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Cite this: Chem. Commun., 2014, 50, 1579
Multifunctional nanoparticles via host–guest interactions: a universal platform for targeted imaging and light-regulated gene delivery†
Received 22nd October 2013, Accepted 20th November 2013
Wenyu Li, Jianwei Du, Kun Zheng, Peng Zhang, Qiaoling Hu and Youxiang Wang*
DOI: 10.1039/c3cc48098d www.rsc.org/chemcomm
Host–guest assembly provides a universal platform to construct responsive carrier systems for targeted imaging and controllable gene delivery. The best advantage of this strategy is that systems are very easy to handle, do not involve tedious chemical reactions and can be flexibly optimized by changing the functional tags responding to a request.
Nanobiotechnology has been advancing rapidly toward biomedical applications of nanoparticles in cancer diagnosis and therapy.1 Over the past decade, multifunctional groups have been covalently conjugated to smart carrier systems combining diagnostic detection agents and therapeutic payloads (anticancer drugs, siRNA, etc.), which are exciting for their potential applications in the early detection and treatment of human cancer.2 Many researchers have aspired to develop responsive carrier systems for the controllable release of therapeutic agents that are sensitive to environmental stimuli such as temperature, pH, redox or specific biomolecules.3 However, the covalent synthetic steps are usually complicated and time-consuming. In some cases, multiplex synthetic progress might be completely carried out again on changing one aspect such as an imaging agent or ligand. Recently, supramolecular chemistry has sparked the emergence of the modular assembly of different functional tags such as imaging, targeting, and treating modules. Typical supramolecular host b-cyclodextrin (b-CD) has well-known host– guest interactions with a vast array of hydrophobic compounds, such as azobenzene (Az), adamantane (AD), and cholesterol.4 The light-controlled molecular recognition of CD for Az has been exploited to develop light-responsive drug or gene delivery.5 For example, a ternary supramolecular system was developed based on a light-responsive Az–CD host–guest interaction, which can reversibly capture and release DNA.4b Meanwhile, our recent study also found intracellular dePEGylation could be easily controlled via lightregulated host–guest interactions between b-CD and Az.5c Therefore, host–guest assembly can provide a novel strategy to physically MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, P. R. China. E-mail: [email protected]; Fax: +86 571 87953729; Tel: +86 571 87953729 † Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc48098d
This journal is © The Royal Society of Chemistry 2014
integrate diagnostic imaging agents and responsive gene delivery carriers. Herein, we attempted to construct multifunctional nanoparticles via host–guest interactions between b-CD and its guest molecules (Fig. 1). Branched polyethylenimine (PEI) was selected as a scaffold polymer and b-CDs were conjugated as host units. Galactose (Gal), a targeting moiety of asialoglycoprotein receptor (ASGP-R) overexpressed on the surface of hepatocytes, was conjugated on azobenzenepolyethylene glycol (Az-PEG-Gal). Fluorescein isothiocyanate (FITC) was selected as a fluorescence imaging probe and conjugated on adamantane (AD-FITC). Subsequently, the supramolecular polymer with multi-functions was easily constructed by host–guest assembly and then used to load therapeutic agents (e.g., DNA) through the electrostatic interactions. The PEG shell was expected to equip the multifunctional nanoparticles with excellent stability. Specific recognition promoted the multifunctional nanoparticles to accumulate at the disease site, which facilitated non-invasive bioimaging.1b The light-responsive CD–Az host–guest interaction might facilitate the controllable release of DNA inside the cells. Therefore, the host–guest assembly acted as a flexible, universal platform to construct multifunctional nanoparticles that physically incorporated targeted imaging and controllable gene delivery into one package. Firstly, PEI-CD was synthesized by the substitution reaction of 6-deoxy-(p-toluenesulfonyl)-b-CD (6-OTs-b-CD) with the amine groups of PEI.6 The CD-grafting level confirmed by 1H NMR was 1.5%, indicating that every PEI chain had 9 CD molecules (Fig. S1, see ESI†). As shown in Scheme S1 (see ESI†), Az-PEG-Gal and AD-FITC were synthesized. In addition, it has been documented that the association constant Ka of b-CD to AD or Az was 2–4 104, 1.7 103 M 1, respectively.7 Due to the significant difference in Ka values, AD-FITC was firstly incorporated onto PEI-CD with the molar ratio of AD to CD at 1 : 3, followed by the addition of Az-PEG-Gal to avoid competitive inclusion among different guests. Next, Az-PEG-Gal was incorporated with various molar ratios of Az on Az-PEG-Gal to b-CDs remaining on PEI-CD (Az/RC). The protocol of inclusion in detail was monitored according to our previous study.5c Then, the resulting supramolecular polymers AD-FITC/PEI-CD, AD-FITC/PEI-CD/Az-PEG and AD-FITC/PEI-CD/Az-PEG-Gal were successfully obtained via
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Fig. 1 Illustration of the multifunctional nanoparticles developed via host– guest interaction for targeted imaging and controllable DNA delivery.
host–guest interactions in mild conditions and designated as SP, SPG and SPG-Gal, respectively. Driven by the electrostatic interactions, the multifunctional supramolecular polymers condensed DNA into nanoparticles. The particle sizes and z-potentials were evaluated in detail by Zetasizer Nanoseries. As shown in Fig. S2 (see ESI†), it was found that the sizes and zeta-potentials decreased slightly with Az/RC ratio increment, and finally had a very small z-potential value of about 6 mV. The results suggest that the nanoparticles formed core–shell architectures with a hydrophilic and neutral PEG-Gal shell surrounding the particle core, which provided feasibility of targeted cellular uptake.8 The Az/RC ratio was kept at 4 in the next experiments to maintain sufficient PEG shell for colloidal stability. Fig. 2(a) shows the nanoparticles have a tightly packed structure. The fluorescence image shown in Fig. 2(b) indicates that the particles were observed with bright green fluorescence, suggesting that the multifunctional nanoparticles could be used in intracellular trafficking. Moreover, an agarose gel retardation assay indicated that the incorporation of FITC and PEG-Gal via host–guest interactions did not significantly
Fig. 2 TEM (a) and fluorescence (b) images of SPG-Gal/DNA nanoparticles (N/P = 10). The observation was performed using a 40 objective; agarose gel electrophoresis of PEI/pDNA (c) and SPG-Gal/pDNA (d) nanoparticles (N/P = 10) in the presence of various concentrations of heparin.
1580 | Chem. Commun., 2014, 50, 1579--1581
ChemComm
affect the DNA condensation ability (Fig. S3, see ESI†), which is of benefit for transfection. The long blood circulation of nanoparticles is a prerequisite for cancer diagnosis and gene therapy in vivo.9 Dynamic light scattering (DLS) and TEM revealed that the multifunctional nanoparticles were stable in physiological salt conditions and complete cellular culture media containing 10% fetal bovine serum (Fig. S4, see ESI†), which was ascribed to steric repulsion of sufficient PEG shell. Heparin was used as a counter polyanion to evaluate the in vivo stability against anionic proteins in the bloodstream. As shown in Fig. 2(c) and (d), for PEI/DNA nanoparticles, the free DNA band began to emerge at a heparin concentration of 60 mg mL 1. In contrast, in case of SPG-Gal/ DNA nanoparticles, no free DNA bands were observed even at the heparin concentration of 80 mg mL 1. The improved competition stability can be attributed to the steric repulsion of the PEG shell, preventing the access of counter polyanion. On the other hand, the light-regulated host–guest interaction Az–CD was expected to equip the multifunctional nanoparticles with PEG-detachable ability for facilitated DNA release. Next, the z-potential was investigated after light irradiation at 365 nm for 15 min. It was found that light irradiation significantly increased the z-potential and particle size (Fig. S2, see ESI†), which were comparable to those of SP/DNA nanoparticles. Moreover, after irradiation, the fluorescent intensity of DNA bands was improved in the agarose gel chamber (Fig. S5, see ESI†), which indicated that the 15 min light irradiation was effective for PEG detachment from the multifunctional nanoparticles. The ideal multifunctional nanoparticles should actively target and accumulate at the cancer site, followed by non-invasive bioimaging diagnosis and controllable gene therapy. In this research, HepG2 (human hepatoblastoma cell line) with the ASGP-R overexpressed on the cell surface was used to investigate the specific-targeting capability. The multifunctional nanoparticles incorporated with FITC via host–guest interactions were directly used for cellular uptake, which could be detected by flow cytometry. As shown in Fig. 3(d), the PEG shell hindered the cellular uptake of SPG/DNA nanoparticles compared with that of SP/DNA nanoparticles. However, the cellular uptake profiles of SPG-Gal/DNA nanoparticles were approximately 4 times for 0.5 h and 1.2 times for 4.5 h higher than those of nanoparticles without targeting molecules. The results suggest that the multifunctional nanoparticles were efficiently accumulated and taken up by cancer cells based on specific recognition. As a direct proof of concept, we carried out the bioimaging and intracellular trafficking of the multifunctional nanoparticles by confocal laser scanning microscopy (CLSM). Cy3-DNA was used to better understand intracellular trafficking. As shown in Fig. 3(a) and (b), for SP/DNA nanoparticles, part of Cy3-DNA was observed to enter into the nuclei, although there was some part of overlapped yellow fluorescence inside the cells. In the case of SPG-Gal/DNA nanoparticles, effective endocytosis was achieved via specific recognition, with obvious yellow fluorescence, and mainly distributed in the cytoplasm. Combined with the results of competition stability in heparin, we speculated that the PEG shell hindered the disassembly of nanoparticles inside the cells. The stable CD–AD host–guest interaction was expected to maintain FITC to monitor the distribution of the polycations. Fig. 3(c) indicates that after 15 min light irradiation (l = 365 nm), most of DNA with red fluorescence was
This journal is © The Royal Society of Chemistry 2014
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efficiency than the nanoparticles without targeting molecules. The main reason was that specific recognition increased cellular uptake efficiency. Furthermore, light-stimulus equipped the multifunctional nanoparticles with around 1.5 times higher transfection efficiency than that without light irradiation, which was comparable to SP/DNA nanoparticles. From these results, we confirmed the feasibility of SPG-Gal/DNA nanoparticles as a responsive multifunctional system. In summary, novel multifunctional nanoparticles were successfully constructed via host–guest interactions. The best advantage of this strategy is that the system is very easy to handle, does not involve tedious chemical reactions and can be flexibly optimized by changing the functional tags responding to a request. We believe the host–guest assembly provides a universal platform to construct responsive carrier systems for targeted imaging and controllable gene delivery, which might have potential application in future disease diagnosis and therapy. This work was financially supported by the National Natural Science Foundation of China (21074110, 51273177).
Notes and references
Fig. 3 CLSM images of HepG2 cells exposed to (a) SP/Cy3-DNA, (b) SPGGal/Cy3-DNA, and (c) 15 min light irradiation treatment with SPG-Gal/ Cy3-DNA after 4.5 h incubation, followed by another 12 h incubation in the absence of nanoparticles. The nuclei were stained with DAPI (blue). (d) Cellular uptake profile of HepG2 cells exposed to different nanoparticles for 0.5 h and 4.5 h. (e) In vitro transfection of the luciferase gene into HepG2 cells mediated by different nanoparticles for 48 h. *denotes statistically significant difference at p o 0.05.
localized in the nuclei and polycations with green fluorescence were mainly distributed in the cytoplasm. The results provide direct proof that the light-regulated PEG detachment facilitated DNA release to enter into nuclei. Here, if only FITC was replaced with NIR optical imaging agents such as Cy5.5, the nanoparticles would be easily visualized by NIR optical imaging in vivo to realize a disease diagnostic application. Next, the cytotoxicity of the multifunctional nanoparticles and light irradiation were investigated. As shown in Fig. S6 (see ESI†), the cytotoxicity of the multifunctional nanoparticles was much lower than that of PEI/DNA particles mainly due to the well-known biocompatibility of cyclodextrin and PEG.10 More importantly, light (l = 365 nm) irradiation for 15 min had little effect on cell viability. Encouraged by these results, luciferase plasmid DNA pGL-3 was used as a model reporter gene to investigate the therapeutic ability. As shown in Fig. 3(e), SPG/DNA nanoparticles showed the lowest transfection efficiency, which was attributed to the reduced cellular uptake and hindered ability of intracellular DNA release. However, when incorporated with targeting molecules, SPG-Gal/DNA nanoparticles showed around 2 orders of magnitude higher transfection
This journal is © The Royal Society of Chemistry 2014
1 (a) H. Cabral, N. Nishiyama and K. Kataoka, Acc. Chem. Res., 2011, 44, 999; (b) R. Bardhan, S. Lal, A. Joshi and N. J. Halas, Acc. Chem. Res., 2011, 44, 936. 2 (a) V. Torchilin, Eur. J. Pharm. Biopharm., 2009, 71, 431; (b) A. K. Iyer, J. He and M. M. Amiji, Curr. Med. Chem., 2012, 19, 3230; (c) H. Jung, K. M. Park, J. A. Yang, E. J. Oh, D. W. Lee, K. Park, S. H. Ryu, S. K. Hahn and K. Kim, Biomaterials, 2011, 32, 7687; (d) J. E. Lee, N. Lee, T. Kim, J. Kim and T. Hyeon, Acc. Chem. Res., 2011, 44, 893. 3 (a) P. Zou, Y. K. Yu, Y. A. Wang, Y. Q. Zhong, A. Welton, C. Galban, S. M. Wang and D. X. Sun, Mol. Pharmaceutics, 2010, 7, 1974; (b) D. E. Owens, J. K. Eby, Y. Jian and N. A. Peppas, J. Biomed. Mater. Res., Part A, 2007, 83A, 692; (c) M. A. C. Stuart, W. T. S. Huck, J. Genzer, M. Muller, C. Ober, M. Stamm, G. B. Sukhorukov, I. Szleifer, V. V. Tsukruk, M. Urban, F. Winnik, S. Zauscher, I. Luzinov and S. Minko, Nat. Mater., 2010, 9, 101. 4 (a) J. X. Zhang and P. X. Ma, Adv. Drug Delivery Rev., 2013, 65, 1215; (b) S. K. M. Nalluri, J. Voskuhl, J. B. Bultema, E. J. Boekema and B. J. Ravoo, Angew. Chem., Int. Ed., 2011, 50, 9747; (c) Z. X. Zhang, K. L. Liu and J. Li, Macromolecules, 2011, 44, 1182; (d) Y. Liu, C. Y. Yu, H. B. Jin, B. B. Jiang, X. Y. Zhu, Y. F. Zhou, Z. Y. Lu and D. Y. Yan, J. Am. Chem. Soc., 2013, 135, 4765. 5 (a) W. Xiao, W. H. Chen, X. D. Xu, C. Li, L. Zhang, R. X. Zhuo and X. Z. Zhang, Adv. Mater., 2011, 23, 3526; (b) Q. A. Jin, G. Y. Liu, X. S. Liu and J. A. Ji, Soft Matter, 2010, 6, 5589; (c) W. Y. Li, Y. X. Wang, L. N. Chen, Z. X. Huang, Q. L. Hu and J. Ji, Chem. Commun., 2012, 48, 10126; (d) Y. P. Wang, N. Ma, Z. Q. Wang and X. Zhang, Angew. Chem., Int. Ed., 2007, 46, 2823; (e) D. P. Ferris, Y. L. Zhao, N. M. Khashab, H. A. Khatib, J. F. Stoddart and J. I. Zink, J. Am. Chem. Soc., 2009, 131, 1686. 6 (a) J. X. Zhang, H. L. Sun and P. X. Ma, ACS Nano, 2010, 4, 1049; (b) S. H. Pun, N. C. Bellocq, A. J. Liu, G. Jensen, T. Machemer, E. Quijano, T. Schluep, S. F. Wen, H. Engler, J. Heidel and M. E. Davis, Bioconjugate Chem., 2004, 15, 831. 7 (a) M. Lahav, K. T. Ranjit, E. Katz and I. Willner, Chem. Commun., 1997, 259; (b) M. Weickenmeier and G. Wenz, Macromol. Rapid Commun., 1996, 17, 731; (c) S. K. Osman, F. P. Brandl, G. M. Zayed, J. K. Tessmar and A. M. Gopferich, Polymer, 2011, 52, 4806. 8 (a) B. X. Zhao, Y. Zhao, Y. Huang, L. M. Luo, P. Song, X. Wang, S. Chen, K. F. Yu, X. Zhang and Q. Zhang, Biomaterials, 2012, 33, 2508; (b) S. Takae, K. Miyata, M. Oba, T. Ishii, N. Nishiyama, K. Itaka, Y. Yamasaki, H. Koyama and K. Kataoka, J. Am. Chem. Soc., 2008, 130, 6001. 9 (a) J. R. McCarthy and R. Weissleder, Adv. Drug Delivery Rev., 2008, 60, 1241; (b) S. Santra, C. Kaittanis, J. Grimm and J. M. Perez, Small, 2009, 5, 1862. 10 (a) W. Li, L. Chen, Z. Huang, X. Wu, Y. Zhang, Q. Hu and Y. Wang, Org. Biomol. Chem., 2011, 9, 7799; (b) M. Ogris, S. Brunner, S. Schuller, R. Kircheis and E. Wagner, Gene Ther., 1999, 6, 595.
Chem. Commun., 2014, 50, 1579--1581 | 1581
Published on 21 November 2013. Downloaded by University of California - Irvine on 25/10/2014 06:09:33.
COMMUNICATION
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Cite this: Chem. Commun., 2014, 50, 1579
Multifunctional nanoparticles via host–guest interactions: a universal platform for targeted imaging and light-regulated gene delivery†
Received 22nd October 2013, Accepted 20th November 2013
Wenyu Li, Jianwei Du, Kun Zheng, Peng Zhang, Qiaoling Hu and Youxiang Wang*
DOI: 10.1039/c3cc48098d www.rsc.org/chemcomm
Host–guest assembly provides a universal platform to construct responsive carrier systems for targeted imaging and controllable gene delivery. The best advantage of this strategy is that systems are very easy to handle, do not involve tedious chemical reactions and can be flexibly optimized by changing the functional tags responding to a request.
Nanobiotechnology has been advancing rapidly toward biomedical applications of nanoparticles in cancer diagnosis and therapy.1 Over the past decade, multifunctional groups have been covalently conjugated to smart carrier systems combining diagnostic detection agents and therapeutic payloads (anticancer drugs, siRNA, etc.), which are exciting for their potential applications in the early detection and treatment of human cancer.2 Many researchers have aspired to develop responsive carrier systems for the controllable release of therapeutic agents that are sensitive to environmental stimuli such as temperature, pH, redox or specific biomolecules.3 However, the covalent synthetic steps are usually complicated and time-consuming. In some cases, multiplex synthetic progress might be completely carried out again on changing one aspect such as an imaging agent or ligand. Recently, supramolecular chemistry has sparked the emergence of the modular assembly of different functional tags such as imaging, targeting, and treating modules. Typical supramolecular host b-cyclodextrin (b-CD) has well-known host– guest interactions with a vast array of hydrophobic compounds, such as azobenzene (Az), adamantane (AD), and cholesterol.4 The light-controlled molecular recognition of CD for Az has been exploited to develop light-responsive drug or gene delivery.5 For example, a ternary supramolecular system was developed based on a light-responsive Az–CD host–guest interaction, which can reversibly capture and release DNA.4b Meanwhile, our recent study also found intracellular dePEGylation could be easily controlled via lightregulated host–guest interactions between b-CD and Az.5c Therefore, host–guest assembly can provide a novel strategy to physically MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, P. R. China. E-mail: [email protected]; Fax: +86 571 87953729; Tel: +86 571 87953729 † Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc48098d
This journal is © The Royal Society of Chemistry 2014
integrate diagnostic imaging agents and responsive gene delivery carriers. Herein, we attempted to construct multifunctional nanoparticles via host–guest interactions between b-CD and its guest molecules (Fig. 1). Branched polyethylenimine (PEI) was selected as a scaffold polymer and b-CDs were conjugated as host units. Galactose (Gal), a targeting moiety of asialoglycoprotein receptor (ASGP-R) overexpressed on the surface of hepatocytes, was conjugated on azobenzenepolyethylene glycol (Az-PEG-Gal). Fluorescein isothiocyanate (FITC) was selected as a fluorescence imaging probe and conjugated on adamantane (AD-FITC). Subsequently, the supramolecular polymer with multi-functions was easily constructed by host–guest assembly and then used to load therapeutic agents (e.g., DNA) through the electrostatic interactions. The PEG shell was expected to equip the multifunctional nanoparticles with excellent stability. Specific recognition promoted the multifunctional nanoparticles to accumulate at the disease site, which facilitated non-invasive bioimaging.1b The light-responsive CD–Az host–guest interaction might facilitate the controllable release of DNA inside the cells. Therefore, the host–guest assembly acted as a flexible, universal platform to construct multifunctional nanoparticles that physically incorporated targeted imaging and controllable gene delivery into one package. Firstly, PEI-CD was synthesized by the substitution reaction of 6-deoxy-(p-toluenesulfonyl)-b-CD (6-OTs-b-CD) with the amine groups of PEI.6 The CD-grafting level confirmed by 1H NMR was 1.5%, indicating that every PEI chain had 9 CD molecules (Fig. S1, see ESI†). As shown in Scheme S1 (see ESI†), Az-PEG-Gal and AD-FITC were synthesized. In addition, it has been documented that the association constant Ka of b-CD to AD or Az was 2–4 104, 1.7 103 M 1, respectively.7 Due to the significant difference in Ka values, AD-FITC was firstly incorporated onto PEI-CD with the molar ratio of AD to CD at 1 : 3, followed by the addition of Az-PEG-Gal to avoid competitive inclusion among different guests. Next, Az-PEG-Gal was incorporated with various molar ratios of Az on Az-PEG-Gal to b-CDs remaining on PEI-CD (Az/RC). The protocol of inclusion in detail was monitored according to our previous study.5c Then, the resulting supramolecular polymers AD-FITC/PEI-CD, AD-FITC/PEI-CD/Az-PEG and AD-FITC/PEI-CD/Az-PEG-Gal were successfully obtained via
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Published on 21 November 2013. Downloaded by University of California - Irvine on 25/10/2014 06:09:33.
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Fig. 1 Illustration of the multifunctional nanoparticles developed via host– guest interaction for targeted imaging and controllable DNA delivery.
host–guest interactions in mild conditions and designated as SP, SPG and SPG-Gal, respectively. Driven by the electrostatic interactions, the multifunctional supramolecular polymers condensed DNA into nanoparticles. The particle sizes and z-potentials were evaluated in detail by Zetasizer Nanoseries. As shown in Fig. S2 (see ESI†), it was found that the sizes and zeta-potentials decreased slightly with Az/RC ratio increment, and finally had a very small z-potential value of about 6 mV. The results suggest that the nanoparticles formed core–shell architectures with a hydrophilic and neutral PEG-Gal shell surrounding the particle core, which provided feasibility of targeted cellular uptake.8 The Az/RC ratio was kept at 4 in the next experiments to maintain sufficient PEG shell for colloidal stability. Fig. 2(a) shows the nanoparticles have a tightly packed structure. The fluorescence image shown in Fig. 2(b) indicates that the particles were observed with bright green fluorescence, suggesting that the multifunctional nanoparticles could be used in intracellular trafficking. Moreover, an agarose gel retardation assay indicated that the incorporation of FITC and PEG-Gal via host–guest interactions did not significantly
Fig. 2 TEM (a) and fluorescence (b) images of SPG-Gal/DNA nanoparticles (N/P = 10). The observation was performed using a 40 objective; agarose gel electrophoresis of PEI/pDNA (c) and SPG-Gal/pDNA (d) nanoparticles (N/P = 10) in the presence of various concentrations of heparin.
1580 | Chem. Commun., 2014, 50, 1579--1581
ChemComm
affect the DNA condensation ability (Fig. S3, see ESI†), which is of benefit for transfection. The long blood circulation of nanoparticles is a prerequisite for cancer diagnosis and gene therapy in vivo.9 Dynamic light scattering (DLS) and TEM revealed that the multifunctional nanoparticles were stable in physiological salt conditions and complete cellular culture media containing 10% fetal bovine serum (Fig. S4, see ESI†), which was ascribed to steric repulsion of sufficient PEG shell. Heparin was used as a counter polyanion to evaluate the in vivo stability against anionic proteins in the bloodstream. As shown in Fig. 2(c) and (d), for PEI/DNA nanoparticles, the free DNA band began to emerge at a heparin concentration of 60 mg mL 1. In contrast, in case of SPG-Gal/ DNA nanoparticles, no free DNA bands were observed even at the heparin concentration of 80 mg mL 1. The improved competition stability can be attributed to the steric repulsion of the PEG shell, preventing the access of counter polyanion. On the other hand, the light-regulated host–guest interaction Az–CD was expected to equip the multifunctional nanoparticles with PEG-detachable ability for facilitated DNA release. Next, the z-potential was investigated after light irradiation at 365 nm for 15 min. It was found that light irradiation significantly increased the z-potential and particle size (Fig. S2, see ESI†), which were comparable to those of SP/DNA nanoparticles. Moreover, after irradiation, the fluorescent intensity of DNA bands was improved in the agarose gel chamber (Fig. S5, see ESI†), which indicated that the 15 min light irradiation was effective for PEG detachment from the multifunctional nanoparticles. The ideal multifunctional nanoparticles should actively target and accumulate at the cancer site, followed by non-invasive bioimaging diagnosis and controllable gene therapy. In this research, HepG2 (human hepatoblastoma cell line) with the ASGP-R overexpressed on the cell surface was used to investigate the specific-targeting capability. The multifunctional nanoparticles incorporated with FITC via host–guest interactions were directly used for cellular uptake, which could be detected by flow cytometry. As shown in Fig. 3(d), the PEG shell hindered the cellular uptake of SPG/DNA nanoparticles compared with that of SP/DNA nanoparticles. However, the cellular uptake profiles of SPG-Gal/DNA nanoparticles were approximately 4 times for 0.5 h and 1.2 times for 4.5 h higher than those of nanoparticles without targeting molecules. The results suggest that the multifunctional nanoparticles were efficiently accumulated and taken up by cancer cells based on specific recognition. As a direct proof of concept, we carried out the bioimaging and intracellular trafficking of the multifunctional nanoparticles by confocal laser scanning microscopy (CLSM). Cy3-DNA was used to better understand intracellular trafficking. As shown in Fig. 3(a) and (b), for SP/DNA nanoparticles, part of Cy3-DNA was observed to enter into the nuclei, although there was some part of overlapped yellow fluorescence inside the cells. In the case of SPG-Gal/DNA nanoparticles, effective endocytosis was achieved via specific recognition, with obvious yellow fluorescence, and mainly distributed in the cytoplasm. Combined with the results of competition stability in heparin, we speculated that the PEG shell hindered the disassembly of nanoparticles inside the cells. The stable CD–AD host–guest interaction was expected to maintain FITC to monitor the distribution of the polycations. Fig. 3(c) indicates that after 15 min light irradiation (l = 365 nm), most of DNA with red fluorescence was
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 21 November 2013. Downloaded by University of California - Irvine on 25/10/2014 06:09:33.
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efficiency than the nanoparticles without targeting molecules. The main reason was that specific recognition increased cellular uptake efficiency. Furthermore, light-stimulus equipped the multifunctional nanoparticles with around 1.5 times higher transfection efficiency than that without light irradiation, which was comparable to SP/DNA nanoparticles. From these results, we confirmed the feasibility of SPG-Gal/DNA nanoparticles as a responsive multifunctional system. In summary, novel multifunctional nanoparticles were successfully constructed via host–guest interactions. The best advantage of this strategy is that the system is very easy to handle, does not involve tedious chemical reactions and can be flexibly optimized by changing the functional tags responding to a request. We believe the host–guest assembly provides a universal platform to construct responsive carrier systems for targeted imaging and controllable gene delivery, which might have potential application in future disease diagnosis and therapy. This work was financially supported by the National Natural Science Foundation of China (21074110, 51273177).
Notes and references
Fig. 3 CLSM images of HepG2 cells exposed to (a) SP/Cy3-DNA, (b) SPGGal/Cy3-DNA, and (c) 15 min light irradiation treatment with SPG-Gal/ Cy3-DNA after 4.5 h incubation, followed by another 12 h incubation in the absence of nanoparticles. The nuclei were stained with DAPI (blue). (d) Cellular uptake profile of HepG2 cells exposed to different nanoparticles for 0.5 h and 4.5 h. (e) In vitro transfection of the luciferase gene into HepG2 cells mediated by different nanoparticles for 48 h. *denotes statistically significant difference at p o 0.05.
localized in the nuclei and polycations with green fluorescence were mainly distributed in the cytoplasm. The results provide direct proof that the light-regulated PEG detachment facilitated DNA release to enter into nuclei. Here, if only FITC was replaced with NIR optical imaging agents such as Cy5.5, the nanoparticles would be easily visualized by NIR optical imaging in vivo to realize a disease diagnostic application. Next, the cytotoxicity of the multifunctional nanoparticles and light irradiation were investigated. As shown in Fig. S6 (see ESI†), the cytotoxicity of the multifunctional nanoparticles was much lower than that of PEI/DNA particles mainly due to the well-known biocompatibility of cyclodextrin and PEG.10 More importantly, light (l = 365 nm) irradiation for 15 min had little effect on cell viability. Encouraged by these results, luciferase plasmid DNA pGL-3 was used as a model reporter gene to investigate the therapeutic ability. As shown in Fig. 3(e), SPG/DNA nanoparticles showed the lowest transfection efficiency, which was attributed to the reduced cellular uptake and hindered ability of intracellular DNA release. However, when incorporated with targeting molecules, SPG-Gal/DNA nanoparticles showed around 2 orders of magnitude higher transfection
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