Water-Soluble Polyion Complex Associates of DNA and Poly(ethylene

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Bioconjugate Chem. 1997, 8, 702−707

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Water-Soluble Polyion Complex Associates of DNA and Poly(ethylene glycol)-Poly(L-lysine) Block Copolymer Satoshi Katayose and Kazunori Kataoka* Department of Materials Science and Technology and Research Institute for Biosciences, Science University of Tokyo, Yamazaki 2641, Noda-shi, Chiba 278, Japan. Received February 13, 1997X

Complex formation of poly(ethylene glycol)-poly(L-lysine) (PEG-PLL) AB type block copolymer with salmon testes DNA or Col E1 plasmid DNA in aqueous milieu was studied. The PLL segment of PEG-PLL interacts with nucleic acid through an electrostatic force to form a water-soluble complex associate with a diameter of ca. 50 nm. PEG segments surrounding the core of the polyion complex prevented the complex from precipitation even under stoichiometric conditions, at which the unit ratio of L-lysine in PEG-PLL and phosphate in the DNA is equal. The profile of the thermal melting curve revealed a higher stabilization of DNA structure in PEG-PLL/DNA complexes compared to that in the complex made from DNA and PLL homopolymer with the same molecular weight as the PLL segment in PEG-PLL. This stabilizing effect on the DNA structure may be due to the compartmentalization of DNA into the microenvironment of PEG with low permittivity. The reversible nature of the PEG-PLL/DNA complex was further verified through the addition of polyanion [poly(L-aspartic acid)]: Poly(L-aspartic acid) replaced DNA in the complex with PEG-PLL, resulting in the release of free DNA in the medium. Furthermore, the PEG-PLL/DNA complex showed high resistance against DNase I attack, suggesting DNA protection through the segregation into the core of the associate having PEG palisade.

INTRODUCTION

Polyion complexes between polyelectrolytes with an opposite charge have attracted wide attention in the fields of medicine and biology. For example, polyion complexes of a nucleotide with polycations are considered one of the promising systems for a gene vector (1-5). DNA has a polyanionic character and can be bound to polycations, e.g., poly(L-lysine), through electrostatic interaction. It is well-known that polylysine strongly binds to DNA to induce compaction of the DNA molecule (6-8). However, a soluble and electrically neutral (stoichiometric) complex consisting of poly(L-lysine) and DNA is hardly obtained because charge neutralization usually induces the formation of insoluble precipitates. Recently, we have shown that complexation of DNA with a poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL) led to the formation of water-soluble complex associates in aqueous milieu (9). Use of a cationic block copolymer with a PEG segment as the complexation partner of DNA was based on the results of our systematic study on polymeric micelle drugs (10, 11). Supramolecular association of a block copolymer consisting of PEG and polyamino acids through hydrophobic or electrostatic interaction leads to the formation of core-shell type nanoassociates or micelles in which drug molecules are hydrophobically or electrostatiscally included in the core of the micelle surrounded by the PEG outer shell (12-14). The proper micelle size, ca. ∼50 nm, and high flexibility and hydrophilicity of the outer-shell PEG seem to contribute to the stability of the micelles. Indeed, both long circulation in the blood compartment and exceptionally high accumulation in a solid tumor were evidenced for block copolymer micelles with an entrapped anticancer drug (doxorubicin) (15, 16). These features of block copolymer micelles provide a rationale * Author to whom correspondence should be addressed (telephone +81-471-23-9771; fax +81-471-23-9362). X Abstract published in Advance ACS Abstracts, August 15, 1997.

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for exploring the feasibility of polymeric micelles as a novel vector system for genes and oligonucleotides. It should be noted that water-soluble and narrowly distributed polyion complex micelles have recently been prepared from a pair of PEG-PLL and an oligonucleotide with antisense activity toward the oncogene (c-Ha-ras) by our group (17). Further, transfection activity of plasmid DNA/PEG-PLL was recently reported by Wolfert et al., indicating a promising feature of these block copolymer/nucleotide complexes as a novel vector system (18). PEG-PLL association with DNA is also interesting from the standpoint of modeling the interaction of cationic proteins with DNA through electrostatic interactions, which are responsible for the stability of the higher ordered chromatin structure. Paticularly, study of the exchange reaction of complexed DNA with other polyanion compounds could provide a motivation for exploring a gene expression from the chemical point of view. We report here the physicochemical characteristics of water-soluble nanoassociates formed between salmon testes DNA or Col E1 plasmid DNA and a PEG-PLL block copolymer. The stability of the complex is discussed on the basis of the results of melting curve measurements, which have been widely used to estimate the properties of polylysine/DNA complexes as a model for chromatin (19-29). Further, dissociation of the complex was evidenced through the exchange reaction of complexed DNA with a model polyanion [poly(aspartic acid)]. MATERIALS AND METHODS

Materials. High molecular weight poly(L-lysine) hydrobromide (H-PLL, DP ) 927), low molecular weight poly(L-lysine) hydrobromide (L-PLL, DP ) 19), poly(Laspartic acid) sodium salt (DP ) 105), and DNA (sodium salt from salmon testes) were purchased from Sigma Chemical Co. and used without further purification. Col E1 plasmid DNA (6646 bp, percentage of GC is 48%), which produces colicin E1, was purchased from Nippon Gene Co., Ltd., Japan. DNase I nuclease and EcoRI restriction nuclease were purchased from Takara Shuzo © 1997 American Chemical Society

Polyanion Complex Associates of DNA and PEG−PLL

Co., Ltd., Japan. Col E1 plasmid was linearized using EcoRI restriction nuclease through a standard protocol. R-Methyl-ω-amino poly(ethylene glycol) (MW ) 4300) was a cordial gift from Nippon Oil & Fats Co., Ltd., Japan. The PEG-PLL block copolymer (average degree of PLL segment is 20) was prepared as described before (14). For other reagents, commercial special grade reagents were used without further purification. Dynamic Light Scattering (DLS) and Laser-Doppler Electrophoresis Measurements of PEG-PLL/ DNA Complex. DLS measurements were carried out using a DLS-700 instrument (Otuka Electronics Co, Ltd.). An Ar ion laser (λ0 ) 488 nm) was used as the incident beam. The sample was prepared by direct mixing of each solution of DNA (from salmon testes) and PEG-PLL in 10 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl. PEG-PLL solution of a certain concentration was added to 1.0 mL of 50 µg/mL (1.00 OD at 260 nm) DNA solution to completely compensate for the charge of the DNA (stoichiometric condition). The DNA concentration of the mixture was then adjusted to 25 µg/mL for DLS measurement. Laser-Doppler electrophoresis measurements of the complex associates were carried out using an ELS-800 instrument (Otuka Electronics) in 10 mM phosphate buffer (pH 7.4). From the determined electrophoretic mobility, the zeta-potential (ζ) was calculated according to the Smoluchouski equation as

ζ ) 4πηu/ where η is the viscosity of the solution, u is the electrophoretic mobility, and  is the dielectric constant of the solvent. Measurement of Melting Curve of PEG-PLL/ DNA Complexes. DNA (from salmon testes) was dissolved at a concentration of 1.00 OD in 1 mM sodium phosphate buffer (pH 7.4). To a constant volume of DNA solution were added directly at once varying amounts of PEG-PLL, H-PLL, or L-PLL in an equivolume of phosphate buffer to form a solution with a DNA concentration of 0.50 OD. The ratio of lysine residue to the nucleotide in the mixed solution is designated r ()[lysine residue]/ [nucleotide]). After 2 h of incubation at room temperature, the same volume of methanol was added to each sample. The melting profiles of the complexes were then monitored by absorbance at 260 nm on a Jasco Ubest-50 spectrophotometer with a Peltier EHC363 type cell holder. Samples were heated from 30 to 76 °C. The scanning rate was 1.0 °C/min. Differential Scanning Calorimeter (DSC) Measurement of DNA Complexes. The thermal behavior of the DNA complexes in 10 mM phosphate buffer (pH 7.4) (DNA concentrations were adjusted to 250 µg/mL) was determined using a microdifferential scanning calorimeter (MC-2 differential scanning calorimeter, Microcal, Inc.). One milliliter of PEG-PLL solution with various concentrations was added dropwise to 1.0 mL of DNA solutions (DNA concentration was 500 µg/mL) to form r ) 0.10, 0.20, and 0.30 complexes with vortex stirring. Two hundred fifty micrograms of complexed DNA was used for DSC measurement. The samples were heated from 10 to 105 °C. The heating rate was 1.5 °C/ min. Exchange Reaction of PEG-PLL/Plasmid DNA (pDNA) Complex with Poly(aspartic acid). The PEG-PLL/pDNA complex (r ) 1.0, DNA concentration was 33.3 µg/mL) was prepared through a direct mixing of linearized Col E1 pDNA and PEG-PLL block copolymer in 10 mM phosphate-buffered saline (10 mM sodium phosphate buffer containing 150 mM NaCl, pH 7.4). The

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Figure 1. Size distribution of PEG-PLL/DNA complex determined from DLS measurement (weight-averaged scale).

complex was incubated for 2 h at room temperature, followed by the addition of poly(aspartic acid) [P(Asp)] solutions of various concentrations in 10 mM phosphatebuffered saline. The molar ratio of P(Asp) to DNA in the complexes was 0.50, 1.0, 2.0, 4.0, 10, and 20. The final concentration of pDNA was adjusted to 25 µg/mL. After overnight incubation of the mixtures at room temperature, 25 µL of each sample was analyzed by electrophoresis at 5.6 V/cm with a 0.6% agarose gel in Tris-HCl buffer (pH 7.4) (3.3 mM Tris, 1.7 mM sodium acetate). DNA was visualized by ethidium bromide (0.5 µg/mL of gel). The same experiments were done for the H-PLL (DP ) 927)/pDNA complex to compare the stability with PEGPLL/pDNA complexes. Nuclease Resistance of PEG-PLL/DNA Complex. Salmon testes DNA (0.8 OD) and an equimolar amount of PEG-PLL in 10 mM phosphate buffer (pH 7.4) containing 5 mM magnesium sulfonate were mixed directly to obtain a 0.4 OD solution of PEG-PLL/DNA complex (r ) 1.0). After the addition of 10 units (10 µL) of DNase I to 1 mL of PEG-PLL/DNA complex at 25 °C, the absorbance change at 260 nm was followed to estimate DNA degradation by DNase I. RESULTS AND DISCUSSION

DLS Measurement of PEG-PLL/DNA Complex. On the addition of PEG-PLL to a 1.0 OD solution of DNA in 10 mM phosphate buffer containing 150 mM NaCl at pH 7.4 (phosphate-buffered saline), no turbidity was observed even under stoichiometric condition, when the unit ratio of L-lysine and phosphate was equal (r ) 1.0). This is in sharp constrast to the mixture of H-PLL or L-PLL with DNA, which caused a precipitate regardless of the polymerization degree with increasing r to 1.0. In spite of the transparent appearance of the PEG-PLL/ DNA solution, the light scattering intensity of the solution was increased about 10 times compared to that of the original DNA solution, suggesting the formation of water-soluble nanoassociates through a complexation of DNA with PEG-PLL. Indeed, this was verified by the histogram analysis of the DLS data. Figure 1 shows the weight-averaged size distribution analyzed by the histogram method for the PEG-PLL/DNA complex. Associates of 48.5 nm in diameter were observed with a small fraction of secondary aggregates in the 140 nm region. Size and distribution were similar for all of the associates prepared with varying salt concentration in the range between 0 and 300 mM NaCl in 10 mM phosphate buffer. Recently, atomic force microscope image of the PEG-PLL/DNA complex was reported by Wolfert et al. (18). Their result revealed that PEG-PLL/ DNA complex mainly forms extented toroidal structure,

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Figure 2. Melting curves of PEG-PLL/DNA complexes in 50% (v/v) methanol/1 mM phosphate buffer (pH 7.4).

which has a size exceeding 100 nm. In comparison, a smaller sized complex was dominant in our light scattering measurement. One possible reason for the inconsistency of the complex size might be a difference in the composition of the block copolymers. They used PEGPLL with a longer chain length of PLL segment (DP ) 78). It should be noted that complex size was correlated to PLL chain length in the case of DNA complex with PLL homopolymer (30). The average ζ-potential of the associate was then calculated using the Smoluchouski equation from the value of the electrophoretic mobility [(-0.890 ( 0.321) × 10-5 cm2/V‚s in 10 mM phosphate buffer, pH 7.4] measured by Laser-Doppler electrophoresis (ELS-800, Otsuka Electronics Co.). The ζ-potential thus caluclated was -1.12 ( 0.40 mV (n ) 3). This small absolute value of the ζ-potential was consistent with the formation of a core-shell structure, in which the PEG corona surrounds the complexed DNA in the core to form a microenvironment with relatively low permittivity. Presumably, this local environment with lowered permittivity may facilitate the compaction of complexed DNA to form a collapsed core of the complexed associate. It should be noted that charge neutralization by polycations as well as lowered permittivity of the environment are both known to be essential factors for DNA compaction (8, 31). Measurement of Melting Curve of PEG-PLL/ DNA Complexes. The stability of the complex was then evaluated through melting curve measurement, which has been widely used for the charecterization of DNA/ polylysine complexes. Melting is conveniently monitored by an increase in absorbance (hyperchromic effect) that results from the disruption of base stacking in doublestranded DNA due to the breakage of hydrogen bonds. A biphasic melting profile has been shown for a DNA/ polylysine complex system with an excess DNA ratio (22-28): One at a lower temperature is the melting of free DNA base pairs, and the other at higher temperature is that of base pairs in the region complexed with polylysine. Because the double-stranded structure of DNA is considerably stable under physiological condition, measurements were carried out in methanol-added buffer with low ionic strength (1 mM sodium phosphate buffer) to reduce the transition temperature of the complexed DNA to the measuring temperature range (30-78 °C) (24). Note that the melting temperature of DNA increases with lowered salt counterion due to an increase in electrostatic repulsion between phosphates as well as with methanol addition due to the destabilization of hydrogen bonding of the base pairs. Figure 2 shows the melting profiles of DNA (from salmon testes) at various complex ratios of PEG-PLL

Katayose and Kataoka

Figure 3. Melting curves of DNA complexes (r ) 0.50) in 50% (v/v) methanol/1 mM phosphate buffer (pH 7.4).

to DNA in 1 mM phosphate buffer/methanol 50% (v/v). The melting temperature (Tm) of free DNA was observed at 44 °C. At r ) 1.0, the transition of free DNA was no longer observed, and only the transition due to the denaturation of the complexed DNA was observed at higher temperature (ca. 70 °C), indicating that a stoichiometric complex was formed between DNA and PEG-PLL. In the 0 < r < 1 region, the melting curve is biphasic. Contribution of the free DNA to hyperchromicity decreased with increases in the ratio of PEG-PLL, while the contribution of complexed DNA increased with increases in the ratio of PEG-PLL. These biphasic melting profiles demonstrate that the complexed and free parts of DNA in the PEG-PLL/DNA system could be clearly discriminated by the melting temperature. Thus, migration of PEG-PLL along/between DNA molecules should be extremely slow to observe free and complexed DNA separately in the melting profile. It should be noted that only a single transition temperature was observed for the L-PLL/DNA system and that this averaged transition temperature shifted to a higher region with increasing ratio of L-PLL to DNA. As shown in Figure 3, L-PLL/DNA at r ) 0.5 showed a single transition at the middle point of the transition temperature of free DNA (r ) 0) and complexed DNA (r ) 1.0). This result suggests the fast migration of the constituent molecules in the complex, resulting in the facile dissociation of the complex. That is, only an unstable complex is formed between L-PLL and DNA. The L-PLL molecule seems to be too short to form a stable polyion complex with DNA and may migrate along the DNA molecule like a low molecular weight counterion (24). Thus, the L-PLL/ DNA complex (r ) 0.50) is thought to form a so-called nonstoichiometric complex. Formation of a nonstoichiometric water-soluble complex occurs when a polyelectrolyte with relatively low molecular weight is mixed with an excess amount of an oppositely charged polyelectrolyte with higher molecular weight (32). Compared with the molecular weight of DNA, the polymerization degree of L-PLL is small enough to form a nonstoichiometric complex. In contrast, the PEG-PLL/DNA complex showed the features of a stoichiometric complex even though the length of the poly(lysine) segment in PEGPLL is almost the same as that in L-PLL. The PEG segment of PEG-PLL contributes crucially to improve the stability as well as the solubility of the complex. One plausible factor for stabilizing the DNA complex with PEG-PLL may be a local decrease in permittivity around the DNA molecule due to the PEG chain. Indeed, PEG is known to induce a coil-globule transition of DNA due to decreased permittivity (33). However, to induce the transition of DNA, an extremely high concentration of PEG (>5 mol/L) should be required. In our system, the

Polyanion Complex Associates of DNA and PEG−PLL

Figure 4. DSC curves of PEG-PLL/DNA complexes in 10 mM phosphate buffer (pH 7.4). Scan rate ) 1.5 °C /min; DNA concentration ) 250 µg/mL.

average PEG concentration in the complex solution was too low to change the bulk permittivity of the solution. Nevertheless, binding of the block copolymer to DNA through electrostatic interaction allows an increase in the local concentration of PEG around the DNA molecules to decrease the permittivity of the microenvironment. Charge neutralization of DNA by the poly(L-lysine) segment as well as compartmentalization of complexed DNA in the microenvironment of PEG may lead to the higher stability of the PEG-PLL/DNA complex compared to the corresponding L-PLL/DNA complex system. Although complex stabilization could be achieved by increasing the molecular weight of the poly(L-lysine) homopolymer in the PLL/DNA system, the resultant complex was too low in solubility in water to form a precipitate. This is in sharp contrast to the PEG-PLL/DNA complex system, which shows a high solubility in aqueous milieu. DSC Measurements of DNA Complexes. As demonstrated in the melting curve experiments described above, stabilization of the double-stranded helical structure of DNA complexed with PEG-PLL or H-PLL is achieved through the neutralization of the phosphate group by the -NH3+ group of the PLL segment. Further, the phase transition of DNA into a globule form may occur with the PEG-PLL/DNA complex because of the compartmentalization into the PEG atmosphere with low pemittivity. However, methanol was added for the experiments in the melting curve measurements. Thus, the possibility that added methanol decreases the permittivity to induce coil-globule transition of DNA is not excluded. Therefore, to estimate the contribution of the PEG segment of the PEG-PLL block copolymer to DNA stabilization in aqueous milieu, DSC measurements were done in 10 mM phosphate buffer (pH 7.4) (34). The DSC curve of native DNA (from salmon testes) showed an endothermic peak at 69.4 °C (Figure 4). The DSC curves of PEG-PLL/DNA with various lysine/phosphate ratios (r) showed two endothermic peaks at around 70 and 95 °C, respectively. These peaks are thought to correspond to the denaturation of free and complexed DNA and are consistent with the biphasic melting profile shown in Figure 2. Note that the area of the peak corresponding to complexed DNA increased with an increase in r, which is in line with formation of a stoichiometric complex of DNA with PEG-PLL in a cooperative manner. Only a broad peak was obtained for the L-PLL/DNA complex (data not shown), reflecting fast migration of molecules in the complex between L-PLL and DNA. The enthalpy of denaturation of the complexed DNA was roughly estimated from the area of the peak that appeared in the higher temperature region (second peak).

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Figure 5. Schematic illustrations of dissociation (a) and exchange (b) of polyion complexes: (a) dissociation of the polyion complex scarcely occurs because of multisite binding; (b) exchange reaction of the polyion complex may occur through a cooperative exchange reaction.

The enthalpy change for denaturation of complexed DNA with PEG-PLL was estimated to be 50 kJ (bp mol)-1 °C-1 and that with H-PLL complex was 18 kJ (bp mol)-1 °C-1. This result indicates greater ability of PEG-PLL to stabilize the DNA double-helical structure than H-PLL even though the latter has a polymerization degree 50 times higher than the former, demonstrating the crucial role of the PEG segment in the stabilization process of the complex. PEG segments surrounding electrically neutralized DNA molecules allow formation of a microenvironment with relatively low permittivity. This local environment may facilitate the compaction of DNA to stabilize the complex structure. Exchange Reaction of PEG-PLL/pDNA Complex on the Addition of Poly(aspartic acid). Simple dissociation of an ion complex between a pair of oppositely charged macromolecules with sufficient chain length is hardly believed to occur because of the integrated stabilization through multisite interaction (18, 22, 35). However, exchange reaction with other charged macromolecules is known to take place as schematically shown in Figure 5 (36). It is suggested that this type of exchange reaction may take place in the release of DNA from the complex with polycations and cationic polypeptides in intracellular environment because various types of anionically charged macromolecules, including mRNA, sulfated sugars, and nuclear chromatin, exist as essential cellular components (35). From these aspects, it may be worth estimating the exchange reaction of DNA in complexes with other polyanions. In this study, P(Asp) with a DP of 105 was used as the replacing polyanion. Figure 6 shows the results of the agarose gel electrophoresis for the PEG-PLL/pDNA complex after the addition of various amounts of P(Asp). As a model double-stranded DNA, linearized Col E1 plasmid DNA (6646 bp, 4.4 × 106 Da) was used in the experiment shown in Figure 6 to omit the effect of molecular size distribution of DNA and to gain sensitivity in gel electrophoresis. Linearized plasmid DNA mixed with PEG-PLL in an equimolar unit ratio (r ) 1.0) showed no migration from the slot of the agarose gel because of the quantitative formation of a neutral complex (Figure 6, lane 2). This fact further confirms a stable complex formation between DNA and PEG-PLL block copolymer even in the solution with physiological salt concentration. Interestingly, pDNA in the complexes began to migrate with the addition of 10 times excess amounts of P(Asp), as shown in lane 7 of Figure 6. This is considered to be due to the cooperative exchange reaction of P(Asp) with pDNA in

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Figure 6. Effect of P(Asp) addition on the electrophoretic migration of pDNA from PEG-PLL/pDNA in 0.6% agarose gel: (lane 1) pDNA alone; (lane 2) PEG-PLL/pDNA complex; (lanes 3-8) PEG-PLL/pDNA complexes with progressively increasing proportions of P(Asp) [0.50, 1.0, 2.0, 4.0, 10, and 20 equiv of P(Asp) to pDNA].

Figure 7. Effect of P(Asp) addition on the electrophoretic migration of pDNA from H-PLL/pDNA in 0.6% agarose gel: (lane 1) pDNA alone; (lane 2) H-PLL/pDNA complex; (lanes 3-8) H-PLL/pDNA complexes with progressively increasing proportions of P(Asp) [0.50, 1.0, 2.0, 4.0, 10, and 20 equiv of P(Asp) to pDNA].

the complex (eq 1), allowing the release of pDNA in the medium.

PEG-PLL/pDNA + P(Asp) f PEG-PLL/P(Asp) + pDNA (1) Worth mentioning are the facts that the PEG-PLL/ pDNA complex formation is not irreversible in nature and that DNA in the complex can be substituted by an appropriate counterpolyanion. A similar phenomenon was also observed for the H-PLL/DNA system. pDNA in the H-PLL/pDNA complex was also substituted with a 10 times excess molar amount of P(Asp) (Figure 7, lane 7). Given that the stability of polyion complexes depends only on the molecular weight of the constituent polyelectrolytes, more P(Asp) should be required to release pDNA from the H-PLL/DNA complex compared to the PEGPLL/DNA system because H-PLL (DP ) 927) has an extremely higher degree of polymerization than PEGPLL, having a PLL segment with a DP of only 20. Obviously, this is not the case, and both PEG-PLL/ pDNA and H-PLL/pDNA complexes required the same amount of P(Asp) to release DNA. Thus, from the viewpoint of the exchange reaction, the PEG-PLL/pDNA complex is stable enough, as is the H-PLL/pDNA complex, in spite of the short PLL segment. In the case of the L-PLL/pDNA system, the L-PLL/pDNA complex was dissociated in the electrophoretic field under this buffer condition even without the addition of P(Asp), indicating its unstable nature. From a practical viewpoint, the regulated release of DNA from the complex through replacement by the counterpolyanion is noteworthy because the restricted

Katayose and Kataoka

Figure 8. Degradation profile of DNA and PEG-PLL/DNA complex by DNase I.

release of free DNA is one of the problems of the conventional vector system based on a polyion complex. Decreased molecular weight of PLL may increase the release rate of DNA, yet it may also shift the nature of the complex to a nonstoichiometric one as shown in Figure 3. Use of the PEG-PLL block copolymer system seems to overcome this discrepancy by stabilizing the complex through the compartmentalization into the PEG microenvironment, yet, on the other hand, retaining the ability for replacement with a counterpolyanion. Nuclease Resistance of PEG-PLL/DNA Complex. Stabilization of DNA through PEG-PLL complexation was further studied from the viewpoint of nuclease resistance. By addition of DNase I to native DNA solution, absorbance of the solution was increased immediately due to the fragmentation of the DNA. However, no substantial increase in absorbance was observed for the PEG-PLL/DNA complex system (Figure 8). From a comparison of the slope of the curves reflecting the velocity of the degradation, it was calculated that the apparent activity of DNase I to degrade complexed DNA with PEG-PLL is only 1.5% of that for native DNA. This high nuclease resistance ability of the PEG-PLL/DNA system indicates the stable nature of the PEG-PLL/DNA complex in which the migration of the constituent polymer chain (PEG-PLL and DNA) is restricted. This feature of high resistance toward nuclease attack is surely an advantage of using the PEG-PLL/DNA complex as a reservoir for DNA under physiological circumstances when DNA degradation through nuclease attack readily takes place. ACKNOWLEDGMENT

This research was supported by a Grant-in-Aid for Scientific Research (Priority Area Research Program: Supramoecular Structures), the Ministry of Education, Science, and Culture, Japan. LITERATURE CITED (1) Wu, G. Y., and Wu, C. H. (1987) Recepter-mediated in vitro Gene Transformation by a soluble DNA carrier system. J. Biol. Chem. 262, 4429-4432. (2) Wagner, E., Zenke, M., Cotten, M., Beug, H., and Birnstiel, M. L. (1990) Transferrin-polycation conjugates as carriers for DNA uptake into cells. Proc. Natl. Acad. Sci. U.S.A. 87, 34103414. (3) Behr, J. P., Demeneix, B., Loeffler, J. P., and Perez-Mutul, J. (1989) Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA. Proc. Natl. Acad. Sci. U.S.A. 86, 6982-6986. (4) Boussif, O., Lezoualc’h, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J. P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethyleneimine. Proc. Natl. Acad. Sci. U.S.A. 92, 7297-7301.

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Polyanion Complex Associates of DNA and PEG−PLL (5) Zhou, X., Klibanov, A. L., and Huang, L. (1991) Lipophilic polylysines mediate efficient DNA transfection in mammalian cells. Biochim. Biophys. Acta 1065, 8-14. (6) Haynes, M., Garrett, R. A., and Gratzer, W. B. (1970) Structure of nucleic acid-poly base complexes. Biochemistry 9, 4410-4416. (7) Chattoraj, D. K., Gosule, L. C., and Schellman, J. A. (1978) DNA Condensation with polyamines II. Electron microscopic studies. J. Mol. Biol. 121, 327-337. (8) Manning, S. G. (1978) The molecular theory of polyelectrolyte solutions with application to the electrostatic properties of polynucleotides. Q. Rev. Biophys. 11, 179-246. (9) Katayose, S., and Kataoka, K. (1996) PEG-Poly(lysine) block copolymer as a novel type of synthetic gene vector with supramolecular structure. In Advanced Biomaterials in Biomedical Engineering and Drug Delivery System (N. Ogata, S. W. Kim, J. Feijin, and T. Okano, Eds.) pp 319-320, Springer-Verlag, Tokyo. (10) Kataoka, K., Kwon, G. S., Yokoyama, Y., Okano, T., and Sakurai, Y. (1993) Block copolymer micelles as vehicles for drug delivery. J. Controlled Release 24, 119-132. (11) Kwon, G. S., and Kataoka, K. (1995) Block copolymer micelles as long-circulating drug vehicles. Adv. Drug. Deliv. Rev. 16, 295-309. (12) Yokoyama, Y., Miyauchi, M., Yamada, N., Okano, T., Sakurai, Y., Kataoka, K., and Inoue, S. (1990) Polymer micelles as novel drug carrier: Adriamycin-conjugated poly(ethylene glycol)-poly(aspartic acid) block copolymer. J. Controlled Release 11, 269-278. (13) Kwon, G. S., Naito, N., Yokoyama, Y., Okano, T., Sakurai, Y., and Kataoka, K. (1995) Physical entrapment of adriamycin in AB block copolymer micelles. Pharm. Res. 12, 200-203. (14) Harada, A., and Kataoka, K. (1995) Formation of polyion complex micelles in an aqueous milieu from a pair of oppositely-charged block copolymers with poly(ethylene glycol) segments. Macromolecules 28, 5294-5299. (15) Yokoyama, M., Okano, T., Sakurai, Y., Ekimoto, H., Shibazaki, C., and Kataoka, K. (1991) Toxicity and antitumor activity against solid tumors of micelle-forming polymeric anticancer drug and its extremely long circulation in blood. Cancer Res. 51, 3229-3236. (16) Kwon, G. S., Suwa, S., Yokoyama, M., Okano, T., Sakurai, Y., and Kataoka, K. (1994) Enhanced tumor accumulation and prolonged circulation times of micelle-forming poly(ethylene oxide-aspartate) block copolymer-adriamycin conjugates. J. Contorolled Release 29, 17-23. (17) Kataoka, K., Togawa, H., Harada, A., Yasugi, K., Matsumoto, T., and Katayose, S. (1996) Spontaneous formation of polyion complex micelles with narrow distribution from antisense oligonucleotide and cationic bloock copolymer in physiological saline. Macromolecules 29, 8556-8557. (18) Wolfert, M. A., Schacht, E. H., Toncheva, V., Ulbrich, K., Nazarova, O., and Seymour, L. W. (1996) Characterization of vector for gene therapy formed by self-assembly of DNA with synthetic block co-polymers. Hum. Gene Ther. 7, 21232133. (19) Shapiro, J. T., Leng, M., and Felsenfeld, G. (1969) Deoxyribonucleic acid-polylysine complexes. Structure and nucleotide specificity. Biochemistry 8, 3219-3232. (20) Carroll, D. (1972) Complexes of polylysine with polyuridylic acid and other polynucleotides. Biochemistry 11, 426-433. (21) Chang, C., Weiskopf, M., and Li, H. J. (1973) Conformation studies of nucleoprotein. Circular dichroism of deoxyribonucleic acid base pairs bound by polylysine. Biochemistry 12, 3028-3032.

(22) Fasman, G. D., and Mandel, R. (1976) Chromatin models.Interaction between DNA and polypeptides containing LLysine and L-Valine: Circular dichroism and thermal denaturation studies. Biochemistry 15, 3122-3130. (23) Tuboi, M., Matsuo, K., and Ts’o, P. O. P. (1966) Interaction of poly-L-lysine and nucleic acids. J. Mol. Biol. 15, 256-267. (24) Leng, M., and Felsenfeld, G. (1966) The preferential interactions of polylysine and polyarginine with specific base sequences in DNA. Proc. Natl. Acad. Sci. U.S.A. 56, 13251332. (25) Olins, D. E., Olins, A. L., and Von Hippel, P. H. (1967) Model nucleoprotein complexes: Studies on the interaction of cationic homopolypeptides with DNA. J. Mol. Biol. 24, 157176. (26) Shih, T. Y., and Bonner, J. (1970) Thermal denaturation and template properties of DNA complexes with purifide histone fractions. J. Mol. Biol. 48, 469-487. (27) Li, H. J., Chang, C., Weiskopf, M., Brand, B., and Rotter, A. (1974) Helix-Coil transition in nucleoprotein:Renaturation of polylysine-DNA and polylysine-nucleohistone complexes. Biopolymers 13, 649-667. (28) Li, H. J., Brand, B., Rotter, A., Chang, C., and Weiskopf, M. (1974) Helix-Coil transition in nucleoprotein. Effect of ionic strength on thermal denaturation of polylysine-DNA complexes. Biopolymers 13, 1681-1697. (29) Weiskopf, M. (1977) Poly(L-Lysine)-DNA interaction in NaCl solutions: BfC and Bfψ transitions. Biopolymers 16, 669-684. (30) Wolfert, M. A., and Seymour, L. W. (1996) Atomic force microscopic analysis of the influence of the molecular weight of poly(L)lysine on the size of polyelectrolyte complexes formed with DNA. Gene Ther. 3, 269-273. (31) Yoshikawa, K. (1996) Kinetics of collapse and Decollapse of a single doubble-stranded DNA chain. Macromol. Symp. 106, 367-378. (32) Kabanov, V. A., and Zezin, A. B. (1982) Water-soluble nonstoichiometric polyelectrolyte complexes: A new class of synthetic polyelectrolytes. Sov. Sci. Rev., Sect. B 4, 207-282. (33) Evdokimov, Y. M., Akimenko, N. M., Glukhova, N. E., and Varshavskii, Y. M. (1974) Compact form of DNA in solution communication I. Feature of the absorption spectra of Polyribonucleotides and DNA in aqueous salt solutions containing polyethyleneglycol. Mol. Biol. (Kiev) 8, 396-405. (34) Fujioka, K., Baba, Y., and Kagemoto, A. (1979) Thermal properties of DNA-poly(L-lysine) system. Polym. J. 11, 509513. (35) Erbacher, P., Roche, A. C., Monsigny, M., and Midoux, P. (1995) Glycosylated polylysine/DNA complexes: Gene transfer efficiency in relation with the size and the substitution level of glycosylated polylysines and with the plasmid size. Bioconjugate Chem. 6, 401-410. (36) Bakeev, K. N., Izumrudov, V. A., Kuchanov, S. I., Zezin, A. B., and Kabanov, V. A. (1992) Kinetics and mechanism of interpolyelectrolyte exchange and addition reactions. Macromolecules 25, 4249-4254. (37) Labrt-Moleur, F., Steffan, A. M., Brisson, C., Perron, H., Feugeas, O., Furstenberger, P., Oberling, F., Brambilla, E., and Behr, J. P. (1996) An electron microscopy study into the mechanism of gene transfer with lipopolyamines. Gene Ther. 3, 1010-1017.

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