pubs.acs.org/Langmuir © 2010 American Chemical Society
Thermally Triggered Cellular Uptake of Quantum Dots Immobilized with Poly(N-isopropylacrylamide) and Cell Penetrating Peptide Chunsoo Kim,† Yuhan Lee,† Jee Seon Kim,† Ji Hoon Jeong,‡ and Tae Gwan Park*,† †
Department of Biological Sciences and Graduate School of Nanoscience and Technology, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea, and ‡School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea Received June 30, 2010. Revised Manuscript Received August 6, 2010 Thermally sensitive quantum dots (TSQDs) that exhibit an “on-demand” cellular uptake behavior via temperatureinduced “shielding/deshielding” of cell penetrating peptides (CPP) on the surface were fabricated. Poly(N-isopropylacrylamide) (PNIPAAm) (Mw = 11.5K) and CPP were biotinylated at their terminal ends and co-immobilized on to the surface of streptavidin-coated quantum dots (QDs-Strep) through biotin-streptavidin interaction. The cellular contact of CPP was sterically hindered due to hydrated PNIPAAm chains below the lower critical solution temperature (LCST). In contrast, above the LCST, grafted PNIPAAm chains were collapsed to make CPP moieties resurfaced, leading to increased cellular uptake of QDs. The temperature-controlled “shielding/deshielding” of CPP was further applied for a thermally triggered siRNA delivery system, where biotinylated siRNA was additionally conjugated to the surface of TSQDs. The level of gene silencing was significantly enhanced by increasing temperature above the LCST due to the surface exposure of CPP.
Introduction Recently, a wide range of nanomaterials such as iron oxide, gold, and quantum dots have been exploited in diverse biomedical applications including diagnostic imaging, therapeutic drug delivery, and biosensing due to their high surface-to-volume ratio that enables versatile functional modifications on the surface while maintaining their inherent nanosized properties.1-6 Multifunctional surface modification of organic and inorganic nanoparticles has been an important issue to achieve specific cell/tissue targeting. A variety of nanoparticles have been modified on the surfaces with targeting ligands to promote their interactions with specific cells.7,8 For successful targeted delivery of nanoparticles, it is also important to reduce their nonspecific cellular uptake, which is mostly caused by surface protein adsorption in the bloodstream. The immobilization of poly(ethylene glycol) (PEG) chains on the surface is the most frequently used strategy to reduce the unwanted cellular recognition.8 Since PEG chains have highly hydrated and mobile properties, the PEG immobilization endows a stealth character to the nanoparticles via a sterically repulsive mechanism, resulting in prolonged circulation in the blood. However, PEGylated nanoparticles also showed *To whom correspondence should be addressed: e-mail
[email protected]; Tel þ82-42-350-2621; Fax þ82-42-350-2610.
(1) Nel, A. E.; M€adler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Nature Mater. 2009, 8, 543–557. (2) Aggarwal, P.; Hall, J. B.; McLeland, C. B.; Dobrovolskaia, M. A.; McNeil, S. E. Adv. Drug Delivery Rev. 2009, 61, 428–437. (3) Shubayev, V. I.; Pisanic, T. R.; Jin, S. Adv. Drug Delivery Rev. 2009, 61, 467– 477. (4) De, M.; Ghosh, P. S.; Rotello, V. M. Adv. Mater. 2008, 20, 4225–4241. (5) Smith, A. M.; Duan, H.; Mohs, A. M.; Nie, S. Adv. Drug Delivery Rev. 2008, 60, 1226–1240. (6) Ghosh, P.; Han, G.; De, M.; Kim, C. K.; Rotello, V. M. Adv. Drug Delivery Rev. 2009, 60, 1307–1315. (7) Mok, H.; Park, T. G. Macromol. Biosci. 2009, 9, 731–743. (8) Kim, S.; Kim, J.-H.; Jeon, O.; Kwon, I. C.; Park, K. Eur. J. Pharm. Biopharm. 2009, 71, 420–430. (9) Jeong, J. H.; Mok, H.; Oh, Y.-K.; Park, T. G. Bioconjugate Chem. 2009, 20, 5–14. (10) Li, S.-D.; Chono, S.; Huang, L. J. Controlled Release 2008, 126, 77–84.
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significantly reduced extent of cellular uptake to targeted cells.9-11 To overcome this dilemma, various acid-cleavable and enzymecleavable PEG chains that can be dePEGylated at the desired tissue site have been alternatively used to modify the surface of nanoparticles. A wide variety of stimuli-responsive polymers that show reversible “dissolution/precipitation” behaviors in aqueous solution in response to temperature, pH, electromagnetic field, ultrasonic wave, and light have been extensively studied.12 Among them, poly(N-isopropylacrylamide) (PNIPAAm) is one of the well-known thermoresponsive polymers exhibiting a lower critical solution temperature (LCST) behavior around 32 °C in aqueous solution.13 Below the LCST, PNIPAAm chains are hydrated and elongated, but the polymer chain is dehydrated and aggregated with raising temperature above it. When they are cross-linked, polyNIPAAm hydrogels exhibit reversible swelling and deswelling behaviors around the LCST. It was reported that PNIPAAmgrafted cell culture substrates exhibited thermoresponsive cell attachment-detachment properties, leading to the facile production of tissue engineered “cell sheet”.14,15 In this study, thermally sensitive QDs (TSQDs) modified with PNIPAAm and CPPs were fabricated to demonstrate thermally responsive cellular uptake of nanoparticles. QDs were used as a model nanoparticle to examine the extent of cellular uptake because of their superior optical properties such as stability toward photobleaching, controllable emission bands, broad absorption spectra, and high quantum yields.16,17 CPPs are positively charged (11) Malek, A.; Czubayko, F.; Aigner, A. J. Drug Target. 2008, 16, 124–139. (12) Bawa, P.; Pillay, V.; Choonara, Y. E.; Toit, L. C. Biomed. Mater. 2009, 4, 1–15. (13) Schmaljohann, D. Adv. Drug Delivery Rev. 2006, 58, 1655–1670. (14) Mizutani, A.; Kikuchi, A.; Yamato, M.; Kanazawa, H.; Okano, T. Biomaterials 2008, 29, 2073–2081. (15) Ebara, M.; Yamato, M.; Aoyagi, T.; Kikuchi, A.; Sakai, K.; Okano, T. Biomaterials 1995, 29, 3650–3655. (16) Medintz, I. L.; Tetsuouyeda, H.; Goldman, E. R.; Mattoussi, H. Nature Mater. 2005, 4, 435–446. (17) Gill, R.; Zayats, M.; Willner, I. Angew. Chem., Int. Ed. 2008, 47, 7602–7625.
Published on Web 08/19/2010
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and short amphiphilic peptides, which can be efficiently internalized within cells.18 It has been reported that intracellular delivery of macromolecules and nanoparticles could be facilitated by conjugating a CPP moiety. Although CPPs are potent mediators for intracellular delivery of macromolecules, lack of cell specificity limits their use in systemic delivery to target tissues. In addition, cationic CPPs presented on the surface of nanoparticles may interact with negatively charged blood components such as albumin when localized in the bloodstream. The surfacelocalized CPP peptide can be readily degraded by the attack of extracellular proteases. Surface modification with a hydrophilic polymer such as PEG may reduce the nonspecific adsorption of serum-derived molecules and the enzyme-mediated degradation of CPP molecules. However, high molecular weight PEG may interfere with the proper contact of CPP to the cells, leading to reduced CPP-mediated intracellular transduction of a cargo molecule. Therefore, for cell specific intracellular delivery, external stimuli-responsive shielding/deshielding of CPP on the surface of nanoparticles is highly desirable. It was hypothesized that thermally induced cellular uptake of QDs could be achieved by cografting PNIPAAm chains and CPPs onto the surface. Hydrated and elongated PNIPAAm chains below the LCST sterically shielded the co-immobilized CPPs for cellular recognition, but dehydrated and collapsed PNIPAAm chains above the LCST allowed the CPPs to be surface exposed, resulting in facile cellular interactions. Furthermore, based on the thermally triggered cellular uptake, TSQDs were applied to a small interfering RNA (siRNA) delivery system. The level of green fluorescent protein (GFP) gene silencing was controlled by modulating temperature below and above the LCST.
Materials and Methods Materials. N-Isopropylacrylamide (NIPAAm), 3-mercaptopropionic acid (MPA), cystamine, biotin-maleimide, HABA/Avidin reagent, and anhydrous dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO). 1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) was obtained from Tokyo Chemical Industry Co. (Tokyo, Japan). Streptavidin-coated quantum dots (QDs-Strep, 12 nm) were purchased from Invitrogen (Carlsbad, CA). 2,20 -Azobis(isobutyronitrile) (AIBN) and N,Ndimethylformamide (DMF) were purchased from Junsei Chemical Co. (Tokyo, Japan). Dithiothreitol (DTT) was obtained from Duchefa Biochemie (Haarlem, The Netherlands). N-Hydroxysulfosuccinimide (Sulfo-NHS) was purchased from Pierce Chemical Co. (Rockford, IL). Petroleum ether and diethyl ether were purchased from Daejung Chemicals & Metals Co. (Siheung, South Korea). Biotin-conjugated GFP siRNA (GFP siRNA-biotin) composed of sense (50 -AACUUCAGGGUCAGCUUGCdTdT-30 ) and antisense (50 -GCAAGCUGACCCUGAAGUUdTdT-30 ) strands were purchased from Dharmacon Research Inc. (Lafayette, CO). GFP overexpressing MDA-MB-435 (human breast cancer cell) cells were donated by Samyang Corp. (Daejeon, South Korea). N-terminal biotin-conjugated cell-penetrating peptide (CPP-biotin, biotinYARVRRRGPRR) was synthesized from Peptron Inc. (Daejeon, South Korea). Fetal bovine serum, phosphate buffered saline (PBS) solution, and Dulbecco’s Modified Eagle Medium (DMEM) were purchased from Invitrogen (Carlsbad, CA). Synthesis and Characterization of Semitelechelic Poly(N-isopropylacrylamide) (PNIPAAm-COOH). N-Isopropylacrylamide (NIPAAm) was purified by recrystallization from petroleum ether and dried at room temperature under reduced (18) Zorkoa, M.; Langel, U. Adv. Drug Delivery Rev. 2005, 57, 529–545. (19) Takei, Y. G.; Aoki, T.; Sanui, K.; Ogata, N.; Okano, T.; Sakurait, Y. Bioconjugate Chem. 1993, 4, 42–46.
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pressure. Semitelechelic polymerization of NIPAAm was done as previously reported.19 To a solution of NIPAAm (1 g, 8.84 mmol) dissolved in DMF (8.84 mL), MPA (9.38 mg, 88.4 mmol) and AIBN (0.58 g, 3.54 mmol) were added. The reaction mixture was stirred for 5 h at 70 °C. The reactant was precipitated with diethyl ether and dried. Molecular weight of PNIPAAm-COOH was determined by gel permeation chromatography (GPC, Waters) using a refractive index detector (RI-71, Shodex) in DMF at 25 °C. The transmittance value of PNIPAAm-COOH solution (0.1 wt %) was measured at different temperatures at 500 nm using a spectrophotometer (UV-1601, Shimadzu). Before each measurement, temperature was stabilized at least for 10 min.
Synthesis of Biotin-Conjugated Poly(N-isopropylacrylamide) (PNIPAAm-biotin). To 5 mL of phosphate buffered saline (PBS) solution (pH 7.4) containing PNIPAAm-COOH (100 mg), EDC (38.4 mg, 100 mmol) and Sulfo-NHS (43.6 mg, 100 mmol) in 2.5 mL of PBS solution were added. After activating carboxylic acid groups, cystamine (15.2 mg, 100 mmol) dissolved in 2.5 mL of PBS solution was added. The reaction mixture was stirred for 4 h at room temperature. The reactant was treated with DTT (154.2 mg, 1 mmol) to reduce the disulfide bond of cystamine. After 1 h, the solution was dialyzed (Mw cutoff: 5 kDa) against deionized water for 6 h and then lyophilized. The yield of a thiolmodified product, PNIPAAm-SH, was estimated by using Ellman’s method. To a solution of anhydrous DMSO (1 mL) containing PNIPAAm-SH (50 mg), biotin-maleimide (25 mg, 55.4 mmol) in anhydrous DMSO (1 mL) was added. The reaction mixture was stirred for 12 h and then dialyzed (Mw cutoff: 5 kDa) against deionized water for 2 days. The resulting PNIPAAmbiotin was lyophilized and stored at -20 °C. The yield of PNIPAAm-biotin was determined by HABA/Avidin assay.20
Preparation and Characterization of Quantum Dots Grafted with Poly(N-isopropylacrylamide) and Cell Penetrating Peptides (TSQDs). The number of biotin binding sites
on a single QD-Strep was measured as previously reported.19 QDs-Strep was incubated with CPP-biotin for 15 min to immobilize CPP onto the surface, and then PNIPAAm-biotin was also incubated with the CPP-immobilized QDs for 30 min to prepare TSQDs (molar ratio of QD-Strep:CPP-biotin:PNIPAAmbiotin = 1:10:10). The immobilization of PNIPAAm, CPP, and/ or siRNA onto the surface of QDs-Strep was confirmed by gel retardation assay. The prepared TSQDs were loaded onto 0.5% agarose gel, and electrophoresis was run at 100 V for 40 min. The QDs were visualized by using a UV illuminator. Evaluation of Cellular Uptakes. To analyze the extent of cellular uptake quantitatively, the prepared TSQDs (5.0 pmol) were treated to GFP overexpressing MDA-MB-435 cells seeded at a density of 1.5 105 cells per well in a 12-well plate. After 1 h further incubation at 25 and 37 °C, the cells were washed with PBS solution twice and treated with a cell lysis solution (1% TritonX100 in PBS solution). The relative amounts of TSQDs within cells were determined by measuring the fluorescence intensity of QDs using a spectrofluorophotometer (SLM-AMINCO 8100, SLM Instrument Inc., Rochester, NY) with an excitation and an emission wavelength of 488 and 509 nm, respectively. To visualize the cellular uptake, TSQDs (2.0 pmol) were treated to GFP overexpressing MDA-MB-435 cells incubated for 24 h at a density of 1.0 105 cells per well in a chamber slide. After 1 h further incubation at 25 and 37 °C, the cells were washed with PBS solution twice and were fixed by 1% paraformaldehyde for 30 min. The prepared samples were visualized by confocal microscopy (LSM510, Carl-Zeiss Inc.). Transfection. GFP siRNA-biotin (50.0 pmol) was additionally incubated for 30 min with TSQDs (5.0 pmol) to formulate GFP siRNA-immobilized TSQDs (TSQDs/GFP siRNA). The TSQDs/GFP siRNA were treated to GFP overexpressing (20) Mok, H.; Bae, K. H.; Ahn, C.-H.; Park, T. G. Langmuir 2009, 25, 1645– 1650.
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Figure 1. (a) Scheme for synthesizing PNIPAAm-biotin. (b) Schematic illustration of TSQDs and their CPP “shielding/deshielding” behavior for modulating cellular uptake in response to temperature. MDA-MB-435 cells seeded at a density of 1.5 105 cells per well in a 12-well plate in a serum-free condition. After further 4 h incubation at 25 and 37 °C, the transfection medium was replaced with a fresh serum-free medium, and the cells were incubated for 2 days. To evaluate the GFP gene silencing effect, the transfected cells were harvested by treating with a cell lysis solution (1% TritonX-100 in PBS solution). The cell lysates were analyzed by a spectrofluorophotometer (SLM-AMINCO 8100, SLM Instrument) with an excitation and an emission wavelength at 488 and 509 nm, respectively.
Results and Discussion Rationally designed and multifunctional nanoparticles could overcome several biological delivery barriers, such as a body surveillance system and transport across cellular membrane, for cell specific delivery and offer concurrent therapeutic and diagnostic (theragnostic) modalities. Generally, theragnostic nanoparticles include single or multimodal probes for molecular imaging or sensor, therapeutically active agents for the direct treatment of a target disease, and an appropriate surface modifier for the maintenance of stable colloidal property in the presence of serum-derived components. It has long been reported that surface modification with hydrophilic polymers such as PEG can not only greatly enhance colloidal stability of nanoparticles in the bloodstream but also improve their tissue distribution and absorption behaviors. In addition, cell or tissue specific moieties were often introduced onto the surface of the nanoparticles for preferential accumulation in target cells. In most cases, however, efficient ligand-mediated targeting could be only achieved by hiding the ligand under the protection of hydrophilic layers before reaching target cells because some ligands could also be recognized by other tissues. For example, folic acid, a targeting ligand popularly employed for targeting cancer cells, could also be recognized and taken up in the liver due to the presence of excessive folate receptors.21 In addition, ligands such as peptide or nucleic acid-based ligands and aptamers (21) Vives, E.; Brodin, P.; Lebleu, B. J. Biol. Chem. 1997, 272, 16010–16017.
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may be susceptible to attack of extracellular proteases or nucleases,22 respectively, unless they are properly protected or rapidly distributed to a target site. In this study, a multifunctional nanoparticle-based theragnostic delivery system that triggers ondemand cellular uptake at the desired site by applying stimulus was fabricated using a thermosensitive polymer, PNIPAAm. QDs grafted with PNIPAAm and CPP are expected to show prolonged circulation in the bloodstream but exhibit enhanced intracellular transduction of QDs within target cells by selectively resurfacing CPP moieties by a thermal stimulus above the LCST. As shown in Figure 1a, PNIPAAm-COOH was prepared by radical polymerization of NIPAAm with MPA using AIBN as an initiator.19 Mn and Mw of PNIPAAm-COOH were calculated to be 7K and 11.5K (PDI = 1.6), respectively. To prepare thiolterminated PNIPAAm (PNIPAAm-SH), PNIPAAm was conjugated cystamine, and the disulfide bond was reduced. The degree of thiol modification was 64.2 ( 7.8%, as determined from Ellman’s method. Biotin-terminated PNIPAAm (PNIPAAm-biotin) was prepared by conjugating biotin-maleimide and PNIPAAm-SH via Michael-type addition. The HABA/Avidin assay revealed that the degree of biotin modification was 42.5 ( 3.4%. To design thermally responsive CPP “shielding/deshielding” QDs for controlled cellular uptake, QDs immobilized with streptavidin (the number of biotin binding site per QD-Strep was 28.6 ( 4.0) were coated with PNIPAAm and CPPs via biotin-streptavidin interactions. Since the biotin-avidin binding is strong (Kd ∼ 10-15 M), it was assumed that PNIPAAm and CPPs could be stably and stoichiometrically immobilized onto the surface of QDs. CPPs are positively charged and short amphiphilic peptides, which can efficiently penetrate cell membranes with translocating different cargos into cells.18 Although the cell penetration mechanism of CPPs is not clearly understood, a number of previous studies showed efficient cell penetration at low temperature (22) Oehlke, J.; Scheller, A.; Wiesner, B.; Krause, E.; Beyermann, M.; Klauschenz, E.; Melzig, M.; Bienert, M. Biochim. Biophys. Acta 1998, 1414, 127–139. (23) Torchilin, V. P.; Rammohan, R.; Weissig, V.; Levchenko, T. S. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 8786–8791.
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Figure 2. Gel retardation assay for various TSQDs.
(0-4 °C) and even in the presence of different endocytosis inhibitors.18,23-25 Among various CPPs, a human transcription factor Hph-1 derived sequence (YARVRRRGPRR) was used in this study. The Hph-1 is known to efficiently facilitate the transduction of a large protein in vitro and in vivo with fast accumulation near the nucleus and in the cytoplasm of the treated cells.26 It was previously shown that many nanoparticles coated with CPPs easily penetrated into the cellular membrane in vivo and in vitro, enhancing their cellular uptake.27-30 By introducing additional PNIPAAm chains onto the surface of QDs/CPP, thermally responsive CPP “shielding/deshielding” QDs could be attained for their “on-demand” cellular uptake by modulating temperature. A schematic illustration for the cellular uptake behavior of TSQDs below and above the LCST is shown in Figure 1b. Below the LCST, PNIPAAm chains on the surface of TSQDs are an extended and hydrophilic state, suppressing their cellular uptake by sterically shielding the CPPs. Above the LCST, PNIPAAm chains become dehydrated and collapsed, resulting in facilitating the cellular uptake of TSQDs by resurfacing the CPPs. The immobilization of CPP-biotin, siRNA-biotin, and PNIPAAm-biotin onto the surface of streptavidin-coated QDs (QDsStrep) was confirmed by gel retardation assay. Figure 2a shows that CPP-biotin was successfully immobilized to the surface of QDs-Strep. Because QDs-Strep is negatively charged due to the presence of streptavidin, QDs-Strep showed a migration tendency (24) Choi, J.-M.; Ahn, M.-H.; Chae, W.-J.; Jung, Y.-G.; Park, J.-C.; Song, H.-M.; Kim, Y.-E.; Shin, J.-A.; Park, C.-S.; Park, J.-W.; Park, T.-K.; Lee, J.-H.; Seo, B.-F.; Kim, K.-D.; Kim, E.-S.; Lee, D.-H.; Lee, S.-K.; Lee, S.-K. Nature Med. 2006, 12, 574–579. (25) Lo, S. L.; Wang, S. Biomaterials 2008, 29, 2408–2414. (26) Song, H. P.; Yang, J. Y.; Lo, S. L.; Wang, Y.; Fan, W. M.; Tang, X. S. Biomaterials 2010, 31, 769–778. (27) Wang, Y.; Liu, X.; Nakamura, K.; Chen, L.; Rusckowski, M.; Hnatowich, D. J. Cancer Biother. Radiopharm. 2009, 24, 573–578. (28) Pujals, S.; Bastffls, N. G.; Pereiro, E.; Lopez-Iglesias, C.; Puntes, V. F.; Kogan, M. J.; Giralt, E. ChemBioChem 2009, 10, 1025–1031. (29) Medintz, I. L.; Pons, T.; Delehanty, J. B.; Susumu, K.; Brunel, F. M.; Dawson, P. E.; Mattoussi, H. Bioconjugate Chem. 2008, 19, 1785–1795. (30) Derfus, A. M.; Chen, A. A.; Min, D.-H.; Ruoslahti, E.; Bhatia, S. N. Bioconjugate Chem. 2007, 18, 1391–1396.
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toward the (þ) electrode. After the immobilization of CPPs onto the surface, QDs/CPP migrated toward the (-) electrode. The gel retardation results for siRNA-biotin immobilization onto the surface of QDs-Strep are shown in Figure 2b. The result displayed increased migration length of QDs modified with increasing amount of negatively charged siRNA-biotin, suggesting that siRNA-biotin was stably bound to the surface of QDs. The observed smearing effect of gel band intensity resulted from a heterogeneous population of siRNA bound QDs-Strep with different degrees of siRNA immobilization. To confirm the grafting effect of PNIPAAm-biotin, different amounts of PNIPAAm-biotin were grafted onto the surface of QDs/CPP, and then siRNA-biotin was immobilized. Because of the shielded charge and increased hydrodynamic size of QDs by immobilized PNIPAAm chains, Figure 2c shows that the mobility of TSQDs/ siRNA was decreased as the grafting amount of PNIPAAm was increased, indirectly suggesting the shielding effect of PNIPAAm chains on the surface of QDs. To quantitatively analyze the extent of cellular uptake of TSQDs below and above the LCST, GFP-overexpressing MDA-MB-435 cells were treated with various TSQDs, and the cells were lysed to measure the intracellular fluorescence intensity of QDs. As shown Figure 3a, when QDs/CPP (QD/CPP = 1/10) incubated at 37 °C were used as a control (100% cellular uptake), the relative cellular uptake extents of TSQDs (QD/CPP/PNIPAAm = 1/10/10) at 25 and 37 °C were 52.4 ( 6.7% and 86.2 ( 4.8%, respectively. The control QD/CPP without PNIPAAm exhibited about 9.6% increment of cellular uptake when raising temperature from 25 to 37 °C, which could be attributed to the enhanced thermal collision frequency of QDs/CPP onto the cell membrane. The significant recovery of cellular uptake above the LCST suggests that CPPs were indeed sterically shielded by surface extended PNIPAAm chains below the LCST but were deshielded above it. It should be noted that the relatively high basal level of cellular uptake at 25 °C (52.4 ( 6.7%) might be caused by nonspecific endocytosis. The co-immobilization of PNIPAAm and siRNA on the surface of bare QDs-Strep showed similarly low extents of cellular uptake below and above the Langmuir 2010, 26(18), 14965–14969
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The additional immobilization of siRNA might reduce the cellular uptake, since siRNA and CPP could be partially complexed via charge interactions to mask CPP exposure. For the visualization of cellular uptake, confocal images were obtained as shown in Figure 3b. For QDs/CPP, there was no significant difference between the extents of cellular uptake between 37 and 25 °C. In contrast, TSQDs showed considerably reduced cellular uptake at 25 °C as compared to 37 °C. The confocal images were consistent with the observed quantitative analysis. GFP siRNA-biotin (50.0 pmol) was additionally immobilized to TSQDs (QD/CPP/polyNIPAAm = 1/10/10) to test whether intracellularly delivered siRNA showed thermally responsive gene silencing behaviors. As shown in Figure 3a, TSQDs/GFP siRNA (QD/CPP/polyNIPAAm/siRNA = 1/10/10/10) showed the lower extent of cellular uptake as compared with TSQDs without siRNA, resulting from increased surface negative charges and charge interactions between immobilized siRNAs and CPPs. However, the thermally responsive cellular uptake tendency was not changed by introducing GFP siRNA to TSQDs. The cells treated only with naked siRNA-biotin were used as a control of 0% gene silencing. As shown Figure 3c, the relative GFP knockdown levels of TSQDs-GFP siRNA at 25 and 37 °C were 4.2 ( 1.8% and 19.9 ( 0.9%, respectively. The levels of GFP silencing in this study are comparable with those of previous study using QDs-based GFP siRNA delivery. On the basis of the observed results of thermally responsive cellular uptake and gene silencing using co-immobilized PNIPAAm and CPP on the surface of nanoparticles, other pH-, photo-, and metabolite-sensitive “on-demand” delivery systems could be molecularly designed. A variety of hydrophilic polymers that show a reversible solubility change behavior in response to an external stimulus can be similarly grafted on the surface, leading to the “shielding/deshielding” of CPP for modulating cellular uptake. For practical applications, it would be important to synthesize thermo- or pH-sensitive water-soluble polymers that exhibit a sharp solubility transition near body temperature or physiological pH. Locally induced hyperthermia or naturally occurring acidic pH environment near the tumor tissue could be applied or utilized for the treatment of cancer.
Conclusion
Figure 3. (a) Quantitative analysis of cellular uptake for various TSQDs. (b) Confocal images of GFP overexpressing MDA-MB435 cells treated with QDs/CPP, QDs/PNIPAAm, and TSQDs after incubating at 37 and 25 °C for 1 h. (c) GFP silencing efficiencies of siRNA only (control), QDs/CPP/GFP siRNA, and TSQDs/GFP siRNA for GFP overexpressing MDA-MB 435 cells.
LCST. When PNIPAAm and siRNA were co-immobilized onto the surface of QDs-CPP (TSQDs), enhanced cellular uptake could be seen above the LCST, although the extent of cellular uptake was slightly reduced as compared to those without siRNA.
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We developed PNIPAAm and CPP co-immobilized QDs to modulate cellular uptake via thermosensitive “shielding/deshielding” of CPPs on the surface below and above the phase transition temperature. Extended and collapsed PNIPAAm chains enabled co-immobilized CPPs to hide and expose for cellular interaction in response to temperature. By further immobilizing siRNA, gene silencing activity could be further modulated by temperature. A variety of organic/inorganic nanoparticles could be similarly conjugated with PNIPAAm, CPP, and siRNA on the surface to confer heat-sensitive “on-demand” cellular uptake and gene silencing. Acknowledgment. This study was supported by the Intelligent Drug Delivery system grant and the program from the Ministry of Education, Science and Technology, Republic of Korea.
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