Polycationic Graft Copolymers as Carriers for Oligonucleotide Delivery

Vladimír Šubr, Čestmír Koňák, Richard Laga, and Karel Ulbrich ... Tomáš Etrych , Blanka Říhová , Fernando Carlos Giacomelli , Petr Štěpánek , Karel Ul...
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Langmuir 2001, 17, 3096-3102

Polycationic Graft Copolymers as Carriers for Oligonucleotide Delivery. Complexes of Oligonucleotides with Polycationic Graft Copolymers Herbert Dautzenberg* and Arkadi Zintchenko Max-Planck-Institut fu¨ r Kolloid- und Grenzfla¨ chenforschung, Am Mu¨ hlenberg, D-14476 Golm, Germany

C ˇ estmı´r Konˇa´k, Toma´sˇ Reschel, Vladimı´r Sˇ ubr, and Karel Ulbrich Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, CZ-162 06 Prague 6, Czech Republic Received December 20, 2000. In Final Form: February 19, 2001 Self-assembling systems based on ionic complexes of oligonucleotides (36 base pairs) and model oligophosphates (polymerization degree of 35) with high molecular poly(L-lysine) molecules (Mw ) 134 000) grafted with short poly[N-(2-hydroxypropyl)methacrylamide] chains (Mw ) 7000) were studied as systems suitable for gene therapy applications. Poly(L-lysine) and poly(trimethylammonioethyl methacrylate chloride) homopolycations were used for comparison. The physicochemical properties of polyelectrolyte complexes (PEC) were examined by static and dynamic light scattering methods. While PECs prepared with homopolycations tend to aggregate, particularly at high degrees of charge conversions, the complexes prepared with graft copolymers are soluble at any charge conversions in aqueous solutions. The complexes prepared with an excess of oligophosphates were found to be stable in physiological salt conditions and in the bovine serum albumin solutions (1 mg/mL). A formation of PEC/albumin complexes and large aggregates was observed for uncompensated PECs with cationic excess.

* To whom correspondence should be addressed. E-mail: [email protected].

from the bloodstream was rapid with a plasma half-life of less than 5 min.9 To avoid the problem of aggregation and to improve the biocompatibility of these synthetic vectors, polycationic block copolymer carriers were introduced that use a polycation conjugated to a hydrophilic nonionic polymer10 such as poly(ethylene glycol) (PEG) and poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA); e.g., poly(ethylenimine-block-ethylene glycol),11 poly(L-lysine-blockethylene glycol),12,13 poly(spermine-block-ethylene glycol),14 and poly(trimethylammonioethyl methacrylate chlorideblock-N-(2-hydroxypropyl)methacrylamide).13 The copolymers and oligonucleotides self-assemble into micelle like aggregates in which the oligonucleotide chains are incorporated into the polyion core. These species are soluble and have a small diameter, low surface charge, and narrow size distribution. They are stable to the physiological saline solution, and oligonucleotides incorporated into them are protected from enzymatic digestion in biological fluids. However after 30 min, the copolymer carriers are cleared from the bloodstream.13 Thus, the oligonucleotidecopolymer complexes are not stable enough for prolonged circulation.

(1) Helene, C. Eur. J. Cancer 1991, 27, 1466. (2) Stein, C. A.; Cheng, Y.-C. Science 1993, 261, 1004. (3) Yacyshyn, B. R.; Bowen-Yacyshyn, M. B.; Jewell, L.; Tami, J. A.; Bennett, C. F.; Kisner, D. L.; Shanahan, Jr., W. R. Gastroenterology 1998, 114, 1133. (4) Webb, A.; Cunningham, D.; Cotter, F.; Clarke, P. A.; Di Stefano, F.; Ross, P.; Corbo, M.; Dziewanowska, Z. The Lancet 1997, 349, 1137. (5) Gao, X.; Huang, L. Biochem. Biophys. Res. Commun. 1991, 179, 280. (6) Bennett, C. F.; Chiang, M.-Y.; Chan, H.; Shoemacer, J. E. E.; Mirabelli, C. K. Mol. Pharmacol. 1992, 41, 1023. (7) Trubetskoy, V. S.; Torchilin, V. P.; Kennel, S.; Huang, L. Biochim. Biophys. Acta 1992, 1131, 311. (8) Bunnell, B. A.; Ashari, F. K.; Wilson, J. M. Somatic Cell Mol. Genet. 1992, 18, 559.

(9) Mahato, R. I.; Takemura, S.; Akamatsu, K.; Nishikawa, M.; Takakura, Y.; Hashida, M. Biochem. Pharmacol. 1997, 53, 887. (10) Seymour, L. W.; Kataoka, K.; Kabanov, A. V. In Self-Assembling Complexes for Gene Delivery: From Laboratory to Clinical Trials; Kabanov, K. V., Felgner, P. L., Seymour, L. W., Eds.; JohnWiley & Sons: New York, 1998; p 219. (11) Vinogradov, S. V.; Bronich, T. K.; Kabanov, A. V. Bioconjugate Chem. 1998, 9, 805. (12) Kataoka, K.; Togawa, H.; Harada, H.; Yasugi, K.; Matsumoto, T.; Katayose, S. Macromolecules 1996, 29, 8556. (13) Read, M. L.; Dash, P. R.; Clark, A.; Howard, K.; Oupicky´, D.; Toncheva, V.; Alpar, H. O.; Schacht, E. H.; Ulbrich, K.; Seymour, L. W. Eur. J. Pharm. Sci. 2000, 10, 169. (14) Kabanov, A. V.; Vinografov, S. V.; Suzdaltseva, Yu. G.; Alakhov, V. Yu. Bioconjugate Chem. 1995, 6, 639.

Introduction In the past decade there has been extensive research in an application of antisense oligonucleotides (ON) for the selective inhibition of gene expression and viral reproduction.1-4 However, the therapeutical applications of such oligonucleotides are currently limited by their low physiological stability, rapid elimination from the body, slow cellular uptake, and lack of tissue specificity. In an attempt to overcome these problems, synthetic vectors based on polycations such as cationic liposomes5,6 and poly(L-lysine) (PLL)7,8 are being developed as delivery systems for oligonucleotides. These positively charged carriers are electrostatically binding with negatively charged oligonucleotides. The carriers provide an enhanced stability against nuclease degradation, improved uptake of oligonucleotides into cells, and increased antisense activity in vitro. On the other hand, there is a problem with the carriers that they are often poorly water-soluble and tend to aggregate particularly at high degrees of charge conversion. Moreover, clearance of oligonucleotide carriers

10.1021/la001779t CCC: $20.00 © 2001 American Chemical Society Published on Web 04/14/2001

Polycationic Graft Copolymers

To improve the stability of the carriers, astromal poly(propylene imine) dendrimers of five generations were used very recently.15,16 In this study we have examined the use of high molecular PLL molecules grafted with short PHPMA chains for self-assembly with oligonucleotides and model oligophosphates in order to improve their stability and biocompatibility. PLL and poly(trimethylammonioethyl methacrylate chloride) (PTMAEM) homopolycations were used for comparison. The physicochemical properties of these complexes have been examined by static and dynamic light scattering methods. The stability to 0.15 N sodium chloride solution and bovine serum albumin interaction was tested. Experimental Section 1. Materials. 1.1. Poly(L-lysine hydrobromide) (PLL) (Mw ) 134 000) was obtained from SIGMA. 1.2. Graft Copolymer of Poly(L-lysine hydrobromide) with Poly(N-(2-hydroxypropyl)methacrylamide) (GPLL). 1.2.1. Preparation of N-(2-hydroxypropyl)methacrylamide (HPMA). N-(2-Hydroxypropyl)methacrylamide (HPMA) was prepared by reaction of methacryloyl chloride with 1-amino-2-propanol, as described earlier.17 1.2.2. Synthesis of PHPMA with Succinimidyloxycarbonyl End Group (PHPMA-COOSu). 1.2.2.1. Synthesis of PHPMA with Carboxylic End Group (PHPMA-COOH). Poly[N-2-(hydroxypropyl)methacrylamide] terminated with one carboxylic end group was prepared by solution radical polymerization of HPMA in the presence of 3-sulfonylpropionic acid as a chain transfer agent (T). HPMA (13.6 g) was dissolved in acetone (120 mL, 0.79 mol/ L). Initiator AIBN (65.2 mg, 0.004 17 mol % relative to monomer) and 3-sulfonylpropionic acid (220 mg, 0.0217 mol % relative to monomer) were then added. The solution was introduced into an ampule and bubbled through with nitrogen. The ampule was sealed, and polymerization was carried out at 50 °C for 24 h. The precipitate of polymer was dissolved in methanol and isolated by precipitation into an excess of acetone-diethyl ether mixture (3:1). The polymer was purified by precipitation from methanol solution into excess of acetone-diethyl ether. The yield was 4.8 g. The polymer was characterized by fast protein liquid chromatography (FPLC), Mw ) 7000 and Mn ) 4100, and titration of carboxylic end groups, Mn,T ) 4300. 1.2.2.2. Synthesis of PHPMA with Succinimidyloxycarbonyl End Groups (PHPMA-COOSu). PHPMA-COOH (1 g) was dissolved in DMF (3 mL), 10-fold molar excess of N-hydroxysuccinimide (0.269 g), and dicyclohexylcarbodiimide (0.481 g) were added at 5 °C. The reaction was catalyzed by 4-(dimethylamino)pyridine (0.059 g). The reaction mixture was stirred, and the reaction was carried out at 5 °C for 22 h. The polymer was isolated by precipitation into acetone-diethyl ether (3:1) and dried in a vacuum. 1.2.3. Synthesis of the Graft Copolymer (GPLL). PHPMACOOSu (0.348 g, Mw ) 7000) was dissolved in DMSO (15 mL) and added to a solution of PLL (0.232 g, Mw ) 134 000) and triethylamine (0.162 g) dissolved in DMSO (50 mL). The reaction mixture was stirred at 20 °C for 48 h. The volume of the mixture was reduced to 5 mL, and methanol (15 mL) was added. The polymer was isolated by precipitation into excess of diethyl ether, and the product was purified by dialysis. Yield was 0.467 g. The content of HPMA in the graft copolymer was obtained by 1H NMR analysis (60.6 wt % HPMA). Molecular weight of the copolymer estimated using 1H NMR data is given in Table 1. The structure of the GPLL copolymer is shown in Figure 1. 1.3. Oligonucleotides from Herring Sperm (OHS) were prepared by a fractionation of “crude oligonucleotides” prepared by (15) Kabanov, V. A.; Zezin, A. B.; Rogacheva, V. B.; Gulyaeva, Zh. G.; Zansochova, M. F.; Joosten, J. G. H.; Brackman, J. Macromolecules 1998, 31, 5142. (16) Kabanov, V. A.; Zezin, A. B.; Rogacheva, V. B.; Gulyaeva, Zh. G.; Zansochova, M. F.; Joosten, J. G. H.; Brackman, J. Macromolecules 1999, 32, 1904. (17) Strohalm, J.; Kopecˇek, J. Angew. Makromol. Chem. 1978, 70, 109.

Langmuir, Vol. 17, No. 10, 2001 3097 Table 1. Characteristics of Polymers polymera

Mw

PTMAEM OPP GPLL

371 000d 3 632 (cal) 334 000 (cal)j 314 000k 13 500c 134 000f

OHS PLL

no. of bp or PP groups 35 ( 4f 36c

M (g/charge)

dn/dc

206.9b 107.8b 560b

0.175h 0.117h 0.175 (cal)i

325g 225e

0.185g 0.188h

a Abbreviations: PTMAEM ) poly(2-(trimethylammonio)ethyl methacrylate), OPP ) sodium phosphate glass, GPLL ) PLL-graftPHPMA (poly(N-(2-hydroxypropyl)methacrylamide), OHS ) oligonucleotides (herring sperm), PLL ) poly(L-lysine). b Colloid titration. c Electrophoresis. d GPC. e Potentiometric chloride titration. f Sigma. g DNA. h Brice-Phoenix differential refractometer. i Calculated as a weight-average of PHPMA (dn/dc ) 0.167) and PLL increments. j Calculated from Mw of PLL taking into account the grafting. k Measured by SLS.

Figure 1. Structure of the graft copolymer PLL-g-PHPMA (GPLL). degradation of deoxyribonucleic acid (DNA) from herring sperm (Sigma D-3159). The crude oligonucleotides were fractionated on A ¨ kta Explorer (Pharmacia) using a Superose 6 column. Distilled water was used as the mobile phase. The flow rate was 0.5 mL/min. Concentrations were detected by UV at 280 nm. Figure 2 shows the chromatogram of the starting DNA and an isolated fraction. The molecular weight was determined on the same chromatographic system connected with a multiangle light scattering detector DAWN-DSP-F (Wyatt Technology Corp., Santa Barbara, CA). Molecular characteristics of OHS are given in Table 1. The size of oligonucleotides was also estimated by electrophoresis using pUC 19/Sau 3A calibration (standards 78-1080 bp). Horizontal electrophoresis was performed using 1% agarose (Serva) gel in 0.1 M Tris-boric acid buffer, pH 8, at 4 V/cm. Ethidium bromide method was used for staining. The result is given in Table 1. 1.4. Oligophosphates (OPP) with 35 phosphate (PP) groups and bovine serum albumin (BSA) were purchased from Sigma. 1.5. 2-(Trimethylammonio)ethyl methacrylate (TMAEM) was synthesized by the method described in the previous work,18 and the weight-average molecular weight of its polymer (PTMAEM) was estimated by GPC (for the result see Table 1). The molar mass per charge of the polyelectrolytes was estimated for a variety of used polymers by colloidal titration19,20 with poly(diallyldimethylammonium chloride) (PDAD(18) Konˇa´k, C ˇ .; Mrkvicˇkova´, L.; Nazarova, O.; Ulbrich, K. Supramol. Sci. 1998, 5, 67. (19) Wassner, K.-H.; Schroeder, U.; Horn, D. Makromol. Chem. 1991, 192, 553. (20) Dautzenberg, H. In Physical Chemistry of Polyelectrolytes; Radeva, Ts., Ed.; Marcel Dekker: New York, in press.

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Dautzenberg et al. where MPA is the molecular mass per charge unit of the polyanion, MPC is the molecular mass per charge unit of the polycation, MNaCl is the molar mass of the released salt, VPC 0 is the initial volume of the polycation solution, VPC d is the volume of the added PA polyanion solution, and cPC 0 and c0 are initial concentration of the polycation and polyanion solutions, respectively. In this case also the refractive index increment of the complexes changes with the mixing ratio. Assuming additivity of the contributions of the components, one obtains

vc )

vPCmPC + vPAmPAX mPC + mPAX

(3)

where vc, vPC, and vPA are the refractive index increments of the complex and the components, respectively. Assuming a 1:1 stoichiometry, these expressions are valid up to X ) 1; after that X ) 1 has to be used. The static light scattering data were analyzed by a Zimm plot: 2

RG q2 Kc 1 ) + 3Mw R(q) Mw

Figure 2. Chromatogram of starting ‘‘crude oligonucleotides” and used isolated fraction. MAC) or poly(styrenesulfonate) of known charge density as counterpart and toluidine blue as indicator. The results are given in Table 1. 2. Preparation Conditions. For static light scattering the complexes were prepared directly in the scattering cell. Ten milliliters of the polycationic solutions was put into the cells. Then the polyanion solutions of twice of the monomolar concentration of the polycation solutions were slowly added under gentle stirring up to the desired mixing ratio. All solutions were made dust-free by filtration through cellulose acetate membranes of 0.2 µm pore size (Sartorius, Germany). This order of mixing was consciously used to obtain complexes on a low level of aggregation. It is well-known from the work of Tsuchida21 and Kabanov22 that starting with an excess of the high molecular weight polyelectrolyte under appropriate salt conditions soluble complexes are formed, where the short chain component is uniformly distributed among the long chain one. But even in the case of highly aggregating systems, low amounts of salt may lower the level of aggregation by nearly 2 orders of magnitude.23 For dynamic light scattering the complexes were prepared in an analogous way. 3. Static Light Scattering (SLS). Static light scattering measurements were carried out with a Sofica 42000 instrument (Wippler and Scheibling, Strasbourg, France). The Sofica instrument was equipped with an intensity stabilized 1 mW He-Ne laser (Spectra Physics) as light source and a PC for data recording. The accuracy of the measurements was better than 1%. The scattering curves were measured after each step of dosage. The refractive index increments, dn/dc, of the individual components of the complexes were measured with a Brice-Phoenix differential refractometer at the light wavelength of 633 nm, or they were taken from the literature. The dn/dc values used are collected in Table 1. The calculation of the concentrations cc(X) and refractive index increments νc(X) of the complexes as a function of the molar mixing ratio X were carried out on the basis of the above-described model of complex formation. Then it follows for the complex concentration cc(X) in dependence on the molar mixing ratio X of anionic nPA to cationic groups nPC:

X)

cc(X) ) cPC 0

(

)(

PA cPA nPA 0 Vd MPC ) PC PC PC PA n M c0 V0

VPC 0 VPC 0

+

VPA d

(

)

(1)

)

MPC + XMPA - XMNaCl MPC

(2)

(4)

where R(q) is the Rayleigh ratio of the scattering intensity, q ) (4π/λ) sin Θ/2, λ is the wavelength in the medium, Θ is the scattering angle between the incident and the scattered beam, K is a contrast factor containing the optical parameters, c is the complex concentration, Mw is the weight-average of the molar mass of the complex particles, and RG is their radius of gyration (calculated from the z-average of the square). The concentration dependence was neglected, what seems to be justified because of the low concentrations of the PEC solutions (∼10-5 g/mL). Extrapolation to zero scattering angle was carried out by linear or quadratic fits of the scattering curves. 4. Dynamic Light Scattering (DLS). Polarized DLS measurements were made in the angular range 30-135° using a light scattering apparatus equipped with an He-Ne (632.8 nm) and Ar ion laser (514.5 nm) and an ALV 5000, multibit, multitau autocorrelator covering approximately 10 decades in delay time τ. The most of the measurements were realized at the scattering angle θ ) 90°. The inverse Laplace transform using the REPES24 method of constrained regularization, which is similar in many respects to the inversion routine CONTIN,25 was used for analysis of time autocorrelation functions. REPES directly minimizes the sum of the squared differences between the experimental and calculated intensity-time correlation functions using nonlinear programming. This method uses an equidistant logarithmic grid with fixed components (here a grid 10 components per decade) and determines their amplitudes. As a result, a scattered light intensity distribution function A(τ) of decay times is obtained which can be easily transformed in a distribution function of hydrodynamic sizes. The time autocorrelation functions were also fitted supposing the Pearson distribution of characteristic relaxation times, τc:24

z(τc) ) τopτc-p-1 exp(-τc /τo)/Γ(p)

(5)

where τo and p are parameters, and Γ(p) is the gamma function of parameter p. The Pearson distribution was chosen for the simplicity of its mathematical treatment. The average hydrodynamic radius, RH, was calculated from the diffusion coefficient, D, using the Stokes-Einstein equation:

RH ) kT/6πηD

(6)

where k is the Boltzmann constant, T is the absolute temperature, and η (0.894 cP) is the viscosity of water at 25 °C. The (21) Tsuchida, E.; Osada, Y.; Ohno, H. J. Macromol. Sci. 1980, B17 (4), 683. (22) Kabanov, V. A.; Zezin, A. B. Pure Appl. Chem. 1984, 56, 343. (23) Dautzenberg, H. Macromolecules 1997, 30, 7810. (24) Stepa´nek, P. In Dynamic Light Scattering; Brown, W., Ed.; Clarendon Press: Oxford, 1993; p 177. (25) Provencher, S. W. Comput. Phys. Commun. 1982, 27, 213.

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Figure 3. Dependence of the mass Mw (a), the size RG (b), and the structural density F (c) of complexes between two homopolycations (PTMAEM, PLL) and the oligophosphate (OPP) on the mixing ratio X as indicated in the insert. The concentrations of starting polycation solution was 2.5 × 10-4 M. (d) Zimm plot of the scattering curves of the complexes PLL/OPP in relation to the mixing ratio X (from top to bottom X ) 0.1 (0.1) 1). experimental error of RH determination for the complexes was typically about 3%.

Results and Discussion As already mentioned, the formation of polyelectrolyte complexes (PEC) is very sensitive to the presence of salt.23 While in deionized water PEC formation is mainly governed by the kinetics of the process and very high levels of aggregation are reached, small amounts of salt decrease these levels drastically. The lower level of aggregation is obviously caused by the screening effect of the salt. This leads to higher chain flexibility and supports rearrangement processes, making the conformational adaptation of chains easier and shifting the system more to thermodynamic equilibrium. On the other hand, higher ionic strength results in macroscopic flocculation. Therefore, as standard condition for the preparation of PECs in this work, we used NaCl solutions of an ionic strength of 1 × 10-2 mol/L, where a significantly lower level of aggregation was observed. 1. Effect of Polymer Structure. At first we studied the complex formation between the homopolycations PTMAEM and PLL and the oligophosphate OPP in relation to the mixing ratio X. The dependence of the particle mass Mw and the size RG of these complexes is represented in parts a and b of Figure 3, respectively. The concentration of the starting solutions was 2.5 × 10-4 monomol/L (calculated with the mass per charge M given in Table 1). To demonstrate the quality of light scattering measurements, the Zimm plot of the scattering curves of the complexes PLL/OPP is given in Figure 3d.

The complexes of both polycations show a high level of aggregation (100-1000 polycation chains), which strongly increases with rising X. Macroscopic flocculation occurred approaching X ) 1. The masses of PEC prepared with PLL bearing primary amino groups in side chains are systematically smaller than those prepared with PTMAEM bearing quaternary amino groups in side chains. Also, the values of the radius of gyration indicate some differences in complex formation of PLL and PTMAEM with OPP. Despite big differences in Mw, the radii are in the same range, but with different tendencies with increasing X. Using the model of spheres with a radius equal to the radius of gyration, we estimated the structural densities of the PECs (Figure 3c). Although being a rough approximation, it reflects the big difference in the degree of swelling of the PECs by nearly an order of magnitude. The solution of the polycations was normally the starting one. For the complex PTMAEM/OPP the reverse order of mixing led already at X ) 0.5 to macroscopic flocculation. The study of complex formation between the graft copolymer and oligoanions (OPP and OHS) revealed a completely different behavior. The Mw values are near to the value of the grafted poly(L-lysine) and increase only marginal with rising X (Figure 4a). The 1:1 mixing region could be passed without flocculation, and further addition of the oligoanions resulted in stable PECs with a particle mass near 1 million. From Mw of the clusters their aggregation number, N can be estimated. Assuming full charge compensation of GPLL molecules by polyanions at the maximum (X ≈ 1.1), the molecular weight of single

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Figure 5. Mean weight fractions, FH, of polymers in the PEC particle (polymer density) plotted as a function of the mixing ratio X for the system GPLL/OPP; the concentration of the starting polycation solution was 1 × 10-3 M.

Figure 4. (a) Dependence of Mw of complexes between a graft copolymer (GPLL), and both the oligophosphate (OPP) and oligonucleotide (OHS) on the mixing ratio X as indicated in the inset. The concentrations of starting polycation solutions were 1 × 10-3 and 2.5 × 10-4 M (indicated in the inset). (b) Dependence of RH and ∆RH/RH of complexes on the mixing ratio X for the system GPLL/OPP. The concentration of the starting polycation solution was 1 × 10-3 M.

GPLL complexes (Mw ) MwPC(MPA + MPC - MNaCl)/MPC) should be about 3.6 × 105 (OPP) and 4.9 × 105 g/mol (OHS), if we use the MwPC given by the supplier (Sigma-Aldrich). However, we found a somewhat lower value of MwPC in our experiments after sharp filtration through 0.2 µm filters. Then, N ) Mw(cluster)/Mw(single GPLL) ≈ 2-3. Thus, a very low aggregation number is observed, and we can state that single GPLL clusters are dominating in the solution. That enables us to control the number of oligoanions per PEC by the ratio of charged groups of the cationic copolymer and the oligoanions. These results are independent of the copolymer concentration in the investigated range (Figure 4a). We also did not find any significant differences between complexes prepared at different contents of NaCl (0.005-0.02 M). Complexes with OPP or OHS showed a quite similar behavior. Thus, oligophosphates can serve as model molecules for physicochemical investigations instead of the much more expensive oligonucleotides. Since the size of these clusters are too small for a reliable determination of the radii of gyration by static light scattering, the hydrodynamic radius RH of the complexes was estimated by DLS. The results for the GPLL/OPP system are given in Figure 4b. RH decreases with X up to X ) 1, and then a small increase (probably due to a small additional aggregation) is observed. Interestingly, also the polydispersity of the PECs decreases to be smallest about X ) 1. To characterize the particle structure, the mean weight fractions, FH, of polymers in the PEC particle

(structural density of PEC) were calculated from the hydrodynamic volumes of particles VH (in cm3) and from their corresponding molecular weights Mw (in g mol-1); FH ) Mw/NAVH, where NA is Avogadro’s number. RH values were used for estimation of VH. The results are shown in Figure 5. According to our expectations, FH increases on approaching the charge compensation point to be more or less saturated for higher X values. The value of FH ≈ 0.2 at higher X is comparable with FH values found for micelles in organic solvents.26 The above experimental results clearly show that graft copolymers (polycations grafted with water-soluble polymers) are more suitable for preparation of stable clusters than homopolycations. The reason for this is in the chemical copolymer structure. Hydrophilic blocks of graft copolymers (e.g., PHPMA blocks), which are not directly involved in the PEC formation, compensate the aggregation effect of hydrophobic polymer sites and protect PEC against spontaneous aggregation. In this study, the oligoanions due to complex binding and condensation on the GPLL backbone are protected by the grafted hydrophilic chains against further aggregation. This is a reversed process to that observed in complexation of DNA27,28 or high molecular weight polycations29 with short oppositely charged copolymers. The polyelectrolyte blocks of the short copolymers induce a condensation of DNA or other large polyelectrolytes, and hydrophilic blocks then act against aggregation. In the limit of very low DNA concentrations single DNA complexes could be formed which collapsed DNA is packed with short hydrophilic blocks.28 2. Time Stability of PECs with GPLL. To judge the time stability of the complexes between GPLL and OPP, we prepared PECs in the range of X between 0.25 and 1.5 in 0.25 steps (in 0.01 N NaCl, concentration of the starting solution 1 × 10-3 monomol/L) and measured the systems by SLS over 4 days. For all complexes only negligible changes of the scattering curves were observed, proving a high stability of the PECs. 3. Salt Stability of the Complexes. A combination of static and dynamic light scattering techniques was also used to examine the stability of complexes after addition of NaCl. Complexes were prepared in 0.01 N NaCl and (26) Tuzar, T.; Plesˇtil, J.; Konˇa´k, C ˇ .; Hlavata´, D.; Sikora, A. Makromol. Chem. 1983, 184, 2111. (27) Wolfert, M. A.; Schacht, E.; Toncheva, V.; Ulbrich, K.; Nazarova, O.; Seymour, L. W. Human Gene Therapy 1996, 7, 2123. (28) Oupicky´, D.; Konˇa´k, C ˇ .; Ulbrich, K. Mater. Sci. Eng. 1999, C275, 356. (29) Dautzenberg, H. Macromol. Chem. Phys. 2000, 201, 1765.

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Figure 6. Time dependence of Mw after an addition of albumin (1 mg/mL) for PECs prepared at X ) 1 in 0.01 N NaCl. Table 2. Time Dependence of RH after BSA Addition (1 mg/mL) X )1

X ) 1.25

time/min

RH/nm

time/min

RH/nm

0 80 180 1140

17.1 16.8 18.9 20.3

0 30 290 1215

14.4 15.6 15.5 15.5

then adjusted by salt solution to 0.15 N. Static light scattering revealed that complexes prepared at X ) 1 and 1.25 are stable for at least 48 h. The Mw measured in course of 2 days did not show any systematic increase. Dynamic light scattering monitored a small increase of RH from 16.0 to 16.7 nm after 48 h in solutions of complexes prepared at X ) 1, which is in the limit of accuracy. The increase of RH could also be due to a screening of electrostatic interactions, resulting in a swelling of the PECs. In the case of complexes prepared at X ) 0.75, Mw value has slowly increased on time to be by 20% higher after 3 h. Thus, uncompensated complexes slowly aggregate in 0.15 N NaCl solution. 4. Interaction of Complexes with Albumin. Light scattering techniques can be also used for a better understanding of an effect of albumin (BSA) on properties of oligonucleotide complexes. GPLL/OPP complexes used for the experiments were prepared by a standard way at three mixing ratios X ) 0.75, 1, and 1.25 in 0.01 N NaCl. Then 1 mg/mL of albumin was added to the complex solutions, which is an amount sufficient for a compensation of all hypothetical positive charges in solution. Figure 6 demonstrates the effect of albumin on Mw of complexes formed at X ) 1. Since the concentration of complexes (g/mL) in solution with added albumin is not exactly known, an apparent molecular weight of the complexes was calculated under the assumption that incorporation of BSA into the complexes is low. In other words, the concentration of complexes was supposed to be unchanged by the BSA addition. The scattered light intensity related to complexes (Ic) was evaluated by subtraction of the scattered intensity of BSA (IA) from the total scattering intensity (IT) of the solution; IC ) IT - IA was used for Mw estimation. It can be seen in Figure 6 that Mw after an incubation period (70 min) starts to increase on time to be saturated after 1000 min at the Mw value 1.3 times higher than the starting value of complexes without albumin. No systematic Mw changes were observed in experiments with complexes prepared at X ) 1.25 in the course of 2000 min. From the practical standpoint, the complexes should remain in circulation until they reach

Figure 7. Changes of RH distribution A(RH) due to addition of albumin (1 mg/mL) and subsequently by NaCl addition (up to 0.15 M) for a variety of incubation times as indicated in the inset.

their target site. As the required half-life in the bloodstream is several hours,30 the observed stability of complexes prepared at X ) 1.25 should be sufficient. Time development of RH is shown for both kinds of complexes in Table 2. The increase of RH after the albumin addition is partly due to a superposition of the dynamics of albumin molecules and their aggregates over the dynamic of GPLL/ OPP complexes. A pronounced initial fast increase of Mw from 6.1 × 105 to 1.5 × 108 was observed with complexes prepared at X ) 0.75. Mw increased further on time to be saturated after 60 min at Mw ) 3.7 × 108. We suppose that this increase is provoked mostly by an interaction of albumin with positively charged PECs. The PEC/BSA complexes bring up an additional scattering to that of GPLL/OPP complexes. The formation of such large polycation/albumin complexes was observed in a system DNA/polycationic copolymers after albumin addition.31 After a subsequent NaCl addition (up to 0.15 M) to the solution, Mw decreased from 3.7 × 108 to 2.2 × 108. We assume that this decrease is mainly caused by breakup of PEC/BSA aggregates that are not stable in the presence of a low-molecular-weight electrolyte (NaCl) due to only weak interactions. Since the static light scattering provides an integral information on all kinds of scatters in solution, dynamic light scattering was used for a separation of individual contributions. The effect of albumin on the distribution of the apparent hydrodynamic radius, A(RH), is demonstrated for complex solutions formed at X ) 0.75 in Figure 7. A(RH) distributions were evaluated by the REPES method from intensity-time correlation functions measured at the scattering angle of 90°. The starting complexes prepared in 0.01 N NaCl are small rather monodisperse particles. After BSA addition a formation of large particles (about 250 nm) is observed in several minutes. There are, probably, complexes of albumin with positively charged GPLL/OPP complexes (PEC/BSA). Besides those large (30) Dash, P. R. Ph.D. Thesis, University of Birmingham, 1998. (31) Oupicky´, D.; Konˇa´k, C ˇ .; Dash, P. R.; Seymmour, L. E.; Ulbrich, K. Bioconjugate Chem. 1999, 10, 764.

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complexes, original GPLL/OPP complexes are surviving (smaller peak at 13 nm). That is an interesting result since the amount of albumin used in the experiment is sufficient to interact with all GPLL/OPP complexes present in solution. We assume that the interaction of BSA is controlled by a content of hydrophilic grafts on the molecule. Thus, the chemical heterogeneity of copolymers can play a role. Uncompensated complexes with a small amount of PHPMA grafts can behave as polycations and form with albumin large complexes. The complexes with higher amount of HPMA grafts are sterically protected against this interaction. The large albumin complexes further aggregated on time (the third peak at large values of RH). The addition of NaCl (up to 0.15 M) partly destroyed the aggregates (see the bottom of Figure 7). As a result, the short peak of GPLL/OPP complexes and middle peak of PEC/BSA complexes increased compared with the peak of big aggregates. The concentration of NaCl was not high enough to destroy all aggregates and PEC/BSA complexes. In the same distribution a new peak at RH < 10 nm occurred, indicating free albumin in the system. Since an addition of 0.15 M NaCl partly destroyed the aggregates, one can expect that an opposite order of mixing may suppress the albumin-induced aggregation. Therefore, first NaCl and then BSA was added up to a concentration of 1 mg/mL under vigorous stirring for 3 h (saturation). According to our expectation, the formation of aggregates was substantially suppressed. The fast initial increase in Mw from 5.9 × 105 to 8.3 × 105 observed after BSA addition is 2 orders of magnitude smaller than in the previous experiment. No further change of Mw value was observed in the course of 2000 min. No complexes of albumin with positively charged GPLL/OPP complexes and large aggregates were found in DLS experiments. Thus, the physiological saline solution substantially decreases an interaction of BSA with the complexes and prevents a formation of PEC/BSA complexes. The fast increase of Mw after BSA addition is probably due to a sorption of BSA molecules into GPLL/OPP complexes. Conclusions An application of high molecular PLL molecules grafted with short PHPMA chains for self-assembly with oligonucleotides and model oligophosphates was investigated

Dautzenberg et al.

by static and dynamic light scattering methods. PLL and PTMAEM homopolycations were used for comparison. The stability to physiological salt conditions and serum albumin interaction was tested. The most important results are the following: For both the homopolycations, a high and with X strongly increasing level of aggregation (100-1000 polycation chains) was observed. Macroscopic flocculation occurred approaching X ) 1. Contrary to that, Mw values of PECs prepared with the graft copolymer were near to the value of the grafted poly(L-lysine) and increased only marginally with rising X to 1:1 mixing region. Further addition of the oligoanions resulted in stable PECs with a particle mass near 1 million. From Mw of the clusters their aggregation number, N ≈ 3, was estimated assuming full charge compensation of GPLL molecules by polyanions (X ≈ 1.1). Thus, the graft copolymers (polycations grafted with water-soluble polymers) were found to be more suitable for preparation of stable small clusters than homopolycations. Oligophosphates can serve as model molecules for physicochemical investigations instead of the much more expensive oligonucleotides. Complexes prepared at X ) 1 and 1.25 are stable for at least 48 h under physiological salt conditions (0.15 N NaCl) while uncompensated complexes slowly aggregate. The complexes prepared with an excess of oligophosphates (X ) 1.25) were found to be stable in the bovine serum albumin solution (1 mg/mL). A formation of PEC/BSA complexes and of large aggregates was observed for uncompensated PEC. The presence of 0.15 N NaCl in solution substantially decreases an interaction of BSA with the complexes and prevents a formation of large PEC/ BSA complexes. As the required half-life in the bloodstream is several hours,28 the observed stability of the complexes should be sufficient for applications. Acknowledgment. Support of the German-Czech program for Bilateral Cooperation in Science and Technology Program under Grant ME 362, of the EU under Grant ERBIC20CT97005, of the Grant Agency of the Czech Republic under Grant 307/96/K226, and of the International Bureau of the BMBF, Germany (Project CZE 99/ 006), is gratefully acknowledged. LA001779T