APRIL 2008 Volume 19, Number 4 Copyright 2008 by the American Chemical Society
COMMUNICATIONS Enhanced Intracellular Delivery of Quantum Dot and Adenovirus Nanoparticles Triggered by Acidic pH via Surface Charge Reversal Hyejung Mok, Ji Won Park, and Tae Gwan Park* Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea. Received December 14, 2007; Revised Manuscript Received February 14, 2008
Quantum dot (QD) and adenovirus (ADV) nanoparticles were surface-modified with graft copolymers that exhibited a charge reversal behavior under acidic condition. Poly(L-lysine) (PLL) was grafted with multiple biotin-PEG chains (biotin-PEG-PLL graft copolymer), and the remaining primary amine groups in the PLL backbone were postmodified using citraconic anhydride, a pH-sensitive primary amine blocker, to generate carboxylate groups. The surfaces of streptavidin-conjugated QDs were modified with citraconylated biotin-PEG-PLL copolymer, producing net negatively charged QD nanoparticles. Under acidic conditions, citraconylated amide linkages were cleaved, resulting in the recovery of positively charged amine groups with subsequent alteration of surface charge values. Intracellular delivery of QD nanoparticles was greatly enhanced in an acidic pH condition due to the surface charge reversal. The surface of avidin-conjugated adenovirus (ADV-Avi) encoding an exogenous green fluorescent protein (GFP) gene was also modified in the same fashion. The expression extent of GFP was significantly increased at more acidic pH than pH 7.4. This study demonstrates that various nanosized drug carriers, imaging agents, and viruses could be surface-engineered to enhance their cellular uptake specifically at a low pH microenvironment like solid tumor tissue.
For the past decade, significant endeavors have been devoted to surface modification of various nanosized drug and imaging agent carriers such as iron oxide nanoparticles, quantum dots, liposomes, polymeric micelles, polymeric nanoparticles, and viruses to target them specifically to desired cells and tissues (1-7). In particular, targeted delivery systems for tumor cells are highly desirable to enhance therapeutic and diagnostic efficacy with reducing undesirable side effects. Targeting ligands including peptides, antibodies, carbohydrates, and aptamers, which specifically bind to the receptors overexpressed only in cancer cells, have been popularly tethered on the surface of * Corresponding author. Tel: +82-42-869-2621; fax: +82-42-8692610. E-mail address:
[email protected].
nanocarriers to deliver them to the tumor tissue in a more specific manner. As alternative approaches, minute physiological changes in the environment of tumor tissues different from that of normal tissues have been utilized as triggering signals to nanocarriers for cancer treatment and tumor imaging (8-10). It is well-known that human and rodent solid tumor tissues exhibit a more acidic microenvironment than normal tissues, and their extracellular pH ranges from 5.8 to 7.4 (11, 12). Thus, a variety of pH-responsive nanoparticles were widely explored to increase the extent of cellular uptake only at the acidic tumor region. A wide array of pH-sensitive moieties, such as polyhistidine, β-amino esters, and sulfonamide, and acidic-cleavable linkages such as acetal, hydrazone, and orthoester have been employed in combination to produce multifunctional nanocar-
10.1021/bc700464m CCC: $40.75 2008 American Chemical Society Published on Web 03/26/2008
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Figure 1. (A) The relative percent of primary amine after amine-blocking reaction by citraconic anhydride at various citraconic anhydride/amine molar ratios. (B) The relative percent of remaining amine group in the citraconylated biotin-PEG-PLL after incubation at pH 5.8 (triangle), 6.6 (square), and 7.4 (circle) at 37 °C as a function of incubation time. The error bars mean standard deviation.
Figure 2. Confocal microscopy images of intracellular QD coated with citraconylated biotin-PEG-PLL and incubated for 3 h at (B) pH 7.4, (C) pH 6.6, and (D) pH 5.8 conditions for HeLa cells. The cells treated with (A) QD coated with as-prepared citraconylated biotin-PEG-PLL and (E) QD coated with noncitraconylated biotin-PEG-PLL were used as negative and positive controls, respectively.
riers that enabled the release of encapsulated or conjugated chemical and biological drugs in response to acidic pH (13-16). It was recently demonstrated that cationic diblock polymers containing primary amine groups, when conjugated with citraconic anhydride, showed charge reversal from negative to positive by cleavage of citraconic amide linkage (16). The charge reversal diblock copolymers were used as nanocontainers for releasing cationic enzymes only in acidic pH. In this communication, we demonstrated that QD nanoparticles surfacemodified with amine-blocked biotin-PEG-grafted PLL copolymer by citraconylation could be delivered efficiently into cells under acidic condition via a surface charge reversal from negative to positive. ADV was also similarly modified to selectively enhance viral gene expression under the acidic condition. A copolymer of biotin-poly(ethylene glycol) (PEG) grafted poly(L-lysine) (PLL) (biotin-PEG-PLL) was synthesized by reacting primary amine groups of PLL (average Mw: 1100, as determined from matrix-assisted laser desorption/ionization timeof flight (MALDI-TOF) mass spectroscopy, Supporting Information Figure S1) with a biotin-PEG-N-hydroxysuccinimide derivative (biotin-PEG-NHS). The resultant graft copolymer had ca. 2.5 biotinylated PEG chains per PLL backbone as characterized by 1H NMR (Supporting Information Figure S2). The remaining primary amine groups of the PLL in the graft copolymer were further modified with a pH-specific amine blocker, citraconic anhydride. Citraconic anhydride has been
known to be able to change the net charge of cationic polymers and peptides by replacing primary amine groups with carboxylate groups (17). After citraconylation reaction for 2 h at various molar ratios of citraconic anhydride/amine, the amount of remaining amine group in the PLL was measured by fluorescamine assay. As shown in Figure 1A, ∼60% of total amine groups in the biotin-PEG-PLL was modified at the citraconic anhydride/amine molar ratio of 0.7, suggesting that the net charge of biotin-PEG-PLL under that condition was slightly negative. The biotin-PEG-PLL copolymer with a citraconylation substitution degree of 60% was used for further study. To show the charge regeneration of primary amine groups in the citraconylated conjugate at different pH conditions, the amount of primary amine groups was quantitatively analyzed by fluorescamine assay after incubating at different pH conditions. Previously, it was reported that the amide bond formed by citraconylation reaction with primary amine group could be specifically cleaved in acidic pH due to a neighboring carboxylic acid group (16, 17). When the citraconylated conjugate was incubated at pH 5.8, 6.6, and 7.4, the relative amounts of exposed amine groups were 87.1 ( 2.4%, 68.1 ( 2.3%, and 42.7 ( 5.0% after 5 h incubation, respectively. The extent of regenerated amine group was significantly increased at pH 5.8 and 6.6 in a time-dependent manner, indicating that the net charge in the conjugate was recovered from negative to positive only in the acidic conditions.
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Figure 3. (A) Surface charge on ADV coated with citraconylated biotin-PEG-PLL incubated at pH 5.8, 6.6, and 7.4. (B) Confocal microscopy images of adenoviral gene, GFP expression for HeLa cells transfected with ADV coated with citraconylated biotin-PEG-PLL (3.4 × 103 per viral particles) after incubating at pH 5.8 and 7.4, respectively. (C) The extent of relative GFP expression (%) for HeLa cells transfected with ADV coated with various amounts of citraconylated biotin-PEG-PLL copolymer after incubating at pH 5.8 and 7.4. Naked ADV was used as a control in all experiments. Scheme 1. (A) Synthesis of Citraconylated Biotin-PEG-PLL Copolymer and Its Charge Reversal at Acidic pH; (B) Surface Coating with Citraconylated Biotin-PEG-PLL onto Streptaividin-Quantum Dot and Avidin-Adenovirus Nanoparticles
The resultant citraconylated biotin-PEG-PLL graft copolymer was immobilized onto the surface of streptavidin-conjugated
QDs (QD-stp, 5-20 nm) via biotin-streptavidin interaction. Dynamic light scattering (DLS) measurement showed that QD-
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stp and citraconylated biotin-PEG-PLL/QD-stp nanoparticles have hydrodynamic sizes of 12.0 ( 1.6 nm and 14.8 ( 5.8 nm, respectively. After incubating in buffer solutions of different pH values 5.8, 6.6, and 7.4 for 3 h, citraconylated biotin-PEGPLL/QD-stp nanoparticles were treated to HeLa cells to show the intracellular uptake extent of QD. Cellular images visualized by confocal microscopy revealed that QD nanoparticles incubated at pH 5.8 and pH 6.6 were more significantly taken up by the cells than those at pH 7.4 (Figure 2). The enhanced intracellular uptake in the acidic condition was also analyzed using flow cytometry. As shown in Supporting Information Figure S3, a red fluorescent peak for QD in pH 5.8 was noticeably shifted to the right-hand side, compared to that at pH 7.4, suggesting that more efficient intracellular uptake occurred in the acidic condition. It is obvious that the net charge of the surface-anchored citraconylated conjugate was reversed from negative to positive, thereby facilitating nonspecific endocytic uptake of QD nanoparticles by increasing their charge interactions with the cell membrane. It was possible that some portion of the regenerated cationic PLL backbones was likely to be adsorbed onto the anionic surface of bare QD-stp nanoparticles via electrostatic interactions, resulting in the formation of multiple PEG loops as illustrated in Scheme 1 B. ADV, a naturally evolved and well-defined nanoparticle, is approximately 60-90 nm in diameter. Although ADV shows high gene transfection efficiency in mammalian cells, its use as a gene carrier has been limited mainly due to nonspecific transfection and innate immune responses. To confer targetspecific delivery capacity and reduced immune response, ADV was often PEGylated by tethering various targeting ligands to specific cells (6, 18, 19). In this study, surface charge reversal by citraconylated conjugate in response to acidic pH was also applied to ADV encoding for green fluorescent protein (GFP). To immobilize the citraconylated biotin-PEG-PLL copolymer onto the viral surface, avidin (avi) was preconjugated onto the ADV surface using the EDC/NHS reaction. The citraconylated biotin-PEG-PLL/ADV-avi nanoparticles were prepared via biotin-avidin interaction in a similar manner to QD-stp. Surface charge values after incubating at pH 5.8, 6.6, and 7.4 for 5 h were determined by a dynamic light scattering technique (Figure 3A). The surface charge value of naked ADV was about -20 mV at pH 7.4 due to the presence of anionic viral coat proteins (4). When citraconylated biotin-PEG-PLL/ADV-avi complex nanoparticles were incubated at pH 7.4, 6.6, and 5.8, the surface charge values became -12.3 ( 0.8, -4.7 ( 0.9, and -2.3 ( 1.2 mV, respectively. This suggests that the charge reversal on the surface of ADV occurred in response to acidic pH. The GFP expression level of ADV was determined as a function of pH using confocal microscopy as shown in Figure 3B. The cells transfected with citraconylated biotin-PEG-PLL/ ADV-avi nanoparticles at pH 7.4 showed significantly reduced extent of GFP gene expression, compared to those at pH 5.8. It is known that naked ADV nanoparticles were transported into cells via coxsackievirus-adenovirus receptor (CAR) mediated endocytosis (19, 20). Surface-exposed fiber proteins on ADV were initially docked to the CAR on the cell membrane for cellular entry. After modifying the surface of ADV-avi with citraconylated biotin-PEG-PLL copolymers, the CAR-mediated mechanism would play a minor role in cellular uptake, because most of the fiber proteins on the surface were likely to be chemically conjugated with avidin and, more importantly, to be sterically inaccessible to the CAR by the presence of immobilized biotin-PEG-PLL copolymers. Thus, the citraconylated biotin-PEG-PLL/ADV-avi nanoparticles might be taken up by cells via a nonspecific endocytic pathway. The levels of GFP expression treated with ADV-avi coated with different amounts of citraconylated biotin-PEG-PLL were quantitatively
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analyzed at pH 7.4 and pH 5.8 using naked ADV as a control (GFP expression: 100%), as shown in Figure 3C. The relative extents of GFP expression for the cells transfected by ADVavi coated with 0.9 × 103, 1.7 × 103, and 3.4 × 103 citraconylated biotin-PEG-PLL copolymers per viral particle were 122.2 ( 9.7%, 101.7 ( 2.5%, and 89.2 ( 7.8%, respectively, at pH 5.8, but they were 91.7 ( 7.2%, 72.8 ( 12.8%, and 63.8 ( 5.8% at pH 7.4 under the same condition. The results imply that the surface charge reversal of surfaceengineered ADV nanoparticles, triggered by acidic pH condition, enhanced the extent of nonspecific cellular uptake and subsequently maintained the extent of viral gene expression to be similar to that of naked ADV. Since the surface charge value of fully regenerated biotin-PEG-PLL/ADV-avi nanoparticles at pH 5.8 was still negative at -2.3 ( 1.2 mV as shown in Figure 3A, it was postulated that the recovered cationic PLL chains were in part bound onto the anionic surface of ADV-avi by charge interactions, making PEG loops as shown in Scheme 1B. Although complete surface charge reversal (a net charge change from negative to positive) of citraconylated biotin-PEGPLL/ADV-avi was not observed at acidic pH due to the rearrangement of PEG chains, a significant change in the surface charge value of ADV toward a positive direction could lead to enhanced cellular uptake and gene expression. In conclusion, we demonstrated that QD and ADV nanoparticles immobilized by citraconylated biotin-PEG-PLL exhibited surface charge reversal at acidic pH. It was possible to deliver them into cells specifically under acidic conditions. The current targeting strategy is expected to have a wide range of applications for delivery of imaging and therapeutic agents to solid tumors that are known to have an acidic microenvironment.
ACKNOWLEDGMENT This study was supported by grants from the National Research Laboratory project, the Ministry of Science and Technology, and the National Cancer Center, Republic of Korea (0620240-1). Supporting Information Available: Detailed materials and methods. This material is available free of charge via the Internet at http://pubs.acs.org.
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