Self-Assembling Micelle-like Nanoparticles with Detachable

Jan 21, 2014 - Fax: 82-32-820-4829. E-mail: [email protected]. ... Ji Hee Kang , Gantumur Battogtokh , and Young Tag Ko. Biomacromolecules 2017 ...
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Self-Assembling Micelle-like Nanoparticles with Detachable Envelopes for Enhanced Delivery of Nucleic Acid Therapeutics Gantumur Battogtokh and Young Tag Ko* College of Pharmacy, Gachon University, Incheon, South Korea 406-799 S Supporting Information *

ABSTRACT: In spite of the great potential of nucleic acids as therapeutic agents, the clinical application of nucleic acid therapeutics requires the development of effective systemic delivery strategies. In an effort to develop effective nucleic acid delivery systems suitable for clinical application, we previously reported a self-assembling micelle-like nanoparticle that was based on phospholipid−polyethylenimine conjugates, i.e., “micelle-like nanoparticles” (MNPs). In this study, we aimed to improve the system by enhancing the efficiency of intracellular delivery of the payload via pH-responsive detachment of the monolayer envelope and release of the nucleic acid therapeutics upon reaching the target tissues with an acidic pH, e.g., tumors. The acid-cleavable phospholipid−polyethylenimine conjugate was synthesized via hydrazone bond, and acid-cleavable MNPs were then prepared and characterized as before. We evaluated the acid-cleavable MNP construct for in vitro and in vivo nucleic acid delivery efficiency using cultured tumor cells and tumor-bearing mice. The acid-cleavable nanocarrier showed an enhanced cellular delivery at pH 6.5 as compared to pH 7.4, whereas the noncleavable nanocarrier did not show any differences. Tail vein injections also led to enhanced intracellular uptake of the acid-cleavable nanocarrier compared to the noncleavable nanocarrier into tumor cells of tumor-bearing mice although no significant difference was observed in total tumor accumulation. KEYWORDS: self-assembling nanoparticles, nucleic acid therapeutics, polyethylenimine, phospholipid, deshielding, micelle-like nanoparticle

1. INTRODUCTION Nucleic acid materials can be used for the therapeutic modulation of gene expression in the form of siRNA and antisense and decoy oligonucleotides (ODN), as well as for the replacement of defective or missing genes by the expression of exogenously introduced genes. However, most nucleic acid therapeutics have not been shown to be applicable for systemic application due to their very short half-life in the bloodstream and inability to cross cell membranes.1 Because the delivery of nucleic acid therapeutics to disseminated and widespread disease sites such as metastasized tumors and inflamed tissues can only be achieved by systemic administration, the development of efficient delivery systems suitable for systemic application is crucial to the success of nucleic acid-based therapies.2,3 Among nonviral nucleic acid delivery systems, nanoparticulate systems of polycationic polyethylenimines (PEI) have been of great interest due to their highest positive charge density among synthetic polycations that enables the effective condensation of nucleic acids by electrostatic interaction.4−6 In vivo stability of those systems during systemic blood circulation has been achieved mainly by steric stabilization with polyethylene glycol (PEG) shielding using PEG-grafted PEI7 and lipid-grafted PEI such as cetylated PEI8 and cholesteryl-PEI.9 PEG shielding, however, is providing in vivo stability only at the expense of transfection efficiency or intracellular uptake.10−12 As an attempt to retrieve the inherent © 2014 American Chemical Society

cellular uptake capability of the complex between PEI and nucleic acids once it reached the target tissues while maintaining in vivo stability during blood circulation, detachable PEG shielding via acid-cleavable PEG in the shells has been suggested. These particles demonstrated enhanced delivery efficiency as compared to stable PEG shielding in acidic cellular microenvironments such as the extracellular environment in tumors.13−16 We previously reported a long-circulating nanoparticulate nucleic acid delivery system suitable for systemic application, i.e., micelle-like nanoparticles (MNPs) based on covalent conjugates between phospholipid (PL) and PEI.17 We hypothesize that, via a self-assembly process, the PEI moiety will drive the formation of a dense polyplex core, while the PL moiety will form a lipid barrier surrounding the core, resulting in MNPs. The PL−PEI conjugate allows for a simple one-step DNA loading procedure with high DNA loading. The lipid barrier will provide an otherwise unstable polyplex with in vivo stability and prolonged systemic circulation time. We successfully demonstrate that the MNPs had properties suitable for in vivo application such as a long circulation time and low Received: Revised: Accepted: Published: 904

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from Sigma-Aldrich Korea (Seoul, South Korea) unless otherwise stated. 2.2. Synthesis of PL−PEI Conjugates. Stable PL−PEI conjugate (PL-am-PEI) was synthesized as previously described.17 The extent of conjugation was determined to be 1:1 (molar ratio of PEI to the lipid) on the basis of the ratio of the ethylene (−CH2CH2−) signal (2.4−2.9 ppm) of the PEI main chain to the methyl (−CH3) signal of the PL head (3.35 ppm) on the 1H NMR spectrum. 1H NMR (in CDCl3, 600 MHz) δ (ppm): 5.14 (br s, NH of PEI), 3.35 (t, N[CH3]3 of PL), 2.4−2.9 (m, −CH2CH2−, ethylene protons of PEI), 1.25 (m, −CH2− [alkane] of PL), 0.88 (t, CH3 of PL). Fourier transform infrared (FT-IR) (ν, cm−1): 3273.3 (m, N−H str, amines and imines of PEI), 2922.7 and 2851.5 (s, CH str, alkanes (−CH2−) of PL), 1711.3 m, CO str, ester of PL), 1565.7 (m, NH def, amido II band), 1462.6 (s, −CH3 def of [CH3]3N-), and 1088.3 and 1057.3 (s, P−O−C str). Acid-cleavable PL−PEI conjugate (PL-hz-PEI) was synthesized based on previously described methods, with minor modifications.15 Briefly, to oxidized AldoPC (10 mg, 13 μmol) dissolved in 1 mL of dimethylformamide (DMF) was added 10 mg (34 μmol) of succinimidyl 6-hydraziniumnicotinate hydrochloride (SHNH, Solulink) in DMF (0.5 mL), and the mixture was stirred at room temperature (rt) under argon for 2 h. The reaction mixture was then incubated with 350 mg (2.6 mmol) of benzyloxybenzaldehyde polystyrene beads (presoaked with 2 mL of DMF) at rt for 4 h. The beads were removed by filtration, and the supernatant was used for further reaction. First, 24 mg of branched PEI (molecular weight: 1.8 kDa; 13 μmol, Polysciences) in 2 mL of dichloromethane and 10 μL of triethylamine were mixed. Activated AldoPC in 2 mL of DMF was slowly added to this solution, and the mixture was stirred overnight at rt. Dichloromethane was first removed by using a rotary evaporator, and DMF was then removed under high vacuum for 3 h. The residue was suspended in 2 mL of water. The solution was purified by membrane dialysis against dH2O (MWCO 2,000 Da) with a Slide-A-Lyzer Dialysis Cassette (Thermo Scientific) and freeze-dried. The final product of purple color was obtained with a yield of 88.6% (34.3 mg). The structure was confirmed by 1H NMR (Bruker, 600 MHz) and FT-IR (Bruker). Chemical shifts were referenced to solvent resonance signals. The extent of conjugation was determined to be 1:1 (molar ratio of PEI to the lipid) according to the ratio of the ethylene (−CH2CH2−) signal (2.3−2.9 ppm) of the PEI main chain to the methyl (−CH3) signal of the PL head (3.18 ppm) on the 1H NMR spectrum. 1H NMR (in DMSO-d6/ CDCl3, 600 MHz) δ (ppm): 8.6 8.05 and 7.5 (m, C−H, protons on the pyridine ring); 6.95 (m, NH of hydrazone bond), 5.08 (br s, NH of PEI), 3.18 (t, −N(CH3)3 of PL), 2.3− 2.9 (m, −CH2CH2−, ethylene protons of PEI), 1.18 (m, −CH2− [alkane] of PL), 0.805 (t, CH3 of PL). FT-IR (ν, cm−1): 3270.6 (m, N−H str, amines and imines of PEI), 2922 and 2850 (s, CH str, alkanes [−CH2−] of PL), 1736.8 (m, C O str, ester of PL), 1643 and 1599.7 (s, CC + CN str, pyridine ring), 1556.22 (m, NH def, amido II band), 1463 (s, −CH3 def of [CH3]3N−), 1301 (s, C−N vb, pyridine ring), and 1087.3 and 1059.5 (s, P−O−C str). 2.3. Analysis of pH-Dependent Cleavage of PL-hz-PEI. The pH-sensitivity of the PL-hz-PEI conjugate was analyzed using size exclusion chromatography by high-performance liquid chromatography (Agilent Technologies, Folsom, CA) as previously described.14 The PL-hz-PEI solution in chloroform was dried under a stream of argon gas to form a thin film.

reticuloendothelial system uptake and, thus, were capable of delivering the loaded plasmid DNA to tumor tissues. In this study, we aimed to improve the nanocarrier system by enhancing the intracellular delivery efficiency of the payload upon reaching target tissues with an acidic pH, e.g., tumors. It was hypothesized that the intracellular uptake at the acidic tissues can be achieved by means of pH-responsive detachment of the monolayer envelope and release of the core complexes between nucleic acid therapeutics and PEI (Figure 1). Acid-

Figure 1. Schematic representation of acid-cleavable micelle-like nanoparticles (cMNPs) with polyethylenimine/oligonucleotide complex core surrounded by a phospholipid monolayer envelope.

cleavable MNPs (cMNPs) would have in vivo behavior similar to that of the stable MNPs (sMNPs) during systemic blood circulation. Upon accumulation in tumor tissues, however, the barrier would be detached under the action of the acidic local pH characteristic of hypoxic areas and expose the core complexes allowing for the subsequent intracellular entry via endocytosis, endosome escape, and cargo release via the “proton sponge” mechanism.18 To test this hypothesis, an acidcleavable PL−PEI conjugate was synthesized via hydrazone bond and used to prepare MNPs, resulting in cMNPs. The hydrazone linkage has proven to be very compatible with the requirements and kinetics for intracellular endosomal dePEGylation of polyplexes.19 We used low molecular weight PEI20,21 with a molecular weight of 2.7 kDa and 20-mer doublestranded ODN containing the nuclear factor κB cis element22 as model nucleic acid therapeutics. We evaluated the cMNP construct for in vitro and in vivo nucleic acid delivery efficiency using cultured tumor cells and tumor-bearing mice.

2. MATERIALS AND METHODS 2.1. Materials. The sequence of the ODN (5′-CCTTGAAGGGATTTCCCTCC-3′ and 3′-GGAACTTCCCTAAAGGGAGG-5′) contained the nuclear factor κB cis element.22 Single-stranded 20-mer ODNs and corresponding 5′-fluorescent dye-conjugated ODNs were purchased from Bioneer Corp. (Chonan, South Korea). Double-stranded ODNs were prepared by annealing equimolar amounts of the singlestranded ODNs at a final concentration of 1 μg/μL. For tracer experiments, ODN was radioactively labeled by a 5′-endlabeling technique using [γ-32P] ATP (PerkinElmer Life and Analytical Sciences, Waltham, MA) and T4 PNK (Promega, Madison, WI) as previously described.23 1-Palmitoyl-2-oleoylsn-glycero-3-phosphocholine, 1,2-disrearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy[polyethylene glycol]-2000) (PEG-PE), cholesterol, oxidized PL, 1-palmitoyl-2-azelaoyl-snglycero-3-phosphocholine (AzPC), and 1-palmitoyl-2-(9′-oxononanoyl)-sn-glycero-3-phosphocholine (AldoPC) were obtained from Avanti Polar Lipids (Alabaster, AL). Branched PEI with a molecular weight of 1.8 kDa was purchased from Polysciences, Inc. (Warrington, PA) and dissolved in water to a final concentration of 1.0 μg/μL. All materials were bought 905

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The film was hydrated with HEPES-buffered saline (HBS; 10 mM HEPES, 150 mM NaCl) either at pH 7.4 or at pH 6.5 at a concentration of 0.4 mg/mL to form micelles. After incubation for varying times (0, 1, and 3 h) at 37 °C, trinitrobenzenesulfonic acid, which reacts with free amine in PEI, was added to the micelle and run through a gel permeation column (ZenixSEC100, 4.6 × 150 mm, Waters Corp.) using HBS as an eluent at a flow rate of 1.0 mL/min and a UV detector (340 nm). Eventually, the pH sensitivity was determined by the appearance of a new peak that originated from the interaction between the cleaved PEI and trinitrobenzenesulfonic acid. 2.4. Preparation of sMNPs and cMNPs. The complexation capability of PL-hz-PEI was determined by agarose gel electrophoresis as previously described.17,24 sMNPs and cMNPs were prepared with PL-am-PEI and PL-hz-PEI, respectively, as described previously.17,24 In brief, either PLam-PEI or PL-hz-PEI (130 μg as PEI) and ODN (100 μg) corresponding to an N/P ratio of 10 were individually diluted in HEPES-buffered glucose to a final volume of 250 μL. The PL−PEI solutions were quickly added to ODN solutions and vortexed. Dry lipid films were separately prepared from the mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, cholesterol, and PEG-PE (42 μg, 21 μg, 15 μg [3:3:0.3 mol/ mol]) and hydrated with 500 μL of HEPES-buffered glucose. The lipid suspensions were incubated with the preformed PL− PEI/ODN complexes for 24 h at rt. Alternatively, the PL−PEI/ ODN complexes were added directly to the lipid films. The resulting suspensions of MNPs were stored at 4 °C until use. Both sMNPs and cMNPs were characterized with respect to size distribution, zeta potential, nanoscale structure, and colloidal stability, as previously described.17,24 2.5. Characterization of MNPs. For size and zeta potentials, the MNPs were diluted with HBS to obtain an optimal scattering intensity. Hydrodynamic diameter and zeta potential were measured by dynamic light scattering and electrophoretic light scattering (Laser Doppler) using a zetapotential and particle size analyzer (ELSZ-1000, Otsuka Electronics Co, Osaka, Japan). Scattered light was detected at 23 °C at an angle of 90°. A viscosity value of 0.933 mPa and a refractive index of 1.333 were used for data analysis. The instrument was routinely calibrated using a latex microsphere suspension. In addition, the MNPs were diluted in HBS at pH 7.4 or pH 6.5; the hydrodynamic diameters were then measured at various time points to analyze the pH-dependent cleavage of the hydrazone bond. The colloidal stability of the MNP particles against salt-induced aggregation was determined by monitoring the hydrodynamic diameter of the MNPs in 0.15 M NaCl. NaCl (5 M) was added to the MNPs in HEPESbuffered glucose to a final concentration of 0.15 M while the size was determined as described above. For cryo-transmission electron microscopy, MNPs were applied as a drop on Lacey Formvar carbon-coated grids (Ted Pella, Redding, CA). After 30 s at rt, excess sample was removed by blotting with filter paper. The grids were then rapidly frozen in liquid ethane and observed under cryogenic conditions using a cryoholder (626DH, Gatan) in a cryotransmission electron microscope (CryoTecnai F20, FEI). 2.6. In Vitro Cellular Uptake by Laser Scanning Confocal Microscopy (LSCM) and FACS. Murine melanoma cells (B16F10) were grown to confluence in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (Gibco) on coverslips (12 mm, Fisher Scientific) at 37 °C and 5% CO2.

The sMNPs and cMNPs prepared with Rhodamine-labeled PE (Rh-PE) and Alexa488-labeled ODN (A488-ODN) were subject to either SBS (10 mM sodium borate, 150 mM NaCl, pH 6.5) or HBS (10 mM HEPES, 150 mM NaCl, pH 7.4) for 1 h, then diluted to a final concentration of 1 μM ODN with 0.5 mL of 10% fetal bovine serum-supplemented Dulbecco’s modified Eagle’s medium cell culture medium and added to the cells. After 1 h incubation at 37 °C, cells were washed 4 times with ice-cold phosphate-buffered saline (PBS) and fixed with 2% (w/v) paraformaldehyde in PBS. The coverslips with fixed cells were mounted with a mounting medium (Fluoromount, Sigma-Aldrich) and observed with a laser scanning confocal microscope (A1Plus, Nikon). Alternatively, the cells, treated as described above, were harvested with 0.25% Trypsin-EDTA and analyzed by flow cytometry (FACS Caliber, BD). 2.7. Cytotoxicity Assay. B16F10 cells (4 × 103) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum in 96-well plates overnight. The cells were treated by replacing the medium with serum-free medium (100 μL) containing a serial dilution of each formulation (up to 50 μg/mL of PEI). After 4 h of incubation, the cells were washed twice with PBS and complete medium (100 μL) was added to each well. After 24 h of incubation, 110 μL of medium containing 10 μL of water-soluble tetrazolium (WST) solution (Ez-Cytox Cell Viability Assay Kit, DoGen, South Korea) was added to each well and the plates were incubated for 2 h. The absorbance at 460 nm was measured for each well using a microplate spectrophotometer (Epoch, BioTek Instruments, Winooski, VT). Relative cell viability was calculated as ratio of the viability of treated cells and that of cells treated only with medium (controls). 2.8. In Vivo Tumor Accumulation in Tumor-Bearing Mice. All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee at Gachon University. Male C57BL/6 mice (OrientBio, South Korea) were inoculated subcutaneously in the left flank with 1 × 106 B16F10 cells 14 days before treatment. On the day of the experiment, MNPs loaded with 32 P-ODN in a 100 μL injection volume corresponding to ∼2 μCi 32P activity and 20 μg of ODN were administered by tail vein injection. After 60 min, the animals were killed by cervical dislocation and organ samples (blood, liver, spleen) and tumor tissues were taken. The samples were solubilized with Soluene350 (PerkinElmer Life and Analytical Sciences, Waltham, MA) and mixed with Hionic-Fluor liquid scintillation cocktail (PerkinElmer Life and Analytical Sciences). The radioactivity of the samples was measured by using a liquid scintillation counter (LS 6000SC, Beckman, Fullerton, CA). The radioactivity was expressed as percentage of the injected dose (% ID/g). Alternatively, double fluorescently labeled MNPs with Rh-PE and A488-ODN were prepared and injected via the jugular vein by iv bolus (100 μL, 20.0 μg of ODN). After 60 min, animals were killed by transcardial perfusion of 5 mL of ice-cold TRIS-buffered saline (10 mM TRIS, 150 mM NaCl), followed by 50 mL of 4% paraformaldehyde in TBS at a flow rate of 4 mL/min. The tumor was removed, and sections of 20 μm thickness were prepared in 10 mM PBS by using a vibratome (Leica V1000S, Germany) and collected on coverslips. After counterstaining of nuclei with DAPI, the sections were observed with a confocal microscope (A1Plus, Nikon). 906

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3. RESULTS 3.1. Synthesis and Characterization of PL-am-PEI and PL-hz-PEI. Stable conjugates of PEI to the distal end of oxidized PL via an amide bond were prepared as previously described.17 In this study, for the synthesis of PL-hz-PEI, the cationic PEI was conjugated to the distal end of oxidized PL via an acid-cleavable hydrazone bond using the heterobifunctional cross-linker SHNH (Figure 2). First, the aldehyde group of the

Figure 2. Synthesis scheme of acid-cleavable phospholipid−polyethylenimine conjugates (PL-hz-PEI).

oxidized AldoPC was activated with SHNH. The reaction was carried out with 2.6 equiv of SHNH in anhydrous DMF. Unreacted SHNH was removed by immobilization onto benzyloxybenzaldehyde containing polystyrene beads. The activated PL-SHNH was then reacted with PEI to form PLhz-PEI. The structure of PL-hz-PEI was confirmed by the appearance of peaks at 8.60, 8.05, and 7.5 ppm from the pyridine ring in the linker in addition to the main peaks of PEI and PE in the 1H NMR spectrum (Figure S1 in the Supporting Information). In FT-IR spectra (Figure S1 in the Supporting Information), specific peaks from the PL-hz-PEI conjugate were also observed at 1643 and 1599 cm−1 for the CC, CN (stretching) of the pyridine ring in the linker as compared with those of the FT-IR spectrum of PL-am-PEI. The pH-dependent cleavage of PL-hz-PEI was determined by the appearance of free PEI upon size exclusion chromatography analysis (Figure 3). Intact PL-hz-PEI was eluted at a void volume, and the free PEI from the cleavage of PL-hz-PEI was eluted at 4.7 min. It was determined that about 30% of PEI was

Figure 3. pH-dependent cleavage of phospholipid−hydrazone− polyethylenimine (PL-hz-PEI) conjugates; SEC chromatograms of free PEI (A) and PL-hz-PEI at pH 7.4 (B) and pH 6.5 (C) after 3 h incubation.

cleaved after 1 h of incubation and more than 90% after 3 h of incubation at pH 6.5. The cleavage of PL-hz-PEI was much slower at pH 7.4. After 3 h of incubation at pH 7.4, cleavage of the free PEI was 2-fold less than that at pH 6.5. In addition, hydrodynamic diameters of cMNPs at pH 6.5 drastically increased after 30 min of incubation, whereas those of sMNPs at pH 6.5 and cMNPs and sMNPs at pH 7.4 did not increase (data not shown). These data indicate that PL-hz-PEI particles with hydrazone bond are readily cleavable in an acidic environment and, thus, cMNPs are able to detach the PL 907

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free lipids, which were determined on the basis of the number of lipid molecules required for a complete monolayer envelope, were adopted. A mixture of free lipids comprising 1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine, cholesterol, and PEG-PE (3:3:0.3 mol/mol) was separately prepared as an aqueous suspension, which was then incubated with the preformed PLam-PEI/ODN or PL-hz-PEI/ODN complexes. Spontaneous formation of a monolayer envelope was proposed to be driven by the hydrophobic interaction between the lipid moieties of PL-am-PEI and free lipids (postinsertion technique) and confirmed by freeze−fracture TEM. The characteristic hardcore structure with monolayer envelope was further supported by cryogenic transmission electron microscopy, which revealed well-developed spherical nanoparticles with core and shell structures (Figure 4b). The cMNPs showed a size distribution and zeta potential comparable to those of sMNPs (Table 1). The average

envelope and expose the PEI/ODN polyplex core in an acidic tumor microenvironment. 3.2. Preparation and Characterization of MNPs. A procedure for generating sMNP was established previously.17 The previously established procedure for the preparation of sMNPs was applied to prepare cMNPs. First, the complexation capacity of PL-hz-PEI was confirmed to be comparable to that of PL-am-PEI and free PEI (Figure 4a). The optimal N/P ratio,

Table 1. Size Distributions and Zeta Potentials of Stable Micelle-like Nanoparticles (sMNPs) and Acid-Cleavable Micelle-like Nanoparticles (cMNPs) (Mean ± SEM, n = 5) samples PL-am-PEI/ODNa PL-hz-PEI/ODNb sMNPs cMNPs

size distribution (nm)

zeta potential (mV)

± ± ± ±

20.2 ± 1.38 18.9 ± 2.47 −2.1 ± 0.86 −0.59 ± 0.82

178.9 181.9 204.2 213.9

5.12 4.67 0.79 3.52

a PL-am-PEI/ODN: phospholipid−polyethylenimine/oligonucleotide complex. bPL-hz-PEI/ODN: acid-cleavable phospholipid−polyethylenimine/oligonucleotide complex.

hydrodynamic diameter of sMNPs and cMNPs is 204.2 ± 0.79 nm and 213.9 ± 3.52 nm, respectively, whereas that of PL-amPEI/ODN and PL-hz-PEI/ODN complexes is 178.9 ± 5.12 nm and 181.9 ± 4.67 nm, respectively. The zeta potential of sMNPs and cMNPs is −2.1 ± 0.86 mV (mean ± SEM, n = 5) and −0.59 ± 0.82 mV (mean ± SEM, n = 5), respectively, whereas that of PL-am-PEI/ODN and PL-hz-PEI/ODN complexes is 20.2 ± 1.38 mV and 18.9 ± 2.47 mV, respectively. The neutral surface charge of MNPs also suggested the presence of a lipid layer that provided charge shielding of the otherwise positive PEI/ODN core. As sMNPs, the cMNPs showed colloidal stability against saltinduced aggregation. Upon NaCl addition, the intermediate PL-am-PEI/ODN or PL-hz-PEI/ODN complexes without lipid monolayer envelope showed continuous increases in the average diameters up to >1000 nm at around 24 h. On the contrary, sMNPs and cMNPs remained stable with no significant aggregation for 24 h (Figure 4c), demonstrating the stabilizing effect of the lipid monolayer envelope against salt-induced aggregation. 3.3. In Vitro Behavior of MNPs. In order to analyze whether cMNPs have a better ability to deliver ODN into cells at an acidic pH, e.g., the tumor extracellular environment, we evaluated the cellular uptake of MNPs by B16F10 cells at pH 7.4 and pH 6.5 using LSCM and flow cytometry (FACS). The higher cellular uptake or association of cMNPs at a lower pH was confirmed by LSCM (Figure 5a). cMNPs showed about 1.5-fold higher signal intensity of Alexa-488 labeled ODN at pH 6.5 than at pH 7.4 while sMNPs showed no difference (ImageJ 1.47v). FACS analysis also revealed that cMNPs led to a 3-fold higher cellular uptake of the payload ODN at pH 6.5 than at pH 7.4, whereas there was no difference in the uptake between

Figure 4. Analysis of acid-cleavable micelle-like nanoparticles (cMNPs) in comparison to stable MNPs (sMNPs): (a) agarose gel electrophoresis of PL-hz-PEI/ODN complexes at varying weight ratios; (b) cryo-TEM analysis of cMNPs; (c) colloidal stability of cMNPs against salt-induced aggregation (mean ± SEM; n = 3).

calculated assuming that 43.1 g/mol corresponds to each repeating unit of PEI containing one amine and 330 g/mol corresponds to each repeating unit of ODN containing one phosphate, was determined on the basis of the amounts of amine required to completely inhibit ODN migration on an agarose gel and determined to be about 10, which is where all ODN is bound to the complexes. Thus, an N/P ratio of 10, the same as that applied for PL-am-PEI, was used for the next enveloping steps. The composition and amounts of enveloping 908

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Figure 6. Cytotoxicity of acid-cleavable micelle-like nanoparticles (cMNPs) in murine melanoma B16F10 cells in comparison to stable micelle-like nanoparticles (sMNPs).

was also measured and compared to those of sMNPs. Tumors and major organs (liver, spleen, lung, kidney, brain, and blood) were collected at 1 h post injection. The radioactivity of the samples was measured by using a liquid scintillation counter and expressed as percentage of the injected dose per gram of tissue (% ID/g). It is very likely that cMNPs, as sMNPs, show prolonged blood circulation. In this study, we focused on demonstrating the benefits of acid-cleavability and deshielding. Thus, the extent of tumor accumulation after iv injection was measured as % ID/g tumor tissue. As expected, cMNPs showed an organ distribution profile similar to that of sMNPs. Both cMNPs and sMNPs led to a significant level of accumulation in organs of the reticuloendothelial system. Only 4.6% ID/g liver and 7.5% ID/g spleen were observed after iv injection of cMNPs. Similarly, 5.4% ID/g liver and 7.0% ID/g spleen were observed after iv injection of sMNPs. About 5.6% ID/g and 4.5% ID/g were found in tumor tissues for cMNPs and sMNPs, respectively (Figure 7a). The difference, however, was not significant (p = 0.1350). The fluorescence microscopy data of frozen tumor sections from in vivo grown tumors are shown (Figure 7b). Tumor sections of animals injected with sMNPs and cMNPs loaded with A488-ODN and Rh-PE were analyzed by LSCM. Intravenous injection of cMNPs led to thoroughly diffused bright fluorescence signals, whereas sMNPs led to particulate and discrete fluorescence signals. Although no significant difference was observed in the total amounts of accumulation measured by using the isotope tracer, intracellular trafficking observed by confocal microscopy revealed that cMNPs were widespread throughout the tissues. The extents of ODN accumulation in tumor tissues were estimated quantitatively from the green signal intensities using image analysis software (ImageJ 1.47v). cMNPs showed 1.6-fold higher signal intensity of Alexa-488 labeled ODN than sMNPs, which demonstrates higher intracellular accumulation. This observation indicates deshielding and disassembly of the cMNP particles, whereas sMNPs were readily found as intact particles.

Figure 5. In vitro behavior of acid-cleavable micelle-like nanoparticles in murine melanoma B16(F10) cells in comparison to stable micellelike nanoparticles: (a) LSCM analysis (green, Alexa488-ODN; red, Rh-PE; blue, DAPI); (b) FACS analysis (black, control; red, sMNPs at pH 7.4; brown, sMNPs at pH 6.5; blue, cMNPs at pH 7.4; purple, cMNPs at pH 6.5).

pH 6.5 and pH 7.4 for sMNPs (Figure 5b). These results indicate that cMNPs could effectively deliver ODN into tumors that have an acidic extracellular environment. The cytotoxicity of sMNPs and cMNPs was evaluated toward B16F10 cells. The cells were treated with MNPs or PEI/ODN polyplexes at different PEI concentrations. The relative cell viability was expressed as percentage of control cells treated with medium. In contrast to naked PEI/ODN polyplexes, MNPs showed minimal cytotoxicity after 24 h of incubation following 4 h of treatments. The cell viability for both sMNPs and cMNPs was more than 80% at a PEI concentration of 50 μg/mL, whereas that for naked PEI/ODN complexes was only 50% (Figure 6). No difference in cell viability was observed between pH 6.5 and 7.4. This result is understandable in light of our data showing a neutral surface charge on MNPs compared to the strong positive charge on the surface of PEI/ ODN complexes. 3.4. In Vivo Behavior and Tumor Accumulation. To demonstrate the enhanced capability of cMNPs in delivering payload nucleic acid therapeutics to target tissues such as tumors, tumor accumulation of ODN was measured using cMNPs loaded with 32P-ODN in tumor-bearing mice and compared to that of sMNPs. The radioactivity in major organs after iv bolus administration of cMNPs loaded with 32P-ODN

4. DISCUSSION We previously demonstrated that self-assembling MNPs of PLam-PEI conjugates provide an efficient gene delivery system for systemic application.17 We were able to achieve advantages by combining polyplexes with a sterically stabilized lipid membrane. The PL-am-PEI conjugate enabled the one-step self-assembly of MNPs with the PEI/ODN polyplex core and a lipid envelope by simultaneous condensation and lipid envelope formation with a loading efficiency of 100% and a loading 909

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intracellular uptake and trafficking similar to that of PEI-based polyplex systems. Ideal nucleic acid delivery systems should be able to accumulate in the target organ or tissue during circulation, to enter target cells delivering the nucleic acid payloads to the cytoplasm by escaping the endocytic pathway and lysosomal degradation, and to release the cargo and assist its trafficking to target intracellular compartments. Therefore, the delivery systems must overcome multiple extracellular and intracellular barriers: (1) adhesion on the cell surface, (2) cellular entry (internalization), and (3) escape from the endosome (cytosolic release).3,25 A number of studies have proven that pHresponsive polymeric nanoparticulates facilitated intracellular delivery of payloads into low acidic environments such as the extracellular microenvironment of tumors and the endosomal microenvironment.26−29 Several nucleic acid delivery systems of pH-sensitive polymers were reported to have enhanced transfection efficiency because of the detachment of the polyethylene glycol shell from polymeric nanoparticles, i.e., dePEGylation in the endosomal or extracellular tumor environment through acid-cleavable bonds between PEG chains and cationic polymers.14,18 Our cMNP was constructed so as to enable detachment of the lipid envelope in an acidic environment by using an acidcleavable hydrazone bond between PL and cationic PEI, thereby providing deshielding of the lipid envelope and exposing the polyplex core to the extracellular space of tumor tissues, which could enter the tumor cell. Here, we hypothesized that pH-responsive MNPs with detachable lipid envelope provide enhanced intracellular uptake. We prepared a micelle-like nanoparticle (MNP) by using PL-hz-PEI. The nanocarrier was constructed using a chemical conjugate of PL and PEI (PL-hz-PEI) obtained via a hydrazone bond, which has been known to easily hydrolyze in an acidic environment and which has also been widely used for the preparation of pHlabile conjugates.19 The structure of PL-hz-PEI particles was confirmed on the basis of 1H NMR and FT-IR spectra, where specific peaks appeared from the aromatic ring in the SHNH linker forming a hydrazone bond. Therefore, PL-hz-PEI particles could be clearly distinguished from PL-am-PEI particles without hydrazone bond. In order to form a hydrazone bond, we used the SHNH linker that is a cross-linker, which can couple an aldehyde group containing compound with an amino-bearing compound.15 PL-hz-PEI was obtained as a purple color solid, which also originates from the aromatic ring in the linker. The pH-dependent cleavage of PL-hz-PEI was confirmed to occur at a rate of 90% over 3 h at pH 6.5, indicating that PL would detach from the polyplex in an acidic microenvironment. The feasibility of the enhanced nucleic acid delivery to acidic tumor tissues was demonstrated in tumor-bearing mice. Accumulation of the payload in the tumor tissue was accessed following iv administration of MNPs loaded with 32P-ODN. At 1 h post injection, 5.6% of the injected dose (% ID) was observed in tumors from mice treated with cMNPs, whereas 4.5% ID was found in tumors from control mice treated with sMNPs. Because the biodistribution data obtained by using the tracer isotope provide only the total accumulation at both intracellular and extracellular tumor spaces, the intracellular deposition of the payload in the tumor tissue was further accessed by LSCM following iv administration of MNPs loaded with A488-ODN. Although no significant difference was observed in the total accumulated amounts, cMNPs were

Figure 7. Tumor accumulation of acid-cleavable micelle-like nanoparticles (cMNPs) in tumor-bearing mice in comparison to stable MNPs (sMNPs): (a) organ accumulation of 32P-labeled oligonucleotide (ODN) and (b) tumor penetration of sMNPs or cMNPs containing Alexa488-ODN (green) and Rh-PE (red) with nuclei counterstained with DAPI (blue).

capacity of 30%. It was proposed that the electrostatic interaction of polycationic PEI moieties with polyanionic ODN would drive the formation of dense PL-am-PEI/ODN polyplex cores, while amphiphilic PL moieties, together with added free unmodified PLs and PEG-grafted PLs (PEG-PE), would form a lipid monolayer envelope around the polyplex cores, leading to the formation of ODN-loaded MNPs that are stabilized by a steric barrier of PEG chains and the membrane barrier of the lipid envelope. In this study, we developed and evaluated a new nanoparticulate nucleic acid delivery system capable of detaching the lipid envelope in an acidic environment along with the prolonged blood circulation of sMNPs. The long-circulating and stimulus-responsive system was prepared using PL-hz-PEI conjugates. The cMNPs, similar to sMNPs, were self-assembled in the presence of ODN to form the lipid envelope surrounding the polyplex core. It was demonstrated that the lipid envelope provided the otherwise unstable polyplex with a prolonged blood circulation time. Upon accumulation in target organs/ tissues, i.e., tumor tissues, the lipid envelope was expected to detach under the action of the local acidic pH and expose the polyplex core, thereby enabling the subsequent intracellular entry via endocytosis, endosome escape, and cargo release via the “proton sponge” mechanism. Such nanoparticulate carrier represents a promising nucleic acid delivery system combining in vivo stability similar to that of PEG-stabilized liposomes and 910

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found widespread throughout the intracellular space of tumor cells, indicating deshielding and disassembly of the nanoparticles, whereas sMNPs were readily found as intact particles.

5. CONCLUSION Micelle-like nanoparticles with acid-cleavable monolayer envelope (cMNPs) allowed for a significantly enhanced intracellular accumulation of nucleic acid therapeutics in tumor tissues as compared to stable micelle-like nanoparticle (sMNPs) while providing comparable blood circulation time and RES accumulation. These qualities of cMNPs make them a promising nucleic acid delivery system for in vivo application.

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR and Fourier transform infrared spectra of stable and acid-cleavable PL−PEI. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*College of Pharmacy, Gachon University, 191 Hambakmoero, Yeonsu-gu, Incheon, South Korea 406-799. Tel: 82-32-8996417. Fax: 82-32-820-4829. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program of Korean National Research Foundation (NRF20110007794).

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