NOVEMBEWDECEMBER 1995 Volume 6, Number 6 0 Copyright 1995 by the American Chemical Society
Bioconjugate Chemistry
COMMUNICATIONS Water-Soluble Block Polycations as Carriers for Oligonucleotide Delivery Alexander V. Kabanov,*S+Sergey V. Vinogradov,t Yulia G. Suzdaltseva, a n d Valery Yu. Alakhov*J Moscow Institute of Biotechnology, Inc., and Department of Polymer Sciences, Faculty of Chemistry, Moscow State University, Vorobievy Gory, Moscow 119899,Russia. Received July 17, 1995@
Water-soluble, block copolymeric carriers consisting of polyoxyethylene (PEO) and polyspermine (PSI chains have been developed for the delivery of antisense oligonucleotides (oligo) into the target cells. These copolymers spontaneously form complexes with oligos in aqueous solutions. The PS block electrostatically binds to the oligo, and as a result, the stability of the oligo is increased. Similarly, the polar PEO block provides for the aqueous solubility of the complex. This paper (i) reports the synthesis of the diblock PEO-PS copolymer and (ii) evaluates the effects of the complexes formed between this copolymer and phosphodiester oligo, complementary to the splice junction of herpes simplex virus type 1 immediate early pre-mRNAs 4 and 5 , on the reproduction of this virus in Vero cells. Infectious titer data 22 and 39 h post infection indicates that the copolymer-oligo complex inhibits the reproduction of the virus beyond the detection limit. Conversely, the free oligo inhibits the reproduction of the virus only 22 h postinfection, while 39 h postinfection significant virus titers are observed. The results of this study suggest that the copolymeric complex increases the sequencespecific inhibition effect of oligo on the virus reproduction.
During the past decade antisense oligonucleotides (oligos)have attracted significant attention as promising
* Corresponding authors. address: Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, 600 South 42nd St, Omaha, NE 68198-6025. Tel.: (402) 5599364. Fax: (402) 559-5060. Present address: Centre National de la Recherche Scientifique, Centre de Biophysique Moleculaire, 1A avenue de la Recherche Scientifique, 45071 Orleans, Cedex 2, France. 8 Present address: Supratek Pharma, Inc., and Immunology Research Center, Institute Armand-Frappier, University of Quebec, 513 boulevard des Prairies, Case Postale 100, Laval, Quebec H7N 423, Canada. Abstract published in Advance ACS Abstracts, November 1, 1995. iPresent
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tools for the selective inhibition of gene expression and viral reproduction (1-3). However, the practical application of oligo for disease therapy has been hindered by the followingmajor problems: (i) poor transport into cells; (ii) non-sequence-specific effects on cells; (iii) rapid degradation in vitro and in vivo; and (iv) rapid elimination from the body. One general approach t o improve oligo performance in vitro and in vivo is to use a parenteral drug delivery system, the most comprehensively studied being cationic liposomes (4),lipopolyamines (5),and homopolycations (6). These carriers all spontaneously bind with the negatively charged oligo molecules to form complexes that have the following advantages for the oligo: (i) enhanced stability against nuclease degradation, (ii) enhanced uptake into the cells, and (iii) increased antisense activity in vitro. One problem with
1043-1802/95/2906-0639$09.00/00 1995 American Chemical Society
Kabanov et al.
640 Bioconjugate Chem., Vol. 6, No. 6, 1995 Scheme 1. Synthesis of PS 2 H2N(CH2)3NH(CH2)3NH2 + Br(CHg)4Br
Scheme 2. Synthesis of the PEO-PS copolymer H2N(CH2)3NH((CH2)3NH(CH2)4NH(CH2)3NH]m(CH2)3NH2
i
pN-LchCihohH N
4
HO(CH2CH20]nC(O)HN(CH2)3NH[(CH2)3NH(CH2)4NH(CH2)3NH]~(CH2)3NH2
I
t A,
I,l'-carbonyldiimidazole
HO[CH2CH20]nC(O)HN(CH2)3NH[(CH2)3NH(CH2)4NH(CH2)3NH]m(CH2)3NH-A
H2N(CH2)3NH[(CH2)3NH(CH2)4NH(CH2)3NH]m(CH2)3NH2
these carriers is that these complexes with oligos are often poorly water-soluble and tend to aggregate in aqueous solutions (6). To avoid this problem, we have developed another class of water-soluble block copolymeric carriers consisting of polyoxyethylene (PEO) and polyspermine (PS). The polycationic PS chains are structurally related to spermines that are naturally occurring DNA binding cations. Therefore, the PEOPS copolymers spontaneously form polyelectrolyte complexes via electrostatic interactions with the negatively charged oligos in aqueous solutions. As a result of charge neutralization, the complexed sites are hydrophobic (6). However, the complex remains in aqueous solution due to the solubilizing effect of the PEO chains. The block polycationic complexes with oligos presumably represent amphiphilic block copolymeric compounds in which the water-soluble PEO chains are linked to hydrophobic blocks of the neutralized polycation and oligo. As a result, the complexes exhibit the ability to form micelles in aqueous solutions (7). Therefore, this approach is fundamentally related to the block copolymeric delivery systems that have recently been developed for parenteral administration of various drugs (8-21). This paper reports preliminary data on the synthesis of PEO-PS block polycations and evaluates the effects of oligos complexes with the copolymer on reproduction of herpes simplex virus type 1 (HSV-1) in Vero cells. The synthesis of PEO-PS block polycations requires two stages. During the first stage (Scheme 1)the PS type polycations were synthesized by polycondensation of N-(3-aminopropyl)-1,3-propanediamineand 1,4-dibromobutane. A 6.55 g (50 mmol) portion of N-(3-aminopropyl)-1,3-propanediamine(Aldrich) was reacted with 5.4 g (25 mmol) of 1,4-dibromobutane (Fluka) in 100 mL of 1,4-dioxane for 16 h a t 20 "C. The 1,4-dibromobutane was initially added dropwise to the reaction system during the first hour. The product of this reaction (intermediate 1)spontaneously precipitated from solution as the hydrobromide salt and was filtered and dried twice from a solution of 10% triethylamine in methanol using a rotary evaporator. This evaporation procedure was effective to remove the hydrogen bromide. The intermediate 1was dissolved in 1,4-dioxaneand reacted with 2.7 g (12.5 mmol) of 1,4-dibromobutane, Again, the reaction proceeded for 16 h a t 20 "C, and the resulting products were recovered and dried a s above. These products, which contained PS of varying degrees of polymerization as well as unreacted initial monomer, were neutralized with acetic acid to a pH of 7-8 and fractionated by gel
filtration on a column (3 x 50 cm) using Sephadex G-25 F equilibrated with 0.05 N acetic acid. The concentrations of PS in the fractions were determined gravimetrically. The concentrations of the free amino groups in PS molecules were determined by titrating the fractionated acid (12)and polymers with 2,4,6-trinitrobenzenesulfonic the number-average molecular masses of the PS (MPs) calculated using the relationship: MPS = CP&CNHP, where Cps is the concentration of the PS (g/L), C N His~ the concentration of amino groups (mom), and 2 is the coefficient accounting for two terminal amino groups in the PS molecules. Three PS fractions (1-111) were obtained, having average molecular masses of 980,720, and 490 g/mol, respectively. The weight yields of these fractions were 19.5%, 20.4%, and 22.7%, respectively, while 27.2% accounted for the remaining initial monomers and intermediate 1. By comparing the experimental and calculated molecular masses of the polymers synthesized we concluded that the major PS components in the fractions I1 and I11 contain 12 and 9 N atoms, respectively, while fraction I consists of a mixture of PS with 15 and 18 N atoms (Table 1). During the second stage (Scheme 2) the PS of fraction I1 was conjugated with PEO using the 1,l'-carbonyldiimidazole reaction. A 1.5 g (1mmol) portion of PEO (MW 1500, from Fluka) was dissolved in 8 mL of 1,Cdioxane and reacted with 0.17 g (1mmol) of 1,l'-carbonyldiimidazole (Aldrich) a t 20 "C for 3 h. This reaction modifies one terminal hydroxyl group of PEO; however, the unmodified PEO and PEO modified by both ends are also produced. The reaction system was then supplemented with 1.44 g (2 mmol) of PS of fraction I1 in 8 mL of 1,4dioxane and the mixture incubated at 20 "C for 16 h. This reaction formed conjugates of PEO with PS having various structures; in particular, triblock copolymers PEO-PS-PEO, and PS-PEO-PS, and the diblock copolymer PEO-PS. To separate these products their free amino groups were modified with 2'-deoxyadenosine (A). The reaction was initiated by supplementing the reaction system with 1 g of 2'-deoxyadenosine (Sigma) activated with 0.68 g of 1,l'-carbonylimidazole in 8 mL of 1,4dioxane. (The excess of the reagents was used to ensure the modification of all free amino groups present). This reaction results in modification of the terminal amino groups of PS only, while the hydroxyl groups of PEO remain unmodified. The 2'-deoxyadenosine group was used as a chromophore to detect PS-containing products during further purification of the conjugate. The products were first purified by gel filtration on Sephadex G-25 F resin and then by reversed-phase HPLC on Silasorb
Table 1. Characteristics of PS in Fractions 1-111 and Theoretical Characteristics of the Major Components in These Fractions
fraction no. I I1 I11
content of NHz-groups, umol per mg of PS 2.04 2.77
4.11
MPS, gimoles 985 720
490
m estd for the maior component 4 and 5 3 2
MPS estimated for the maior component, dmol 871and1056 686 501
no. of N atoms estd for the maior component 15 and 18 12
9
Communications IE pre-“A AS5MAcr E4,5SA SSMAcr
HSV-1
Bioconjugate Chem., Vol. 6,No. 6,1995 641 43
5’
CUUCCCGCAGIGAGGAACGUC 3’ UGTC CTCCTTGC 5‘ 3 GGCGTCCTCCTT 5 3’ UCGlTCCTGCCT 5’
3’
Figure 1. Nucleotide sequence of antisense oligos AS5Acr (15) and IE4,5SA (131, nonsense oligo S5MAcr (15),and the target intron-exon boundary of IE of mRNAs 4 and 5. The target sequence complementary to AS5Acr is underlined.
CIS column (9 x 240 mm, 10 pm, NPO “Chromatographia”, Moscow, Russia) using the gradient of acetonitrile (5-40%) in 50 mM triethylammonium acetate buffer (pH 7.5). An independent experiment demonstrated that, under these conditions, the copolymers are well separated from both the unreacted PEO and unconjugated PS chains modified with 2’-deoxyadenosine. Several fractions of the block copolymers were obtained. We describe below only one, lower molecular mass fraction that contained the desired copolymer. The concentration of the 2’-deoxyadenosine groups in the copolymer of this fraction was 0.397 pmol per mg as determined spectrophotometrically by measuring absorbance of 2‘-deoxyadenosine groups a t 260 nm (2’-deoxyadenosine molar absorption coefficient a t 260 nm equals 15 300 units). Since the spectra of this copolymer revealed the characteristic spectra of 2‘-deoxyadenosine linked to the free amino groups of PS, it was unlikely that the polymer consisted of a triblock structure, PEO-PS-PEO, in which both terminal aminogroups of PS are linked to PEO chains. Furthermore, it was also unlikely that it had a triblock structure (A)PS-PEO-PS(A). The molecular mass of such a triblock copolymer would be 1260 g/mol, as determined from the content of 2‘-deoxyadenosine groups in the copolymer assuming that two 2’deoxyadenosine groups are linked to the two end amino groups in the (AIPS-PEO-PS(A) molecule. Further, since the molecular mass of two 2’-deoxyadenosine groups approximates 500 g/mol, this would result in PS-PEOPS chains with a molecular mass of about 760 g/mole which is significantly lower than the molecular mass of the initial PEO. Therefore, we concluded that the copolymer obtained had a diblock structure PEO-PS(A). The molecular mass of this copolymer, as determined from the content of 2’-deoxyadenosine groups, approximates 2520 glmol, which was consistent with the value calculated for the PEO-PS(A) diblock copolymer (= 2470 g/mol). The effects of the PEO-PS(A) copolymer on the performance of antisense oligo was further evaluated using HSV-1 reproduction in Vero cells as a model. For these studies the 12-mer phosphodiester oligo AS5MAcr
having the polynucleotide structure CGTTCCTCCTGU HSV-1 was used. The target for this oligo was first identified by Kulka et al. (13, 14). These authors used methylphosphonate-based oligo IE4,5SA having sequence TTCCTCCTGCGG, complementary to the splice junction immediate early (IE) pre-mRNAs 4 and 5 of HSV-1 (italic types indicates methylphosphonate residues in IE4,5SA; the sequence common for IE4,5SA and AS5MAcr is underlined). The rationale for the choice of this sequence was based on the findings indicating that (i) IE genes play a regulatory role in HSV replication and (ii) RNA splicing may be involved in the control of gene expression (13). The AS5MAcr sequence is two nucleotides shorter a t the 3’ end compared to IE4,5SA (Figure 1). It also has U instead of C a t the 3‘ end, which was necessary to introduce an acridine substituent (15). However, there are also two additional nucleotides a t the 5’ end of AS5MAcr compared to IE4,5SA, which are complementary to the target sequence (Figure 1). The 12-mer SBMAcr with randomized sequence TCCGTCC’ITGCU identical in composition to antisense oligo was used as a “nonsense” oligo control. The unmodified antisense phosphodiester CGTI’CCTCCTGCwas previously reported to be inactive in inhibiting HSV-1 reproduction in concentrations up to 20 p M (15). Therefore, to evaluate the copolymer effects, both antisense and nonsense oligos AS5MAcr and SBMAcr were modified a t the 5‘-end with n-undecyl and a t the 3’-end with acridine moieties (Figure 2). Such double-modified oligos reveal elevated activity and stability compared to unmodified phosphodiesters (15,16).The n-undecyl modification ( I 7) enhances oligo binding and uptake into cells. The acridine moiety intercalates into DNA and enhances binding of oligos with nucleic acid targets (18). In particular, the modified oligo AS5MAcr has previously been reported to effectively inhibit the HSV-1 reproduction in Vero cells in a sequence-specific manner in concentrations of 1-20 pM (15). The synthesis and effects of the AS5MAcr and S5MAcr in the absence of the copolymer are described elsewhere (15). To prepare complexes between the oligos and PEO-PS(A), 2 pmol of the copolymer in 0.5 mL of 0.1 M sodium acetate, pH 4.0 was mixed with 1pmol of oligo in 0.5 mL of the same buffer, and then the system was diluted with RPMI-1640 media to obtain the desired concentration of the complex. The formation of the complexes between the PEO-PS(A) and oligo was confirmed by reversedphase HPLC on a Silasorb c16 column: By using the fluorescence probe (perylene) technique (191,these com-
-0-I-Oyj
c11
0
-o-Poiy(dN)
AH
.----”.$J Ca
Figure 2. Structure of hydrophobically modified oligo derivative. Table 2. Effect of 12-merOligos (11) and Their Complexes with PEO-PS(A) on the Reproduction of HSV-1 in Vero Cells
oligo concn, pM
copolymer concn, pM
control AS5MAcr
0 4
complex of AS5MAcr and PEO-PS(A)
2 4 2
0 0 0
system studied
complex of S5MAcr and PEO-PS(A) PEO-PS(A)
4
infectious titer of HSV-1 (PFU/mL) 22 h 39 h 105 5 x 106 0 105 6 x lo3 106
4
0
2
103 105 105
4 4
0 2
105
5 x 106 not determined
Kabanov et al.
642 Bioconjugate Chem., Vol. 6,No. 6,1995
plexes were shown to form micelles. The critical micelle concentration (cmc) for the complexes was in the order of several pM, which was in the cmc range of amphiphilic block copolymers (19). These results indicate that the binding of oligo to PS chains leads to formation of hydrophobic sites due to the charge neutralization (61, which leads to the complexes self-assembling into micelles. The toxic effects of AS5MAcr and complexes formed between AS5MAcr and PEO-PS(A) were evaluated using HSV-1-infected Vero cells. In these experiments, the synthesis of cell and virus proteins was studied using a pulse-chase technique as previously described (20). The infected cells were incubated with 2, 7.5, and 20 pM of AS5MAcr or oligo-copolymer complex for 6 h after infection, which was a t 0.1 PFU/cell multiplicity. (The uninfected or virus-infected cells which were not treated with oligos or complexes were used in the control experiments.) After incubation, the medium was replaced by fresh Hank's solution containing 20 pCi/mL of [l4C1lactalbumin hydrolysate (Reakhim, Russia), and cells were incubated for 1 h a t 37 "C and then washed and lysed with 0.1% sodium dodecylsulfate. The lysates were analyzed by polyacrylamide gel electrophoresis under Laemmli conditions (211, and autoradiographs were obtained using the R-film. All concentrations of oligo and complex studied inhibited the synthesis of virus-specific proteins beyond the detection limit. Significant inhibition of the synthesis of cell proteins was observed for 20 pM oligo and complex which was indicative of cytotoxic effect. Some inhibition of cell proteins was observed a t 7.5 pM. Both a t 20 and 7.5 mM concentration the inhibition was more pronounced in the case of free oligo compared to the complex, which suggests that the complex is less cytotoxic than free oligo. No cytotoxicity was observed both for the free oligo and complex a t 2 PM* The experiment on HSV-1 reproduction was performed as previously described (15). Briefly, monolayers of Vero cells were infected a t 0.01 PFU/cell multiplicity with the virus. Oligos, PEO-PS(A), or their complexes were added to the cells a t various concentrations 1h prior to the infection. The complex formed between nonsense oligo and the copolymer was used in the control experiments to exclude nonspecific effects. After 8 h of incubation of the infected cells in the presence of oligonucleotides the medium was replaced with the fresh media containing fetal calf serum. The virus infectious titer (PFU/mL) was determined 22 and 39 h post infection on monolayers of Vero cells (22). All experiments were performed in triplicate. The variations in the infectious titers determined were less than 25%. The results of the experiment are presented in Table 2. The treatment with 4 pM AS5MAcr led to a decrease in the virus titer 22 h postinfection; however, substantial virus concentration was observed 39 h postinfection. When complexed with PEO-PS(A) this oligo inhibited virus reproduction beyond the detection limit after both 22 and 39 h postinfection. The copolymer also increased the inhibition effect of 2 pM AS5MAcr 22 and 39 h after infection. In the absence of oligo, the copolymer did not affect the infection. Furthermore, the effect of the copolymer on oligo activity was sequence specific, since no inhibition of the virus was observed with the complexes of nonsense oligo. Therefore, the PEO-PS(A) copolymer significantly prolonged the effect of antisense oligo. The enhanced activity of oligo delivered to the cell in the complexed form was probably due to one or both of the following reasons. Firstly, it may be due to the increase in uptake of the complexed oligos into cells that results in elevation of
intracellular oligo concentration. A similar correlation between the increased uptake and functional activity has been previously reported for the DNA-polycation complexes (23). Secondly, it may be due to stabilization of the complexed oligo against enzymatic degradation that increases the half-life of oligo in cells. The stabilization of nucleic acids incorporated into polyelectrolyte complexes against enzymatic digestion also has been reported (24). The studies on the mechanism of the observed effect of the PEO-PS(A) copolymer on oligo activity are currently in progress in our laboratories. LITERATURE CITED
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