Galactose-PEG dual conjugation of β-(1→3)-d -glucan schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake

ARTICLE IN PRESS

Biomaterials 27 (2006) 1626–1635 www.elsevier.com/locate/biomaterials

Galactose-PEG dual conjugation of b-(1-3)-D-glucan schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake$ Ryouji Karinagaa, Takahisa Anadaa, Jusaku Minaria, Masami Mizua, Kazuya Koumotoa, Junji Fukudaa, Kohji Nakazawaa, Teruaki Hasegawab, Munenori Numatab, Seiji Shinkaib, Kazuo Sakuraia, a

Department of Chemical Process and Environments, The University of Kitakyushu, 1-1, Hibikino, Wakamatu-ku, Kitakyushu, Fukuoka 808-0135, Japan b Faculty of Engineering Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, Fukuoka 812-8581, Japan Received 6 May 2005; accepted 11 August 2005 Available online 19 September 2005

Abstract Antisense oligonucleotides (AS ODNs) are applied to silence a particular gene, and this approach is one of the potential gene therapies. However, naked oligonucleotides are easy to be degraded or absorbed in biological condition. Therefore, we need a carrier to deliver AS ODNs. This paper presents galactose moieties that were conjugated to the side chain of SPG to enhance cellular ingestion through endocytosis mediated by asialoglycoprotein receptor specifically located on parenchymal liver cells. We introduced galactose with two types of chemical bonds; amide and amine, and the amine connection showed lower ingestion and more toxicity than the amide one. Since PEG was known to induce endocytosis escape, we combined PEG and galactose aiming to provide both cellular up-take and subsequent endocytosis escape. We designed lactose or galactose moieties to attach to the end of the PEG chain that connects to the SPG side chain. When the PEG had the molecular weight of 5000–6000, the antisense effect reached the maximum. We believe that this new type of galactose and PEG dual conjugation broaden the horizon in antisense delivery. r 2005 Elsevier Ltd. All rights reserved. Keywords: Antisense; Polysaccharide; Gene transfer; Hepatocyte

1. Introduction Antisense oligonucleotides (AS ODNs) can silence a particular gene by exploiting their exquisite ability to specifically bind to the target mRNA [1,2]. The molecular biological mechanism of antisense therapy include: (1) delivery of AS ODNs to the cells; (2) cellular uptake of AS ODNs; (3) release of AS ODNs to cytosol; (4) binding to a particular mRNA to produce a DNA/RNA duplex; and (5) RNase H cleavage of the duplex to inhibit protein expression [3]. However, two major obstacles must be overcome: the instability of AS ODNs in biological fluids $ This is the 42th paper in the series of ‘‘Polysaccharide/Polynucleotide Complexes’’ study. Corresponding author. Tel.: +81 93 695 3390; fax: +81 93 695 3390. E-mail address: [email protected] (K. Sakurai).

0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.08.023

and low uptake efficiency into the target cells. The instability of AS ODNs is due to hydrolysis mediated by deoxyribonuclease and non-specific protein binding [4,5]. Hydrolysis can be suppressed significantly by oligonucleotide analogues such as phosphorothioates, phosphoramidates, and peptide nucleic acids [1,5–7]. The phosphorothioates represent the leading candidate among the first generation of antisense compounds, and several of them are being tested in phase I/II clinical trials. When AS ODNs made from phosphorothioates are used in vivo, they bind to serum proteins, leading to undesirable biological events [4]. It is reasonable that materials that can complex with phosphorothioates can prevent the AS ODN from participating in undesirable interactions. Cationic lipids can form a complex with AS ODNs and encapsulate them in liposomes; however, this process has drawbacks. For example, cationic liposomes tend to accumulate in the

ARTICLE IN PRESS R. Karinaga et al. / Biomaterials 27 (2006) 1626–1635

reticulo-endothelial system, reducing lifetime in the serum [8]. Synthetic polycations, such as poly(L-lysine) and polyethyleneimine have been studied as AS ODN carriers. Although polycations improve cellular uptake, they possess serious disadvantages, such as toxicity and poor solubility of the resultant polyion complexes [9,10]. Natural polysaccharides are considered an alternative for the design of AS ODN carriers, because they biodegrade into nontoxic components and provide satisfactory solubility. The recent finding that a polysaccharide of schizophyllan can form a complex with polynucleotides suggested that schizophyllan can function as an AS ODN binding site as well as the carrier [11,12]. Schizophyllan is an extracellular polysaccharide produced by the fungus Schizophyllan commune; its main chain consists of b-(1-3)-D-glucan and one b-(1-6)-D-glycosyl side chain that links to the main chain at every three glucose residues (see Fig. 1(a)) [13]. Schizophyllan adopts a triple helical conformation in water and a random coil in dimethyl sulfoxide (DMSO) [14–17]. Addition of water to the DMSO solution (renaturation), can partially retrieve the triple helix structure [18,19]. Recently, Sakurai and Shinkai found that the single chain of schizophyllan (sSPG) forms a macromolecular complex with a homophosphodiester polynucleotide (such as poly(C), poly(A), poly(U), poly(dA), or poly(T)], when the polynucleotide is present during the renaturation process (see Fig. 1(b)) [20,21]. Novel features possessed by this complex include: (1) remarkable stability (large binding constant) and water solubility under physiological conditions, (2) highly stoichiometric complex formation with a stoichiometric number that indicates interaction between two schizophyllan units and three base units (see Fig. 1(b)), (3) priority for hybridization over complexation, in other words, dissociation of the complex followed by hybridization when the sSPG/DNA complex meets the corresponding complementary sequence [e.g., s-SPG/poly(T) meets poly(dA)] [22], and (4) inhibition (or reduction) of non-specific interactions between bound AS ODNs and serum proteins [12]. The use of schizophyllan as an AS ODN carrier provides all four of these advantages. However, schizophyllan itself

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has no ability for cellular uptake and thus, in order to use schizophyllan as an AS ODN carrier, an uptake device is need. In this paper, we introduce galactose into the SPG side chain to enhance uptake by hepatocytes. Carbohydrate-based conjugates allow targeting of a certain class of cell membrane receptors. These receptors recognize a specific carbohydrate motif and internalize them via endocytosis. The asialoglycoprotein receptor (ASGP-R) is located on parenchymal liver cells (e.g., hepatocytes) and recognizes terminal galactose or lactose residues. For lactose, the glucose moiety functions as a tether and the galactose serves as the ligand. Previous studies have demonstrated that the binding affinity of a carbohydrate ligand to ASGP-R is highly influenced by the number and orientation of the sugar residues; however, the terminal galactose residue is the most important moiety [23]. In the past, several different approaches have been used to enable glyco-targeted delivery of nucleic acids to hepatocytes. Wu and Wu developed a DNA carrier system based on a soluble noncovalent complex between nucleic acids and an ASGP-poly-L-lysine conjugate [24,25]. Maier et al. designed a multivalent carbohydrate recognition motif for ASGP-R and showed cell-specific delivery of antisense drugs to parenchymal liver cells [26,27]. These previous studies demonstrate the ability to design cellular targeting systems and the importance of the glucose cluster effect. This study applies the results of the preliminary report by Hasegawa et al. to the SPG/AS ODN delivery system [28,29]. 2. Materials and methods 2.1. Synthesis of galactos-appended schizophyllan We introduced galactose into the side chain of SPG in three different ways as described in Fig. 2. Furthermore, for comparison, we made the glucose-appended SPG. The first compound, Scheme 1 and denoted by Lac-SPG, has a functional group shown by the upper linen in Table 1. 2aminoethyl-b-lactoside was attached to the formyl group that had been converted from the glucosyl side chain. The second one is denoted by GalSPG (middle line in Table 1 and Scheme 2), in which lactonelactone was reacted with the amino group that had been attached to the formyl group. The third one is Gal-PEG-SPG, presented in Table 2 and Scheme 4, which

Fig. 1. (a) Schizophyllan repeating unit. (b) Schematic of complex formation between a polynucleotide and schizophyllan, and a stoichiometric model for the polynucleotide/schizophyllan complex. G and B indicate the main-chain glucose moieties of schizophyllan and the base molecules of polynucleotides, respectively.

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Scheme 1 (i) (ii) OH

HO O

O OH O

HO O

OH

OH

Scheme 2 (i) (iv)

O

HO HO OH

(iii)

OH O

HO O

OH

O

(v)

OH O OH

OH

Scheme 3 (i) (vi)

(iii)

OH

HO O

O

HO

OH

Scheme 4 (i) (vii)

(v)

R R

O

HO HO

OH O

O

HO

OH

O O OH

OH O

HO O

O OH

1-n

O

HO

OH O

O

OH

HO O

O OH

OH

n

(iii)

Fig. 2. Reaction schemes for introduction of functional groups into the side chain of s-SPG. Scheme 1 shows that 2-aminoethyl -b-lactoside was attached to the formyl group converted from the glucosyl side chain. Scheme 2 shows that lactonelactone reacted with the amino group attached to the formyl group. Scheme 4 shows addition of PEG between the amino group and the lactonelactone.

Table 1 Sample codes and the modification levels of lactose and galactose appended SPG and the materials used for comparison Sample code Lacð13:6Þ-SPG

R

Modification level

#

13:6
1:4 mol% 29:0
1:0 mol%

Lacð29:0Þ-SPG 3 Galð2:1Þ-SPG Galð3:2Þ-SPG 7 7 7 Galð8:7Þ-SPG 7 7 Galð6:5Þ-SPG 7 5 Galð11:0Þ  SPG

11:0
0:9 mol%

Glu(4.8)-SPG

4.870.5 mol%

SP(4.6)-SPG

4.670.3 mol%

2:1
0:3 mol% 3:2
0:4 mol% 8:7
0:5 mol% 6:5
0:1 mol%

Table 2 Sample codes of the PEG-galactose (or glucose) conjugated SPG Sample code

R

Modification level M w of PEG 1070.1 mol% 5000

PEG5 K(10)-SPG

M w of PEG Gal-PEG 200-SPG

3

Gal-PEG 600-SPG 7 7 7 Gal-PEG 2 K-SPG 5 Gal-PEG 6 K-SPG Glu-PEG 200-SPG

3

Sample number

200

ð1Þ

600

ð2Þ

2000

ð3Þ

6000

ð4Þ

200

ð5Þ

Glu-PEG 600-SPG 7 7 7 Glu-PEG 2 K-SPG 5

600

ð6Þ

2000

ð7Þ

Glu-PEG 6 K-SPG

6000

ð8Þ

When we made all Gal-PEG SPG and Glu-PEG SPG, we used the same F-SPG as that was used when PEG 5 K(10)-SPG was made. Therefore, the modification levels for all samples are estimated to be 10 mol%. This was confirmed for a few samples.

ARTICLE IN PRESS R. Karinaga et al. / Biomaterials 27 (2006) 1626–1635 has polyethylene glycol (PEG) linker between the amino group and the lactonelactone. 2.1.1. Synthesis of LAC-SPG (Scheme 1) [29] Lac-SPG was prepared through the following procedure. The glucose side chain of SPG (M w ¼ 150 k) was oxidized in a NaIO4 aqueous solution (the molar ratio of NaIO4 to SPG was 8:10). After reacted for 2–5 days at 4 1C, we obtained a formyl-terminated SPG (F-SPG). After reacted F-SPG and aminoethyl-b-lactoside, the resultant imine was reduced with NaBH4 to yield Lac-SPG. The modification levels of lactose were determined with elemental analysis and the results are presented in Table 1. 2.1.2. Synthesis of GAL-SPG and GLU-SPG (Scheme 2) and PEG appending SPG (Scheme 3) [30] A total of 40 ml of 28% ammonia water and 100 mg of sodium cyanoborohydride was added into an F-SPG (100 mg) aqueous solution, and the mixture was stirred for 1 week to give an imine-terminated SPG (Im-SPG). To an Im-SPG/DMSO solution, lactonolactone (or maltonolactone) was added and stirred for 1 week and the product was dialyzed with water to obtain Gal-SPG (or Glu-SPG). The synthesis procedure of lactonolactone (or maltonolactone) was done as follows. 24.2 g of blactose (or a-maltose) was dissolved in mixed solvent (18 ml of H2O and 48 ml of methanol), and 34.4 g of iodine was dissolved in 480 ml of methanol. After these two solutions were mixed, a total of 500 ml of 4 vol% potassium hydrate (methanol solution) was delivered by drops into the mixed solution for 3 h. Subsequently, a white solid was obtained by recrystallization using mixed solvent of H2O and methanol. After the white solid was dissolved in water, this solution was purified by ionexchange column chromatography (Amberlite IR-120B). Methoxypolyethylene glycol amine (M w ¼ 5000, from Sigma) was attached to the side chain of SPG (Scheme 3) and the experimental details were reported in a previous paper [30]. 2.1.3. Synthesis of GAL-PEG-SPG (Scheme 4) b-galactose-terminated PEG was attached to the side chain of SPG by the procedure illustrated in Scheme 4. A total of 20 g of PEG was dissolved in 100 ml of pyridine and p-toluene sulfonyl chloride (equivalent mole of the hydroxyl group of the PEG end) was added to the solution under stirring at room temperature. After pyridine was removed in vacuum, and the resultant di-tosylated compound was dissolved in dimethylformamide, sodium azide was added to the solution. After stirring at 80 1C till the sodium azide was resolved, reduction with palladium carbon gave the amine group. This animated PEG was dissolved in a F-SPG/DMSO solution and the solution was stirred overnight at room temperature. After 200 mg of sodium cyanoborohydride (excess) was added into the solution, the mixture was stirred for 1 week. After dialyzation, lyophilization gave a white solid. This solid was dissolved in DMSO, and 1 g of lactonolactone (or maltonolactone) was added. After the dialysis and lyophilization of the product was carried out, it was purified with chromatography (TOYOPEARL HW-50F, eluent: 0.01 mol dm-3 of aqueous NaOH solution) to give Gal-PEG-SPG (or Glu-PEG-SPG).

2.2. Oligonucleotides sequence and other materials Since short and hetero-ODNs cannot bind to s-SPG, we attached a poly(dA)40 at the 30 ends of 50 -GTG CCG GGG TCT TCG GGC phosphorothioate. The resultant ODN was used as an AS ODN and denoted by AS-c-myb. As a negative control, we used a sense sequence; 50 GCC CGA AGA CCC CGG CAC A40-30 and denoted by S-c-myb, mismatch sequence; 50 -GTC CTG GGG TCG TCG GGC A40-30 and denoted by MS-c-myb, scrambled sequence of AS-c-myb; 50 - TGC TGC GCG TGG TCG GCG-(dA)40-30 and denoted it by Sc-c-myb, respectively. All ODNs were synthesized at Hokkaido System Science (Hokkaido, Japan) and purified with high-pressure liquid chromatography. PBS and the fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco BRL. Minimum essential medium (MEM)

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was obtained from Sigma. Dulbecco’s modified Eagle’s medium (DMEMs) was obtained from Nissui Pharmaceutical Co. Ltd. Bovine serum albumin (BSA) was obtained from Sigma. SlowFade Light Antifade Kit, and eosin-5-isothiocyanate were obtained from Molecular Probe. Fluorescein isothiocyanate-I (FITC-I) was obtained from Wako.

2.3. Complexation of SPG/ODN Complexation between all of the modified SPGs and ODNs was carried out with the established method and the complexation was confirmed with circular dichroic spectroscopy and gel electrophoresis (see Supplementary Information). Unless mentioned, the molar ratio (Ms–SPG/MODN) was controlled to 1.5, where Ms–SPG and MODN are the repeating molar concentrations of s-SPG and nucleotide, respectively. Furthermore, we confirmed that the lactose in Lac-SPG can be recognized by lectin and the binding constant is in the same magnitude as free lactose (Supplementary Information).

2.4. Cell culture and antisense assay The hepatoblastoma cell Hep G2 was obtained from Riken Bioresource Center in Japan. Hep G2 cells were maintained in MEM supplemented with 10% FBS and 1% sodium pyruvate. Hepatocytes were isolated from Wistar rat and incubated in DMEM (Gibco BRL) at 37 1C in fully humidified air containing 5 wt% of CO2 (for details see the Supplementary Information) The cells were seeded in 96-well plates (Falcon) at a density of 2  104 cells/ml (1 well/100 ml); the following day, the medium was changed for a fresh medium, and cells were treated with an appropriate amount of ODN (30 or 50 or 60 mg/ml), ODN/s-SPG complex (containing 30 or 50 or 60 mg/ml ODN), or modified or unmodified s-SPG (60 or 100 or 120 mg/ml). Subsequently, cells were incubated for 3 days before measurement of the cell growth. The cell number was evaluated by use of Cell Counting Kit-8s, WST-8 assay (Dojindo, Japan). All growth studies were carried out at least twice. For convenience, the number of cells was normalized by that of the control and this value was defined as the cell growth. Plates were read on microplate reader Multiscan JX (Thermo Labsystems) using a wavelength of 450 nm in comparison to 650 nm. Antisense effect and cellular up-take were examined with RT-PCR and flow cytometry (see Supplementary Information).

3. Results and discussion 3.1. Antisense assay for synthesized SPG carriers Fig. 3 compares cell growth upon administration of ASc-myb to Hep G2 with the modified SPG samples listed in Table 1. The protooncogene c-myb is preferentially expressed in hematopoietic cells, and AS-c-myb indicate AS ODN corresponding c-myb mRNA [30]. For the GalSPG system, the cell growth decreased with an increase in the modification level, i.e., the antisense effect is enhanced. In contrast, Glu(4.8)-SPG, which was modified with aglucose, showed the same cell growth as SPG. These features suggest that galactose-modified SPG was recognized by ASGP-R on Hep G2 and the bound AS-c-myb was ingested to a greater extent than that of a-glucose modified SPG. The greater ingestion of AS-c-myb is responsible for the difference in cell growth. A comparison of Lac-SPG with Gal-SPG showed that Lac-SPG was less effective in reducing cell growth than Gal-SPG. However, modification levels of Lac(13.6)-SPG and Lac(29.0)-SPG were larger than those of Gal-SPG

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80

80

Cell growth /%

100

Cell growth /%

100

60

40

20

60

40

20

0

0 0

(a)

+ 20 mM Galactose

30

60

0

Control Gal(6.5)-SPG

Naked

30

(b)

AS-c-myb concentration (µg/ml) SPG

Gal(11.0)-SPG

60

AS-c-myb concentration (µg/ml)

G lu(4.8)-SPG

G al(2.1)-SPG

Gal(3.2)-SPG

Lac(13.6)-SPG

Lac(29.0)-SPG

SP(4.6)-SPG

Fig. 3. Comparison of cell growth for all carriers listed in Table 1 (a) in the antisense assay and (b) an inhibition test with galactose. Hep G2 cell numbers were determined with a WST-8 assay using a Cell Counting Kit-8s (Dojindo), after administration of AS-c-myb and incubation of the cells for 3 days. ASc-myb with the carriers was added to 2  103 cells in 96-well plates (Falcon).

Lane 1: Control 1

2

3

4

5

6

7

8

9

Lane 2: Naked Lane 3: Glu(4.8)-SPG

c-myb mRNA (% of control)

Lane 4: Gal(2.1)-SPG 100

62

52

49

38

21

11

15

34

Lane 5: Gal(3.2)-SPG Lane 6: Gal(6.5)-SPG Lane 7: Gal(11.0)-SPG


-actin mRNA (% of control)

100 113

115 104

99

97

108

104

97

Lane 8: Lac(13.6)-SPG Lane 9: Lac(29.0)-SPG

Fig. 4. Comparison of naked AS-c-myb and its complexes with each carrier by RT-PCR, confirming sequence-specific mRNA silencing. Numbers under the gel image indicate relative intensities of the mRNA bands.

samples. As described in Section 2.1, the method of connecting the carbohydrate to the side chain of SPG is different in Lac-SPG and Gal-SPG. In Lac-SPG, the lactose moiety is connected to the side chain by a secondary amine, while Gal-SPG is connected through an amide bond. Thus, Lac-SPG has a cationic nature while Gal-SPG is neutral. This could explain the difference, i.e., the cationic nature might reduce the affinity of lactose for ASGP-R, or the cation may have formed an ionic complex with the anionic AS ODN to disturb release of the bound AS ODN. Examination of the cytotoxicity of the carriers indicated that Lac-SPG was slightly more toxic than Gal-

SPG (see Supplemental Information). This difference can be attributed to the chemical nature of the connection, because amines are generally cytotoxic. Fig. 3b shows an inhibition assay for lactose uptake; cell growth was measured in culture medium containing 20 mM galactose. Results clearly show that addition of galactose eliminates the difference in cell growth among the carriers, indicating that lactose is responsible for the cellular uptake. Furthermore, the ability of AS-c-myb (or its complex) to silence target mRNA in a sequence-specific manner was examined using RT-PCR and the results are presented in Fig. 4. The amount of b-actin mRNA expression was used

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Gal(8.7)-SPG

SPG

Naked

Control

as a reference. As shown in the lower panel of Fig. 4, no differences were found among the samples, indicating that administration of ODN does not influence the amount of b-actin mRNA. In contrast, naked AS-cmyb and complexed AS-c-myb decreased the mRNA amount; some complexes nearly eliminated mRNA. The most dramatic reduction was observed for Gal(6.5)-SPG and Gal(11.0)-SPG, consistent with the proliferation assay results shown in Fig. 3. Therefore, we conclude that the reduction of cell growth and the difference between the carriers shown in Fig. 3a is due to enhancement of AS ODN uptake by lactose- or galactose-modified SPG.

32

Cell number

24

16

8

1631

3.2. Observations of b-galactose uptake into hepatocytes Fig. 5 shows a comparison of cellular uptake of FITClabeled phosphodiester oligo (dA)40 when the ODN was exposed to Hep G2 in the naked state or complexed with SPG or Gal(8.4)-SPG. The SPG complex was ingested to a greater extent than was the naked ODN. This difference is due to the less absorption of complexed ODN by BSA in the medium compared to absorption of the naked ODN; thus, the amount of ODN being able to reach the cellular surface is increased [12]. According to the difference in the fluorescence intensity in Fig. 5, ingestion of Gal(8.4)-SPG was approximately one order of magnitude greater than that of SPG, due to the galactose attached to the carrier. Fig. 6a shows the comparison between microscopic images of eosin-labeled carriers (Gal-SPG, Glu-SPG, and SPG) exposed to rat hepatocytes (spheroid). The red color indicates the eosin-localized region. Gal-SPG produced the largest red-stained region; while Glu-SPG and SPG possessed similar features. Panel b compares microscopic images of Hep G2, spheroid, and monolayer cells after exposure to FITC-labeled Gal-SPG. All cells ingested the Gal-SPG producing a spotty pattern of green color distribution, indicating that these carriers are located in intracellular vesicles, probably endosomes or lysosomes. When the same experiment was conducted with Glu-SPG, no green region observed in any of the cells (data not shown). 3.3. Galactose and PEG dual conjugation to maximize cellular up-take

0 100

101

102

103

Fluorescence intensity / a.u. Fig. 5. Comparison of flow cytometric (FCM) histograms upon administration of FITC-labeled phosphodiester oligo (dA)40 to the naked state and to the complexes with un-modified SPG and Gal(8.7)-SPG.

Previous work has revealed a PEG chain attached to SPG/AS ODN complexes allows the complexes to escape endocytosis, probably due to depletion interactions in the spatially confined vesicles [31]. Consequently, the ingested carrier can release bound AS ODN to the cytosol before transport to the lysosome. Administration of AS ODN

Fig. 6. Confocal laser scanning microscope (CLSM) images of galactose uptake. Treatment of rat primary spheroid hepatocyte with poly(dA) complexed three carriers: (a-1) Gal(8.7)-SPG; (a-2) Glu(4.8)-SPG; (a-3) SPG; SPG was labeled with eosin; (b), treatment of three hepatocyte cells [(b-1) Hep G2, (b-2) rat primary spheroid, (b-3) rat primary monolayer] with poly(dA)/Gal(8.7)-SPG carrier. SPG was labeled with FITC.

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with a carrier having both an RGD peptide and PEG dramatically enhanced the antisense effect. This enhancement was due to the hybrid effect involving RGD-induced endocytic uptake and PEG-induced endosomal escape. One advantage of SPG is that preparation of hybrid carriers is quite easy. SPG/polynucleotide complexes can be prepared from two s-SPG chains and one nucleotide (see Fig 1b). Therefore, preparation of complexes from a mixture of PEG-SPG and RGD-SPG results in complexes containing both PEG and RGD moieties, as demonstrated in a previous report [32]. The same hybrid effect was observed when Gal-SPG/PEG-SPG/AS ODN was administrated to Hep G2 (see Supplemental Information). Encouraged by the enhanced antisense effect found for the Gal-SPG/PEG-SPG system, lactose or galactose moieties were attached to the end of the PEG chain connected to the SPG side chain, as shown in Table 2. This Gal-PEG dual conjugation may possess an advantage over the Gal-SPG/PEG-SPG hybrid because all complexes in the Gal-PEG-SPG system contain both PEG and galactose, producing an enhanced antisense effect, while only half of the Gal-SPG/PEG-SPG/AS ODN complexes contain both moieties. Fig. 7 shows cell growth when Hep G2 was exposed to AS-c-myb with the carriers listed in Table 2 (numbers in parentheses correspond to sample numbers in 4th column of Table 2). In the Gal-PEG

system, the cell growth decreased with increasing PEG chain length (i.e., the antisense effect was enhanced). In contrast, no difference was observed in the Glu-PEG system. Contrary to Hep G2, the melanoma A375 without ASGP-R exhibited less growth with longer PEG for both the Gal-PEG and Glu-PEG systems. As shown, the cell growth of the hybrid system is larger than that of the GalPEG 6K-SPG system (4) and comparable with that of GalPEG 2K-SPG system (3). These results imply that the GalPEG dual conjugation is better than the hybrid system. Figs. 8 and 9 present RT-PCR and FCM results, respectively. Both sets of results indicated that the dual conjugation and hybrid systems are ingested to a greater extent than were those in the Hep G2 system, allowing specific silencing of the c-myb mRNA. However, when we carefully examined and checked reproducibility, the FCM results indicated that the PEG 5 K and Gal-SPG (hybrid) was more ingested by the cells than that of Gal-PEG 6KSPG (dual conjugation), and the RT-PCR showed that the target mRNA was more silenced by the hybrid than by the dual conjugation. These two results seem to contradict the WST results; however, the reason is not clear at this moment. Modification levels of Gal-PEG 6K-SPG, PEG 5K(10)SPG, and Gal(8.7)-SPG were 10, 10, and 8.7 mol%, respectively. When we prepared the hybrid, we mixed

AS-c-myb

Sc-c-myb

Hybrid (1:1)

Hybrid (1:1)

Gal(8.7)-SPG

Gal(8.7)-SPG

Hep G2

(8) (7) (6) (5) (4) (3) (2) (1) Naked Control 30

40

(a)

50 60 70 80 Cell growth / %

90 100

50 60 70 80 Cell growth / %

90 100

0

20

(b)

40

60

80

100

80

100

Cell growth / %

A375

(8) (7) (6) (5) (4) (3) (2) (1) Naked Control 30 (c)

40

0 (d)

20

40

60

Cell growth / %

Fig. 7. Comparison of cell growth among samples listed in Table 2: (a) Hep G2 treated with AS-c-myb; (b) A375 with AS-c-myb; (c) Hep G2 with Sc-cmyb and (c) A375 with Sc-c-myb.

ARTICLE IN PRESS R. Karinaga et al. / Biomaterials 27 (2006) 1626–1635

1

2

3

4

5

6

7

8

100

78

67

71

68

87

48

55

c-myb mRNA (% of control)

β -actin mRNA (% of control)

100 102 105 98 108 100 103 96

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Lane 1: control Lane 2: naked Lane 3: SPG Lane 4: PEG 5K(10)-SPG Lane 5: Glu(4.8)-SPG/PEG 5K(10)-SPG Lane 6: Glu-PEG 6K-SPG Lane 7: Gal(8.7)-SPG/PEG 5K(10)-SPG Lane 8: Gal-PEG 6K-SPG

Fig. 8. FCM confirming that PEG-galactose dual-conjugation and hybrid enhances uptake of Hep G2.

Fig. 9. RT-PCR confirming that PEG-galactose dual conjugation specifically reduces mRNA expression. Hep G2 was treated with 50 mg/ml of AS-c-myb or its complex with each carrier. Other conditions were the same as those indicated for Fig. 4.

those at a PEG 5K(10)–SPG/Gal(8.7)-SPG ratio of 4:6. Therefore, the final Gal content in the hybrid complex was approximately 5–6 mol%; on the other hand, the Gal content of the dual conjugation was 10 mol%. We should have used the same final Gal content to compare the hybrid and dual conjugate; however, we could not increase the Gal modification level more than 11 mol%. Fig. 3 showed that the cell growth seemed to reach the minimum when the Gal modification was 6.5 or more, and we found there was no

essential difference in the antisense effect between the two PEG 5 K(10)-SPG and Gal(8.7)-SPG and PEG 5 K(10)SPG and Gal(11.0)-SPG hybrids. Therefore, we used Gal(8.7)-SPG for all experiments. Fig. 7 shows the PEG chain length dependence of cell growth. For preparation of Gal-PEG-SPG and Glu-PEGSPG, the F-SPG was used. Thus, modification levels achieved were in the same range, meaning that the difference in the Gal-PEG system (1–4) of Fig. 7 should

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be related to the PEG chain length. According to the previous work, ingestion ability reaches a maximum when the molecular weight of the PEG chain is 5000–6000. The same explanation can be applied to the Gal-PEG system for Hep G2. Attachment of glucose instead of galactose resulted in no change in the antisense effect, which is consistent with results shown in Figs. 3–7. When the same assays were conducted for A375, no difference between Gal-PEG and Glu-PEG was found, confirming the targeting capability of Gal-PEG. 4. Conclusion Galactose moieties were conjugated to the side chain of SPG to enhance cellular ingestion through endocytosis mediated by the ASGP-R specifically located on parenchymal liver cells. We introduced galactose via two types of chemical bonds: amide and amine bonds; the amine connection resulted in lower ingestion and greater toxicity compared to the amide bond. Since PEG induces endocytosis escape, PEG and galactose were combined to increase cellular uptake and subsequent endocytosis escape. Lactose or galactose moieties were attached to the end of the PEG chain connected to the SPG side chain. The antisense effect reached a maximum when PEG had a molecular weight of 5000–6000. We believe this new dual conjugation of galactose and PEG will increase opportunities for antisense delivery. Appendix A. Supplementary Information The online version of this article contains additional supplementary data. Please visit doi:10.1016/j.biomaterials.2005.08.023. References [1] Stein CA, Cheng YC. Antisense oligonucleotides as therapeutic agents is the bullet really magical. Science 1993;261:1004–12. [2] Uhlmann E, Peyman A. Antisense oligonucleotides: a new therapeutic principle. Chem Rev 1990;90:543–84. [3] Keith T, Stevenson J, O’Dwyer P. Antisense therapeutics: lessons from early clinical trials. Curr Opin Oncol 2001;13:499–505. [4] Stein CA. Two problems in antisense biotechnology: in vitro delivery and the design of antisense experiments. Biochim Biophys Acta 1999;1489:45–52. [5] Chirila TV, Rakoczy PE, Garrett KL, Lou X, Constable IJ. The use of synthetic polymers for delivery of therapeutic antisense oligodeoxynucleotides. Biomaterials 2002;23:321–42. [6] Nielsen PE, Egholm M, Berg RH, Buchardt O. Sequence-selective recognition of DNA by strand displacement with a thyminesubstituted polyamide. Science 1991;254:1497–500. [7] Agrawal S, Goodchild J, Civeira MP, Thornton AH, Sarin PS, Zamecnik PC. Oligodeoxynucleoside phosphoramidates and phosphorothioates as inhibitors of human immunodeficiency virus. Proc Natl Acad Sci U S A 1988;85:7079–83. [8] Zelphati O, Uyechi LS, Barron LG, Szoka Jr. FC. Effect of serum components on the physico-chemical properties of cationic lipid/ oligonucleotide complexes and on their interactions with cells. Biochim Biophys Acta 1998;1390:119–33.

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