Bioconjugate Chem. 2005, 16, 322−329
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Supramolecular Gene Delivery Vectors Showing Enhanced Transgene Expression and Good Biocompatibility Xintao Shuai,† Thomas Merdan,† Florian Unger, and Thomas Kissel* Department of Pharmaceutics and Biopharmacy, Philipps-University of Marburg, Ketzerbach 63, D-35032 Marburg, Germany. Received June 30, 2004; Revised Manuscript Received January 23, 2005
Soluble supramolecular inclusion complexes were formed by threading R-cyclodextrin (R-CD) molecules over poly(ethylene glycol) (PEG) and poly(-caprolactone) (PCL) chains of ternary block copolymers of PEG, PCL and polyethylenimine (PEI). Characteristic shifts of PCL absorptions in FTIR, 1H NMR and UV spectra strongly suggest that R-CD is threaded over PEG and PCL blocks. Due to the reduced hydrophobic interaction between PCL blocks, the resulting supramolecular complexes displayed a dramatically increased solubility, in comparison with the ternary block copolymers. Their ability to complex DNA was almost as efficient as that of branched PEI 25 kDa, as shown in the ethidium bromide fluorescence quenching experiments. Resulting DNA polyplexes displayed a size of around 200 nm and a neutral surface charge. Microscopy studies in 3T3 fibroblasts revealed an efficient cellular uptake. Transfection efficiencies of inclusion complexes were in the same order of magnitude as PEI. In contrast to PEI a 100× lower toxicity was observed by MTT-assay, allowing the administration of nitrogen-to-phosphate ratios of up to 20. These new gene delivery systems merit further characterization under in vivo conditions.
INTRODUCTION
Various nonviral gene delivery systems consisting of DNA and polycations, dubbed as “polyplexes”, have been investigated (1-3). Poly(ethylenimine), PEI, has attracted considerable interest due to its high positive charge density and consequently strong DNA binding. Another beneficial feature of PEI is the “proton-sponge effect”, which is thought to facilitate endosomal/lysosomal escape of DNA into the cytoplasm (4). PEI with molecular weights >2000 Da seems to be necessary for effective gene delivery (5-7). PEI and PEI/DNA complexes were found to be cytotoxic in molecular weight and composition dependent manner due to their electrostatic interactions with negatively charged cell membranes (5, 7). Copolymers of PEI with hydrophilic macromolecules forming DNA polyplexes with neutral surface charge have extensively been investigated recently (8-12). Most studies utilize nonionic hydrophilic polyethers, e.g. poly(ethylene glycol) (PEG), as a building block to generate block or graft copolymers with linear or branched PEIs. When these copolymers were mixed with pDNA in aqueous solution, the cationic PEI segment of the copolymer first binds DNA via electrostatic interactions and then the neutralized polyions aggregate to form an insoluble core. The nonionic water-soluble PEG chains act as a hydrophilic shell stabilizing the resulting nanoscale polyelectrolyte complexes (8). Polyplexes thus prepared show a lower positive surface charge compared to PEI, and consequently less cytotoxicity was observed. Unfortunately, the transfection efficiency was also decreased. We recently synthesized a new type of ternary blockcopolymers by grafting diblock copolymers of PEG and * To whom correspondence should be addressed. Tel +496421-282-5881. Fax +49-6421-282-7016. E-mail:
[email protected]. † These authors have equally contributed to this work.
PCL onto branched PEI. Intensive hydrogen bonding between PCL and PEI blocks was noted in the bulk materials, and solubility was strongly affected by composition, i.e., block length and graft density. DNA polyplexes formed with soluble copolymers or copolymers possessing a high critical micelle concentration (cmc) showed significant transfection efficiency under in vitro conditions. By contrast, those copolymers with a high graft density and long PCL blocks showed very poor gene expression. In the latter case, access and binding of DNA to PEI was seriously hindered by hydrophobic PCL chains surrounding the hydrophilic PEI head. Hydrogen bonding between PCL and PEI blocks is also believed to be an impediment for the DNA complexation with PEI (13). Therefore, breaking of hydrogen bonds between PCL and PEI as well as the dissolution of PCL block in aqueous media seem to be key factors for improving gene transfection efficiency of these copolymers. In this publication, we present an unusual way to dissolve the PCL blocks of the ternary copolymers in aqueous solution by including them inside the R-cyclodextrin (R-CD) cavities. R-CD is a cyclic oligosaccharide consisting of six glucose units (15). One of its distinctive characters is its amphipathy, i.e., it possesses a hydrophobic cavity of 4.5 Å in diameter with a hydrophilic outer layer supporting many hydroxyl groups (15, 16). Supramolecular inclusion complexes (ICs), organized by noncovalent interactions, can be formed by threading R-CD molecules onto various polymer chains (17-23). The driving force for the threading process is probably due to intermolecular hydrogen bonding between neighboring CDs, as well as steric compatibility and hydrophobic interactions between host and guest molecules. It has been demonstrated that R-CD forms inclusion complexes with both poly(ethylene glycol) (PEG) (17, 18) and PCL (24-28) chains. In most cases, a full coverage of individual polymer chains with R-CD bracelets leads to crystalline water-insoluble inclusion complexes. In
10.1021/bc0498471 CCC: $30.25 © 2005 American Chemical Society Published on Web 02/23/2005
Supramolecular Gene Delivery Vectors
Figure 1. The chemical structures of R-cyclodextrin (a) and copolymer of PEI, PCL and PEG (b).
dilute solutions a partial coverage of PEG or PCL chains with R-CD can be achieved. In the latter case, inclusion complexes with PCL may be completely soluble in water. Here we investigate supramolecular physicochemical and biological properties of supramolecular inclusion complexes as carriers for gene delivery. EXPERIMENTAL SECTION
Materials. -Caprolactone (from Aldrich) was purified by vacuum distillation over CaH2. Monomethoxyl-poly(ethylene glycol) (MPEG) (Aldrich) was dried by azeotropic distillation with dry toluene. The PEI with a molecular weight of 25 kDa (abbreviation: PEI25k) was a gift from BASF (Germany). This PEI is a highly branched molecule. Its contents of primary, secondary, and tertiary amine units are 42%, 33%, and 25% respectively. R-CD was purchased from Aldrich and vacuumdried at 40 °C prior to use. Diblock copolymers of PCL and PEG were synthesized by ring-opening polymerization of -caprolactone and then grafted to PEI, as described in our recent publication (13). The chemical structures of these copolymers and threading of R-CD are shown in Figure 1. Molecular characteristics of polymers used in this study are shown in Table 1. To prepare inclusion complexes, the predetermined amount of R-CD aqueous solution (e.g. 5 mg/ mL, 20 mL) was added stepwise to the copolymer micelle solution (e.g. 4.2 mg/mL, 15 mL) in a glass vial immersed in an ultrasonic water bath at room temperature. Sonication was applied for 30 min during and after the addition of the R-CD solution. The mixed solution was then stirred magnetically overnight at room temperature, frozen at ∼-40 °C, and then freeze-dried to yield a powdered sample (30). pCMV-Luc encoding for luciferase as a reporter gene was purchased from Plasmid Factory, Bielefeld, Germany. NIH/3T3 (Swiss mouse embryo) cell line was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Cells were cultured according to the protocols suggested by the supplier. Cell Culture. NIH/3T3 cells (DSMZ#: ACC 59) were cultured under conditions recommended by the supplier in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum. Spectroscopic Measurements of Copolymers and Inclusion Complexes. FTIR spectral studies were carried out using a Nicolet 510P FTIR spectrometer with a resolution of 2 cm-1. Powder samples were compressed into KBr pellets for FTIR measurements. 1H NMR spectra were recorded on a JEOL GX 400 D spectrometer
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in deuterated water (D2O) at room temperature. UV spectra of solution samples were recorded on a Shimadzu UV-160 UV-visible recording spectrophotometer. Ethidium Bromide Exclusion Assay. DNA condensation was measured by the decrease in ethidium bromide fluorescence, as described earlier (31, 32). The assay was performed in 96-well plates in triplicate using 8 µg of salmon testes DNA was dissolved in 79 µL of water and 50 µL of 60 mM Tris buffer pH 7.4 were added to each well. Volumes were adjusted to 300 µL using water. Subsequently, appropriate volumes of 0.05 mg/mL polymer solutions were added to yield N/P ratios between 0.2 and 4. After 10 min 20 µL of a 0.1 mg/mL ethidium bromide solution were added. Wells were mixed thoroughly, and the fluorescence was measured using a fluorescence plate reader with an excitation wavelength at 518 nm and an emission wavelength of 605 nm. Polyplex Formation. Luciferase plasmid (pCMV-luc) and the appropriate amount of polymer or inclusion complex were dissolved separately in 0.9% sodium chloride solution of pH ) 7. The two solutions were mixed by vigorous pipetting, and complexes were allowed to interact for 10 min before use. Polyplexes were prepared for transfection experiments with 4 µg of plasmid in 300 µL of NaCl 0.9% and the appropriate amount of polymer or conjugate in 300 µL of sodium chloride solution (0.9%, pH ) 7). Photon Correlation Spectroscopy. Hydrodynamic diameters of the DNA/polymer complexes were determined by photon correlation spectroscopy. Measurements of micelle solutions were performed on a Zetasizer 3000 HS from Malvern Instruments, Herrenberg, Germany (10 mW HeNe laser, 633 nm). Scattering light was detected at 90° angle through a 400 µm pinhole at a temperature of 25 °C. For data analysis, the viscosity (0.88 mPa‚s) and the refractive index (1.33) of distilled water at 25 °C were used. The instrument was routinely calibrated using Standard Reference latex particles (AZ 55 Electrophoresis Standard Kit, Malvern Instruments). Measurements were analyzed by the CONTIN algorithm. Values given are the means of 5 runs of 60 s ( standard deviation. Zeta Potential Measurements. pCMV-Luc plasmid (16 µg) was complexed with the appropriate amount of polymer in 800 µL of NaCl 150 mM as described above. Zeta-potential measurements of polyplexes formed at N/P 7 were carried out in the standard capillary electrophoresis cell of the Zetasizer 3000 HS from Malvern Instruments at position 17.0 at 25 °C. Sampling time was set to automatic. Average values were calculated with the data of 5 runs. Fluorescent Labeling of PEI and DNA for Confocal Laser Scanning Microscopy Experiments. Polymer or inclusion complexes (20 mg) were dissolved in 2 mL 0.1 M sodium bicarbonate solution at pH 9. Oregon Green 488 carboxylic acid succinimidyl ester (1 mg, Molecular Probes) was dissolved in 200 µL of dimethyl sulfoxide and added dropwise under stirring to the polymer solution. The mixture was stirred in the dark for 3 h at room temperature before the labeled polymer was purified by ultrafiltration in an Amicon cell (regenerated cellulose membrane, molecular weight cut off 10 000) and washed with 0.1 M borate/1.0 M sodium chloride solution pH 7.5. The washing procedure was performed until no absorption was detectable at 488 nm in the cell outflow. As a final step, the buffer was exchanged with distilled water. Plasmid (pCMV-Luc) was labeled with Cy3 using a commercial kit (Mirrus Label It, Mobitec, Go¨ttingen, Germany). Procedure was performed according to the manufacturer’s manual.
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Table 1. Molecular Characteristics of Polymers Used in This Study molecular weights of blocks (Da) polymersa
PEI
PCL
PEG
grafting densityb
solubilityc (mmol/L)
PEI25k PEI25k-g-(PCL580-b-PEG5k)2.9 PEI25k-g-(PCL2k-b-PEG2k)2.8 PEI25k-g-(PCL1.2k-b-PEG2k)5.1
25000 25000 25000 25000
580 2000 1200
5000 2000 2000
2.9 2.8 5.1
Sd S 0.031 80%) when cells were transfected with inclusion complex-based polyplexes at N/P 20. A part of this reduced toxicity is probably due to the neutral surface charge of complexes; however, our data suggest also that cyclodextrin further improves biocompatibility. Gene Transfection Efficiency of Polyplexes. The transfection efficiency to 3T3 cells with polyplexes based on three copolymers and their inclusion complexes is shown in Figure 9. Although PEI25k-g-(PCL580-b-
Figure 9. Transfection efficiency of polyplexes to 3T3 cells based on (1) PEI25k, (2) PEI25k-g-(PCL580-b-PEG5k)2.9, (3) inclusion complex of PEI25k-g-(PCL580-b-PEG5k)2.9, (4) PEI25kg-(PCL2k-b-PEG2k)2.8, (5) inclusion complex of PEI25k-g-(PCL2kb-PEG2k)2.8, (6) PEI25k-g-(PCL1.2k-b-PEG2k)5.1 and (7) inclusion complex of PEI25k-g-(PCL1.2k-b-PEG2k)5.1.
PEG5k)2.9 was soluble in a wide concentration range and showed quite good performance in DNA condensation and transgene expression (13), threading of R-CD onto the PCL blocks may further enhance the gene transfection of this copolymer-based polyplex by further reducing the hydrophobic hindrance of DNA binding to PEI blocks. Effect of incorporating R-CD on the transgene transfection of polyplexes of other two copolymers appears to be more significant. Surprisingly, at N/P 3 and 7, although PEI25k-g-(PCL1.2k-b-PEG2k)5.1 did not transfect the cells at all, the polyplex based on the inclusion complex of this copolymer exhibited excellent transfection efficiency. Intracellular Uptake of Polyplexes. After incubating the NIH 3T3 fibroblasts for 3 h, a typical confocal laser scanning microscope image of an inclusion complex containing polyplex is shown in Figure 10. The micrograph demonstrates the efficient internalization of polyplexes, despite their neutral surface charge. Both DNA and copolymer colocalize in punctuate vesicles, probably in the lysosomal compartment. The mechanism by which the polyplexes escape from the endo/lysosome is presently under investigation. Most likely, the PEI component of the copolymer acts as proton sponge, while inclusion complex formation modulates complex solubility and cytotoxicity. No significant differences could be observed between the subcellular distribution of IC-containing polyplexes compared to PEI25k (data not shown). CONCLUSION
Upon formation of inclusion complexes between R-CD and preferably the PCL blocks of the ternary copolymers, the copolymers lost their amphipathy and became more hydrophilic. This effect not only led to an increase in water solubility of copolymers but also favored the access and binding of DNA. Consequently, enhanced gene transfection efficiency was detected in transfection ex-
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Figure 10. LSCM micrographs of internalization of fluorescentlabeled polyplex based on inclusion complex of PEI25k-g(PCL2k-b-PEG5k)2.8 into 3T3 fibroblasts after 3 h of incubation. Images assignment: Red: DNA, green: inclusion complex, and blue: nucleus. The image on the right at the bottom is an overlay of the three fluorescent colors and the light microscopic image.
periments, especially for the two copolymers showing distinct CMC. Furthermore these delivery systems showed an excellent biocompatibility, a property that makes them interesting candidates for further in vivo investigations. ACKNOWLEDGMENT
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BC0498471
