was synthesized and modified onto the surface of DOX-loaded NPs to achieve the targeted effect on tumor cells. FA is an essential B vitamin, which could internalize into the cells via a low-affinity reduced folate carrier protein or via high-affinity folate receptors. The best studied of these receptors is FR, a cell surface glycosyl phosphatidylinositolanchored glycoprotein that can internalize bound FA and FA-conjugated compounds via receptor-mediated endocytosis (Paulos et al., 2004). So FA was used as the target moiety which could bind to the FR on the KB cells. A series of PEG-containing ligands, commonly named PEG–PE conjugates, were reported and used for the modification of various vehicles to achieve active-targeting nanocarriers that spontaneously accumulate in specific sites (Lukyanov et al., 2002; Torchilin, 2005). Thus, PEG–PE was used at the anchor and FA–PEG–PE was applied as the target ligand of the system. During the modification procedure, FA–PEG–PE ligands were extensively coated onto the NPs/DOX carriers due to the electric charge and lipophilic interaction, which covered their original surface charge and caused the decrease in zeta potential. The optimization of ligand-to-carrier ratio was carried out by measuring the zeta potential. As shown in Figure 2 and Table 1, the optimum ratio was obtained at 35% (FA–PEG–PE to NPs/DOX, w/w) and the optimum FA-NPs/ DOX had a size of 229 nm and zeta potential of +22 mV. After the optimization of the modification procedure, the FA–PEG–PE to NPs/DOX ratio was determined as 35% and used for further experiments. The DL and EE of the FA-NPs/ DOX and other vectors were determined by a subtraction method. As shown in Table 1, the DL of FA-NPs/DOX and NPs/DOX is around 10% and the EE of both vectors is around
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85%. The results demonstrated that binding of FA–PEG–PE ligand did not detach the DOX from the complexes and the modified vectors are stable. The in vitro release profile (Figure 3) of FA-NPs/DOX showed slightly slower release than NPs/DOX during the first 24 h. This might be due to the hindrance caused by coating of ligand to the release of drugs initially. As time passes, more FA–PEG–PE detached from the vectors, allowing the release of drugs more and more freely. At the end of the release study, the total amount of drugs delivered from the two kinds of vehicles was nearly the same (over 80%). In vitro cytotoxicity analysis was carried out in KB cells. The cell viabilities of FA-NPs/DOX and NPs/DOX over the studied concentration range were between 80% and 100% compared with controls (Figure 4). FA-NPs/DOX showed no higher cytotoxicity than NPs/DOX at all concentrations. In vivo anticancer activity of FA-NPs/DOX and other vectors was evaluated against KB solid tumors in mice. The mice were injected with 10 mg/kg of FA-NPs/DOX, NPs/DOX, NPs, and DOX solution to the tumor site; 0.9% sodium chloride solution was used as a blank control. The tumor growth rate was not found to significantly decrease with free DOX treatment and NPs (the same behavior with 0.9% sodium chloride solution); however, the tumor growth rate was significantly decreased in the group treated with NPs/DOX. Furthermore, FA-NPs/DOX showed higher antitumor effect than free NPs/DOX in vivo. These results indicate that FA-modified DOX-loaded NPs have promising application in antitumor drug delivery and would be very useful in anticancer cancer therapy.
Conclusions This study showed that a targeting ligand (FA–PEG–PE)modified DOX-loaded bioadhesive PLGA nanoparticles can significantly improve the therapeutic efficiency of the vector, and successfully control the tumor growth rate on tumorbearing mice. The conclusion should be that FA could function as an excellent active targeting ligand to improve the cell targeting of the carriers, and the modified DDS could function comprehensively to improve the efficacy of cancer therapy. The system was shown to be an excellent carrier for delivery of DOX, leading to the efficiency of antitumor therapy.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
References Akbarzadeh A, Mikaeili H, Zarghami N, et al. (2012). Preparation and in vitro evaluation of doxorubicin-loaded Fe3O4 magnetic nanoparticles modified with biocompatible copolymers. Int J Nanomed 7: 511–26.
Pharm Biol, 2014; 52(8): 978–982
Brannon-Peppas L, Blanchette JO. (2004). Nanoparticle and targeted systems for cancer therapy. Adv Drug Deliv Rev 56:1649–59. Danhier F, Feron O, Pre´at V. (2010). To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anticancer drug delivery. J Control Release 148:135–46. Elnakat H, Ratnam M. (2004). Distribution, functionality and gene regulation of folate receptor isoforms: Implications in targeted therapy. Adv Drug Deliv Rev 56:1067–84. Fields RJ, Cheng CJ, Quijano E, et al. (2012). Surface modified poly(b amino ester)-containing nanoparticles for plasmid DNA delivery. J Control Release 164:41–8. Gao X, Wang B, Wei X, et al. (2013). Preparation, characterization and application of star-shaped PCL/PEG micelles for the delivery of doxorubicin in the treatment of colon cancer. Int J Nanomed 8: 971–82. Jia Y, Yuan M, Yuan H, et al. (2012). Co-encapsulation of magnetic Fe3O4 nanoparticles and doxorubicin into biodegradable PLGA nanocarriers for intratumoral drug delivery. Int J Nanomed 7: 1697–708. Jiang Z, Sun C, Yin Z, et al. (2012). Comparison of two kinds of nanomedicine for targeted gene therapy: Premodified or postmodified gene delivery systems. Int J Nanomed 7:2019–31. Kong F, Zhou F, Ge L, et al. (2012). Mannosylated liposomes for targeted gene delivery. Int J Nanomed 7:1079–89. Li P, Liu D, Miao L, et al. (2012). A pH-sensitive multifunctional gene carrier assembled via layer-by-layer technique for efficient gene delivery. Int J Nanomed 7:925–39. Liu C, Yu W, Chen Z, et al. (2011). Enhanced gene transfection efficiency in CD13-positive vascular endothelial cells with targeted poly(lactic acid)-poly(ethylene glycol) nanoparticles through caveolae-mediated endocytosis. J Control Release 151:162–75. Lukyanov AN, Gao Z, Mazzola L, Torchilin VP. (2002). Polyethylene glycol-dia-cyllipid micelles demonstrate increased accumulation in subcutaneous tumors in mice. Pharm Res 19:1424–9. Men K, Liu W, Li L, et al. (2012). Delivering instilled hydrophobic drug to the bladder by a cationic nanoparticle and thermo-sensitive hydrogel composite system. Nanoscale 4:6425–33. Morris VB, Sharma CP. (2010). Folate mediated histidine derivative of quaternised chitosan as a gene delivery vector. Int J Pharm 389: 176–85. Parker N, Turk MJ, Westrick E, et al. (2005). Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Anal Biochem 338:284–93. Paulos CM, Reddy JA, Leamon CP, et al. (2004). Ligand binding and kinetics of folate receptor recycling in vivo: Impact on receptormediated drug delivery. Mol Pharmacol 66:1406–14. Torchilin VP. (2005). Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov 4:145–60. Watanabe K, Kaneko M, Maitani Y. (2012). Functional coating of liposomes using a folate-polymer conjugate to target folate receptors. Int J Nanomed 7:3679–88. Wang BL, Gao X, Men K, et al. (2012a). Treating acute cystitis with biodegradable micelle-encapsulated quercetin. Int J Nanomed 7: 2239–47. Wang W, Zhou F, Ge L, et al. (2012b). Transferrin–PEG–PE modified dexamethasone conjugated cationic lipid carrier mediated gene delivery system for tumor-targeted transfection. Int J Nanomed 7: 2513–22. Wu G, Zhou F, Ge L, et al. (2012). Novel mannan–PEG–PE modified bioadhesive PLGA nanoparticles for targeted gene delivery. J Nanomater 2012:981670. Wu M, Gunning W, Ratnam M. (1999). Expression of folate receptor type alpha in relation to cell type, malignancy, and differentiation in ovary, uterus, and cervix. Cancer Epidemiol Biomarkers Prev 8: 775–82. Yamada A, Taniguchi Y, Kawano K, et al. (2008). Design of folatelinked liposomal doxorubicin to its antitumor effect in mice. Clin Cancer Res 14:8161–8.
ORIGINAL ARTICLE
Folate-modified doxorubicin-loaded nanoparticles for tumor-targeted therapy Guicun Wu, Zuozhi Wang, Xiaoshan Bian, Xiaojie Du, and Changhong Wei
Pharmaceutical Biology Downloaded from informahealthcare.com by McMaster University on 02/06/15 For personal use only.
Department of Oncology, Shandong Provincial Crops Hospital, Chinese People’s Armed Forces, Jinan, China
Abstract
Keywords
Context: Polymeric nanoparticles (NPs) have been used frequently as drug delivery vehicles. Surface modification of polymeric NPs with specific ligands defines a new biological identity, which assists in targeting of the nanocarriers to specific cancers cells. Objective: The aim of this study is to develop a kind of modified vector which could target the cancer cells through receptor-mediated pathways to increase the uptake of doxorubicin (DOX). Methods: Folate (FA)-conjugated PEG–PE (FA–PEG–PE) ligands were used to modify the polymeric NPs. The modification rate was optimized and the physical–chemical characteristics, in vitro release, and cytotoxicity of the vehicle were evaluated. The in vivo therapeutic effect of the vectors was evaluated in human nasopharyngeal carcinoma KB cells baring mice by giving each mouse 100 ml of 10 mg/kg different solutions. Results: FA–PEG–PE-modified NPs/DOX (FA-NPs/DOX) have a particle size of 229 nm, and 86% of drug loading quantity. FA-NPs/DOX displayed remarkably higher cytotoxicity (812 mm3 tumor volume after 13 d of injection) than non-modified NPs/DOX (1290 mm3) and free DOX solution (1832 mm3) in vivo. Conclusion: The results demonstrate that the modified drug delivery system (DDS) could function comprehensively to improve the efficacy of cancer therapy. Consequently, the system was shown to be a promising carrier for delivery of DOX, leading to the efficiency of antitumor therapy.
Active targeted, cancer therapy, drug delivery system, folate–PEG–PE
Introduction The tumor-targeting ability of anticancer drugs is relying on the efficient targeted drug delivery systems (DDSs) (Watanabe et al., 2012). Non-viral DDS such as polymeric nanoparticles (Fields et al., 2012), liposomes (Kong et al., 2012), and other carriers (Brannon-Peppas & Blanchette, 2004; Danhier et al., 2010) have been widely developed as they have advantages such as less toxic, low immunogenic, and easily to be modified. Folate (FA) is used as a tumor-targeting ligand, which leads nanoparticles into cancer cells as a result of FA-receptor (FR)-mediated endocytosis, as FRs are over expressed in many human cancer cells, while they show limited expression in normal cells (Elnakat & Ratnam, 2004; Parker et al., 2005; Wu et al., 1999). FA-targeted drug delivery vectors showed enhanced accumulation in folate receptor expressing tumor model (Morris et al., 2010). FA-mediated transfection has been shown to facilitate particles internalization into cells through membrane receptors in vivo. This could improve the
History Received 14 September 2013 Revised 18 November 2013 Accepted 9 December 2013 Published online 9 July 2014
internalization of the vectors hence improving transfection efficiency (Yamada et al., 2008). PEG–phosphatidylethanolamine (PEG–PE) conjugates with various PEG lengths and terminal-targeted moieties can provide extremely stable, long-circulating, and actively targeted nanocarriers which spontaneously accumulate at specific sites (Lukyanov et al., 2002; Torchilin, 2005). These kinds of ligands were also used previously by our groups for surface modification of vehicles to achieve the targeted drug delivery (Jiang et al., 2012; Wang et al., 2012b; Wu et al., 2012). In this study, FA was linked to PEG–PE to form FA–PEG–PE as ligands for the surface modification of nanocarriers. In the present study, a FA-conjugated lipid, FA–PEG–PE, was synthesized and modified on the surface of DOX-loaded bioadhesive PLGA nanoparticles to form NPs/DOX. The in vivo effects were observed on mice-bearing KB human carcinoma cells (KB cells) model. Unmodified NPs (blank NPs), modified NPs not containing DOX (FA-NPs), and unmodified NPs/DOX are applied as controls.
Materials and methods Correspondence: Changhong Wei, Department of Oncology, Shandong Provincial Crops Hospital, Chinese People’s Armed Forces, 12-8 Jiang Shui Quan Road, Jinan 250031, People’s Republic of China. Tel: +86 531 81180828. E-mail: [email protected]
Materials Doxorubicin hydrochloride (DOXHCl), folic acid, and (3-[4,5-dimehyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium
Modified nanoparticles for cancer therapy
DOI: 10.3109/13880209.2013.874533
bromide (MTT) were purchased from Sigma-Aldrich Co., Ltd (St Louis, MO). Poly(D,L-lactic-co-glycolic) (PLGA, 50:50, avg. MW 25 000) was obtained from Shandong Institute of Medical Instrument (Shandong, China). Maleimide–PEG2000–COOH was purchased from Shanghai Yare Biotech Inc. (Shanghai, China). KB cells were obtained from the American Type Culture Collection (Manassas, VA). All other chemicals were of analytical grade or higher. Animals
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BALB/c mice (6-week-old, 20–25 g weight) were housed under standard laboratory conditions. All animal experiments complied with the requirements of the National Act on the Use of Experimental Animals (People’s Republic of China). Synthesis of FA–PEG–PE Maleimide–PEG2000–COOH (100 mg) was dissolved with dimethyl sulfoxide (DMSO) and stirred with PE (50 mg) as a mixture. 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide (EDCHCl) (80 mg) and triethylamine (TEA, 1 equivalent of EDCHCl) were dissolved in DMSO and added dropwise into the mixture in an ice bath, stirred for 12 h to produce maleimide–PEG–CO–NH–PE. The FA was then added to the maleimide–PEG2000–COOH solution and the whole solution was incubated for 1 h at room temperature with gentle stirring to get FA–PEG–PE ligands. The product was dialyzed against Milli-Q water (Millipore, Billerica, MA) for 18 h to form FA–PEG–PE solution. The mixture was centrifuged at 2000 g for 10 min at 4 C, and then resuspended in a phosphatebuffered saline (PBS) (pH 7.4). Preparation of NPs/DOX NPs/DOX complexes were prepared following the methods described previously by our group (solvent displacement technique) (Wu et al., 2012) (Figure 1). Briefly, Carbopol 940 (CP) (Guangzhou Yiming Chemical Materials Co., Ltd., Guangdong, China) was dispersed in distilled water at room temperature and left overnight to swell. The required amount of 1 M NaOH was added to neutralize the dispersion until pH 7.0 was reached and diluted with distilled water to afford a 0.02% (w/v) CP solution. PLGA polymer (50 mg) was accurately weighted and dissolved in 3 ml acetone. DOXHCl (200 mg) was weighted and dissolved in PBS. The organic phase and DOX containing PBS solution were added at the same time, dropwise into the 0.02% CP solution being stirred at 600 rpm at room temperature. When complete evaporation of the organic solvent had occurred, the redundant stabilizers and the nanoparticles were separated by
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ultracentrifugation at 100 g, 4 C for 10 min. The pellet was resuspended in Milli-Q water, washed three times, and filtered through a 0.45 mm membrane. Modification of NPs/DOX with FA–PEG–PE FA–PEG–PE ligands were dissolved in 5 ml of PBS (pH 7.4). Then the solution was added dropwise into 20 ml of NPs/DOX complexes that was stirred at 600 rpm at RT leading to the immediate modification. The obtained complexes was resuspended in Milli-Q water, washed three times, and filtered through a membrane with 0.45 mm pore size to obtain FA-NPs/ DOX (Figure 1). During this procedure, FA–PEG–PE ligands were coated onto the surface of the NPs/DOX continuously which would cause the decrease of zeta potential. To optimize the modification ratio, FA–PEG–PE ligands dissolved in PBS were designed at different weight ratios to the NPs/DOX (w/w) and the zeta potential of complexes was determined. NPs not containing DOX were also modified as the above mentioned method to form modified blank NPs (FA-NPs). Characterization of FA-NPs/DOX Physical–chemical characteristic The mean particle size, polydispersity index (PDI), and zeta potential of NPs, NPs/DOX, and FA-NPs/DOX were analyzed by photon correlation spectroscopy (PCS) with a Zetasizer 3000 (Malvern Instruments, Malvern, England, UK). The average particle size was expressed as volume mean diameter. Drug loading ability encapsulation efficiency assay The drug loading (DL) and encapsulation efficiency (EE) of the FA-NPs/DOX were determined by a subtraction method. Briefly, 0.2 ml of NPs/DOX complexes solution was centrifuged through a filter (EMD Millipore, Billerica, MA) with molecular weight cutoff of 3 kDa. Free DOX could pass through the filter, but FA-NPs/DOX could not pass through the filter. Unincorporated DOX in the solution was quantified by determining the absorbance at 485 nm using a spectrophotometer (Gao et al., 2013). DL and EE were calculated using the following equations: DL ¼ ðconcentration of ½total DOX free DOXÞ ðconcentration of½polymer þ total DOX 1
free DOXÞ
100%
EE ¼ concentration of ðtotal DOX free DOXÞ concentration of total DOX1 100%
Figure 1. Preparation and modification of NPs/DOX complexes.
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In vitro release of FA-NPs/DOX
Statistical analysis
The in vitro release study of FA-NPs/DOX was performed in a modified dialysis method (Men et al., 2012; Wang et al., 2012a). Briefly, 0.5 ml FA-NPs/DOX solution was transferred into a dialysis membrane (MWCO 3000), and 0.5 ml of free DOX solution in water (0.25 mg/ml) was used as the control. Then solutions were dialyzed against 20 ml acetic acid sodium buffer at pH 5.5 and PBS, both containing Tween 20 (0.5%), at 37 C with gentle shaking. A total of 20 ml of the surrounding dialysis medium was removed at predetermined time points for analysis, and 20 ml of fresh buffer at the relevant pH was added to the dialysis medium. The released DOX from FA-NPs/DOX was able to infiltrate through the dialysis bag because the molecular weight of the DOX was less than 3000. The released DOX was quantified by determining absorbance at 485 nm using the spectrophotometer.
All studies were repeated three times and all measurements were carried out in triplicate. Results were reported as means ± SD (SD ¼ standard deviation). Statistical significance was analyzed using Student’s t-test. Differences between experimental groups were considered significant when the p value was less than 0.05 (p50.05).
In vitro cytotoxicity evaluation To examine the cytotoxicity, KB cells were seeded in 48 well plates at 1 104 cells/well and incubated for 24 h to allow cell attachment. The cells were then exposed to a series of FA-NPs and NPs solutions at different concentrations for 48 h. The cell viability was then assessed by MTT assay. About 5 mg/ml of MTT in PBS was then added to each well and the plate was incubated for an additional 4 h at 37 C under the aforementioned 5% CO2 atmosphere. Then the MTT containing medium was removed, and the crystals formed by living cells were dissolved in 100 ml DMSO. Cells without treatment were used as a control. The absorbance at 570 nm of the solution in each well was recorded using a Microplate Reader (Model 680, BIO-RAD, Hercules, CA). The relative cell viability (%) was calculated as (AbssampleAbsblank)/ (AbscontrolAbsblank) 100. In vivo antitumor effect Tumor-bearing mice were prepared by inoculating (s.c.) a suspension of KB cells (1106 cells) into the right armpit of BALB/c mice (Li et al., 2012; Liu et al., 2011). Briefly, the mice were acclimatized at a temperature of 25 ± 2 C and a relative humidity of 70 ± 5% under natural light/dark conditions for 1 week before dosing. Then, the mice were injected subcutaneously in the right armpit with KB cells suspended in PBS. Tumors were permitted to reach 4–5 mm diameter before initiation of the studies. In vivo anticancer activity of FA-NPs/DOX was evaluated against KB solid tumors in mice. Five groups of tumorbearing mice (six per group) were used. The mice were injected with 10 mg/kg of (1) FA-NPs/DOX, (2) NPs/DOX, (3) NPs, (4) free DOX solution, and (5) 0.9% sodium chloride solution (blank control). All drugs were diluted with 0.9% sodium chloride (100 ml), and all were administered through direct intratumoral injection. After drug administration, the mortality was monitored daily and the tumor growth was determined by caliper measurement every 3 d. Tumor volume was calculated as follows (Jia et al., 2012) Tumor volume mm3 ¼ length width2 =2 ð3Þ
Results Optimization of the modification ratio FA–PEG–PE ligands were continuously coated onto the surface of the NPs/DOX. This procedure would mask the cationic surface charge NPs/DOX complexes and cause the decrease of zeta potential. The best ratio of the ligands to the carriers was optimized by measuring the change of zeta potential. As illustrated in Figure 2, the optimized ratio of the FA–PEG–PE to NPs/DOX was 35%. This ratio was determined and used for further experiments. Characterization of FA-NPs/DOX Size, zeta potential, DL, and EE Mean particle size, polydispersity index (PDI), zeta potential, DL, and EE of NPs, NPs/DOX, and FA-NPs/DOX were characterized and summarized in Table 1. In vitro release study The in vitro release profiles of FA-NPs/DOX and unmodified NPs/DOX are illustrated in Figure 3. Both the FA-NPs/DOX and unmodified NPs/DOX reached over 80% drug release at the time point of 72 h. 45 Zeta potential (mV)
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40 35 30 25 20 15 0%
5%
10% 15% 20% 25% 30% 35% 40% 45% 50% 55% Ratios of FA-PEG-PE to NPs/DOX
Figure 2. Optimization of the modification ratios of FA–PEG–PE to NPs/DOX.
Table 1. Particle size, zeta potential, and gene loading quantity of different vectors. Characteristics
Samples
Mean particle size (nm)
NPs 125.6 ± 3.2 NPs/DOX 159.3 ± 4.8 FA-NPs/ 229.2 ± 3.1 DOX
Zeta Polydispersity potential (mV) index (PDI) 0.12 ± 0.02 0.15 ± 0.03 0.16 ± 0.02
DL (%)
EE (%)
41.8 ± 2.8 – – 32.1 ± 2.3 9.8 ± 0.9 86.2 ± 1.3 21.8 ± 3.1 9.5 ± 0.8 85.6 ± 1.6
Modified nanoparticles for cancer therapy
DOI: 10.3109/13880209.2013.874533
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were between 80% and 100% compared with controls (Figure 4).
NPs/DOX 80
FA-NPs/DOX
In vivo drug delivery and antitumor therapy
Cumulative released drug (%)
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In vivo anticancer activity of FA-NPs/DOX was evaluated against KB solid tumors in mice. The in vivo antitumor efficiency of FA-NPs/DOX, NPs/DOX, NPs, and free DOX was observed. The tumor growth curves of each group are presented in Figure 5. Results indicate that NPs/DOX treatment resulted in smaller tumor volume compared with free DOX; FA-NPs/DOX treatment resulted in smaller tumor volume compared with all the others. The results illustrate that (1) encapsulation of DOX in NPs enhanced the anticancer activity of DOX in vivo; and (2) FA-modified NPs/DOX gain better antitumor therapeutic effect than unmodified ones.
60 50 40 30 20 10 0 0
12
24
36
48
60
72
84
96
108 120
Time (h)
Discussion
Figure 3. The in vitro release profiles of FA-NPs/DOX and NPs/DOX.
NPs FA-NPs
Relative cell viability (%)
100 80 60 40 20 0 10
20
50
100
200
400
600
800
Concentrations (µg/ml)
Figure 4. In vitro cytotoxicity evaluation of FA-NPs and NPs. 3000
FA-NPs/DOX NPs/DOX NPs DOX CONTROL
2500 Mean tumor volume (mm3)
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2000
1500
1000
500
0 0
2
4
6
8
10
12
14
Time (days)
Figure 5. The tumor growth curves of FA-NPs/DOX, NPs/DOX, NPs, and free DOX.
In vitro cytotoxicity evaluation In vitro cytotoxicity of FA-NPs and NPs was evaluated in KB cells at different concentrations. The cell viabilities of the vectors over the studied concentration range (10–800 mg/ml)
The intratumoral administration of anticancer drugs represents a growing trend for maximizing local tumor control with minimal systemic toxicity. However, it requires suitable DDS for treatment efficacy (Jia et al., 2012). In the present study, a biodegradable nanocarrier has been developed to reduce or minimize undesired interactions or undesired uptake into normal sites (Akbarzadeh et al., 2012). Moreover, an active targeting ligand was synthesized and modified onto the surface of DOX-loaded NPs to achieve the targeted effect on tumor cells. FA is an essential B vitamin, which could internalize into the cells via a low-affinity reduced folate carrier protein or via high-affinity folate receptors. The best studied of these receptors is FR, a cell surface glycosyl phosphatidylinositolanchored glycoprotein that can internalize bound FA and FA-conjugated compounds via receptor-mediated endocytosis (Paulos et al., 2004). So FA was used as the target moiety which could bind to the FR on the KB cells. A series of PEG-containing ligands, commonly named PEG–PE conjugates, were reported and used for the modification of various vehicles to achieve active-targeting nanocarriers that spontaneously accumulate in specific sites (Lukyanov et al., 2002; Torchilin, 2005). Thus, PEG–PE was used at the anchor and FA–PEG–PE was applied as the target ligand of the system. During the modification procedure, FA–PEG–PE ligands were extensively coated onto the NPs/DOX carriers due to the electric charge and lipophilic interaction, which covered their original surface charge and caused the decrease in zeta potential. The optimization of ligand-to-carrier ratio was carried out by measuring the zeta potential. As shown in Figure 2 and Table 1, the optimum ratio was obtained at 35% (FA–PEG–PE to NPs/DOX, w/w) and the optimum FA-NPs/ DOX had a size of 229 nm and zeta potential of +22 mV. After the optimization of the modification procedure, the FA–PEG–PE to NPs/DOX ratio was determined as 35% and used for further experiments. The DL and EE of the FA-NPs/ DOX and other vectors were determined by a subtraction method. As shown in Table 1, the DL of FA-NPs/DOX and NPs/DOX is around 10% and the EE of both vectors is around
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85%. The results demonstrated that binding of FA–PEG–PE ligand did not detach the DOX from the complexes and the modified vectors are stable. The in vitro release profile (Figure 3) of FA-NPs/DOX showed slightly slower release than NPs/DOX during the first 24 h. This might be due to the hindrance caused by coating of ligand to the release of drugs initially. As time passes, more FA–PEG–PE detached from the vectors, allowing the release of drugs more and more freely. At the end of the release study, the total amount of drugs delivered from the two kinds of vehicles was nearly the same (over 80%). In vitro cytotoxicity analysis was carried out in KB cells. The cell viabilities of FA-NPs/DOX and NPs/DOX over the studied concentration range were between 80% and 100% compared with controls (Figure 4). FA-NPs/DOX showed no higher cytotoxicity than NPs/DOX at all concentrations. In vivo anticancer activity of FA-NPs/DOX and other vectors was evaluated against KB solid tumors in mice. The mice were injected with 10 mg/kg of FA-NPs/DOX, NPs/DOX, NPs, and DOX solution to the tumor site; 0.9% sodium chloride solution was used as a blank control. The tumor growth rate was not found to significantly decrease with free DOX treatment and NPs (the same behavior with 0.9% sodium chloride solution); however, the tumor growth rate was significantly decreased in the group treated with NPs/DOX. Furthermore, FA-NPs/DOX showed higher antitumor effect than free NPs/DOX in vivo. These results indicate that FA-modified DOX-loaded NPs have promising application in antitumor drug delivery and would be very useful in anticancer cancer therapy.
Conclusions This study showed that a targeting ligand (FA–PEG–PE)modified DOX-loaded bioadhesive PLGA nanoparticles can significantly improve the therapeutic efficiency of the vector, and successfully control the tumor growth rate on tumorbearing mice. The conclusion should be that FA could function as an excellent active targeting ligand to improve the cell targeting of the carriers, and the modified DDS could function comprehensively to improve the efficacy of cancer therapy. The system was shown to be an excellent carrier for delivery of DOX, leading to the efficiency of antitumor therapy.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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