Bioconjugate Chem. 2002, 13, 975−984
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Antisense Oligonucleotides Delivered to the Lysosome Escape and Actively Inhibit the Hepatitis B Virus Keith D. Jensen,† Pavla Kopecˇkova´,†,‡ and Jindrˇich Kopecˇek*,†,‡ Department of Pharmaceutics and Pharmaceutical Chemistry and Department of Bioengineering, University of Utah, Salt Lake City, Utah 84112. Received June 12, 2002; Revised Manuscript Received July 3, 2002
The subcellular fate and activity in inhibiting the hepatitis B virus of free and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-phosphorothioate oligonucleotides were studied. Their internalization and subcellular fate were monitored with confocal microscopy. A fraction of the internalized free oligonucleotides escaped into the cytoplasm and nucleus of Hep G2 cells but were not active antiviral agents. Covalently attaching the oligonucleotides to the HPMA copolymers via nondegradable dipeptide GG spacers resulted in sequestering the oligonucleotides in vesicles after internalization. Conjugation of the oligonucleotides to an HPMA copolymer via a lysosomally cleavable tetrapeptide GFLG spacer resulted in release of the oligonucleotide in the lysosome and subsequent translocation into the cytoplasm and nucleus of the cells. The HPMA copolymer-oligonucleotide conjugate possessed antiviral activity, indicating that phosphorothioate oligonucleotides released from the carrier in the lysosome were able to escape into the cytoplasm and nucleus and remain active. The Hep G2 cells appeared to actively internalize the phosphorothioate oligonucleotides as oligonucleotide-HPMA copolymer conjugates were internalized to a greater extent than unconjugated polymers.
INTRODUCTION
One of the most appealing aspects of antisense oligonucleotides is their inherent specificity. They are designed to inhibit the synthesis of a single unwanted protein (reviewed in ref 1). Intense research has overcome many of the obstacles facing antisense therapy except delivery of the oligonucleotide to its target. In order for the antisense oligonucleotide to inhibit the synthesis of the target protein, the oligonucleotide must cross at least one cell membrane and hybridize with its target mRNA in the cytoplasm or nucleus of the diseased cell. Endocytosis appears to be the major pathway by which oligonucleotides enter most cells, although much remains to be learned (2). Because phosphorothioate oligonucleotides are inherently large and charged molecules, it has been presumed that they cannot cross cell membranes without some type of help (e.g., permeation enhancers or microinjection), and degradation by lysosomal enzymes is the expected fate of the oligonucleotides. Yet the literature continues to accumulate positive evidence of antisense activity suggesting that at least a fraction of the oligonucleotides escape before being degraded (3). More information on the subcellular fate of free oligonucleotides is needed as free antisense oligonucleotides approved by the FDA or in clinical trials are typically administered by intravenous injection (4, 5). These oligonucleotides are presumed to be internalized by endocytosis and somehow cross the endosomal/lysosomal membrane before being degraded. It is not known at what * Correspondence should be addressed to this author at the Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, 30 S. 2000 E RM 301, Salt Lake City, UT 84112. Tel: 801-581-4532, Fax: 801-581-3674, E-mail:
[email protected]. † Department of Pharmaceutics and Pharmaceutical Chemistry. ‡ Department of Bioengineering.
stage of endocytosis the antisense oligonucleotides escape into the cytoplasm. In this research, we hypothesized that antisense oligonucleotides delivered in the lysosome would be able to escape into the cytoplasm before being degraded and actively inhibit synthesis of the target protein. To test this hypothesis, we conjugated the oligonucleotides to the HPMA copolymers via lysosomally cleavable spacers to ensure delivery of the oligonucleotides in the lysosome. Using confocal microscopy, we monitored the subcellular fate of the oligonucleotides and polymers, and demonstrated antisense activity by a biochemical assay. EXPERIMENTAL PROCEDURES
Hep G2 (6) cells (human hepatocellular carcinoma) were from American Type Culture Collection (Manassas, VA). Minimum essential media alpha modification (MEMR) and fetal bovine serum (FBS) were from HyClone (Logan, UT). N-Benzoyl-Phe-Val-Arg-p-nitroanilide was from Sigma (St. Louis, MO). A SlowFade Light Antifade Kit and Lissamine rhodamine B (LR) ethylenediamine were from Molecular Probes (Eugene, OR). Sephadex G-25 (PD-10) columns, a Superose 6 (10/30) column, a Superdex 75 (10/30) column, and a Sephadex LH-20 column were from Pharmacia (Piscataway, NJ). Deionized water was used for the preparation of all buffers. All other chemicals were of reagent grade or better. The oligonucleotide was synthesized by the Emory Microchemical Facility (Atlanta, GA). The oligonucleotide was a 21-mer phosphorothioate oligonucleotide whose sequence was derived from work by Wu and Wu (7), 5′TTTATAAGGGTCGATGTCCXX-3′. The oligonucleotide had a primary amine on the 5′-end [5′-amino-modifier C6 (Glen Research, Sterling, VA)] and a fluorescein on the 3′-end [6-Fluorescein CPG (Glen Research)]. The oligonucleotide was purified by reverse-phase HPLC [Brownlee ODS-300 (Perkin-Elmer, Wellesley, MA) C18, 250 × 10 mm column with 7 µm, 300 Å silica with a 50
10.1021/bc025559y CCC: $22.00 © 2002 American Chemical Society Published on Web 08/13/2002
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mM triethylamine acetate (pH 6.9)/acetonitrile gradient buffer system], and the purity and identity were analyzed by capillary electrophoresis (1 predominate peak) and MALDI-TOF MS [molecular weight measured: 7523.29 (7517.40 calcd)]. Size exclusion chromatography (SEC) was used to estimate the molecular weight of the polymers, detect unbound oligonucleotide, and purify the oligonucleotideHPMA copolymer conjugates. The molecular weight of the aminolysed (with aminopropanol) polymer precursors was determined on Superose 6 in PBS buffer using laser light scattering detector MiniDawn (Wyatt Inc., Santa Barbara, CA). Synthesis. Monomers. HPMA (8), N-methacryloylglycylglycylgalactosamine (MA-Gly-Gly-GalN) (9), Nmethacryloylglycylglycine p-nitrophenyl ester (MA-GGONp) (10), and N-methacryloylglycylphenylalanylleucylglycyl p-nitrophenyl ester (MA-GFLG-ONp) (11) were prepared according to previously described procedures. N-Methacryloylglycylglycyl ethylenediamine Lissamine rhodamine B (MA-GG-LR) was prepared by slowly adding a solution of Lissamine rhodamine B ethylenediamine (20 mg, 33 µmol) in DMF (0.2 mL) to MA-GG-ONp (24 mg, 75 µmol) dissolved in DMF (0.3 mL). To this was added diisopropylethylamine (15 µL, 86 µmol) and allowed to stir for 3 h. The DMF was removed in vacuo, and the product was precipitated with acetone/ether. The product was separated on a Sephadex LH-20 column eluted with MeOH and the purity checked by TLC (methylene chloride/methanol/acetic acid 3:1:0.1, Rf ) 0.68). The extinction coefficient of the product was 561 ) 120 000 M-1 cm-1 (methanol). Polymer Conjugate P1 [P-GG-(Oligo-Fl)]. A polymer precursor was prepared by polymerization of the comonomers HPMA and MA-GG-ONp at a molar ratio of 92:8 (acetone, 50 °C, 24 h, AIBN). The polymer (0.47 mmol of ONp/g of polymer, 7.5 mol %, 272 ) 9500 M-1 cm-1 in DMSO, Mw ) 25 kDa, Mw/Mn ) 1.3) was purified by precipitation from methanol into acetone. The polymer precursor (34.3 mg, 16 µmol of ONp) and the oligonucleotide (3.05 mg, 0.41 µmol) were dissolved in anhydrous DMSO (0.2 mL). To this was added diisopropylethylamine (10 µmol) and allowed to stir overnight. The next day, an additional amount of diisopropylethylamine (10 µmol) was added and allowed to stir for 3 h. The reaction mixture was diluted with water, and the product was isolated on a PD-10 column followed by extensive dialysis (cutoff 12-14 kDa). The conjugate was analyzed by SEC (Superose 6 column, PBS, UV and RI detection), which found the oligonucleotide bound to the polymer with no detectable amounts of free oligonucleotide. The amount of bound oligonucleotide was 9.0 µmol/g of polymer [75% efficiency determined from the absorption of fluorescein (494 ) 70 000 M-1 cm-1, 0.1 M borate buffer, pH 9.1, chemical structure in Figure 1A)]. Polymer Conjugate P2 [P-(GG-LR)-(GFLG-(Oligo-Fl))]. Polymer P2 was similar to P1 except that the polymer backbone was labeled with LR and the oligonucleotide was conjugated via the tetrapeptide spacer, GFLG. Briefly, a precursor polymer was prepared by polymerization of the comonomers HPMA, MA-GFLG-ONp, and MA-GG-LR in the molar ratio 95:5:1 (acetone, 50 °C, 24 h, AIBN). The polymer [0.058 mmol of LR/g of polymer (0.90 mol %, 563 ) 1.2 × 105 M-1 cm-1, methanol), 0.22 mmol of ONp/g of polymer (3.6 mol %, 400 ) 1.8 × 104 M-1 cm-1, 0.1 N NaOH), Mw ) 25 kDa, Mw/Mn ) 1.4] was purified by reprecipitation from methanol into acetone. To conjugate the oligonucleotide, the precursor polymer
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(9.5 mg, 2.1 µmol of ONp) and the oligonucleotide (2.8 mg, 0.37 µmol) were dissolved in anhydrous DMSO (70 µL). To this was added diisopropylethylamine (3 µL) and allowed to stir overnight. The next day, an additional amount of diisopropylethylamine (5 µL) was added and allowed to stir for 3 additional hours. The reaction mixture was diluted with water and the product was isolated on a PD-10 column. The product was dialyzed (cutoff 12-14 kDa) extensively, but still contained free oligonucleotide. The conjugate was purified by SEC (Superdex 75 column, PBS), and fractions of the conjugate were collected and dialyzed to remove salts. The purified conjugate (chemical structure in Figure 1B) contained 21 µmol (70% binding efficiency) of fluoresceinlabeled oligonucleotide per gram of conjugate and 35 µmol of LR per gram of conjugate. Cell Culture. Hep G2 cells were cultured in MEM-R media supplemented with 10% FBS in a 37 °C incubator with 5% CO2 (v/v) with humidified air without antibiotics. For all biological solutions, the pH was adjusted to 7.4 using cell culture grade HCl or NaOH as needed. Confocal Fluorescent Microscopy. The preparation of the Hep G2 cells for microscopy was described previously (12). Briefly, after incubation, the cells were fixed with 3% paraformaldehyde for 20 min at room temperature and mounted with SlowFade Light antifade medium. Cells were analyzed on a Zeiss (Thornwood, NY) LSM 510 confocal imaging system with a Zeiss Axioplan 2 microscope (100 × plan-apo objective, NA ) 1.4, oil) and an argon laser (for P1 fluorescein excitation: 488 nm emission, 505 nm long-pass filter; for P2 (LR present), excitation 488 and 543 nm, emission beam splitter 545 nm, green channel emission 500-550 nm, red channel emission 560 nm long-pass). The settings for all the confocal systems were adjusted so that control cells always yielded dark images (i.e., no background fluorescence was visible). To determine if the signals from the two dyes could be separated, two “control” HPMA copolymers labeled with either LR or fluorescein were utilized. Cells were incubated with each of the control polymers, and the microscope settings were adjusted to prevent “bleed-over” of green fluorescence into the red channel. The 8-bit fluorescent images were initially scaled to 256 Gy levels, and colored look-up tables were applied for both dyes. Oligonucleotide Stability. Rat liver tritosomes (isolated lysosomal enzymes) were harvested as previously described (13). The activity of the enzymes was determined by measuring the cleavage of N-benzoyl-Phe-ValArg-p-nitroanilide. The tritosomes (100 µL) and 900 µL of a citrate-phosphate cocktail [citrate-phosphate buffer (pH 5.5), 5 mM glutathione, 0.2% Triton X-100, 0.65 mM EDTA, and 1 mM nitroanilide substrate (final concentrations)] were warmed to 37 °C and mixed, and the absorption at 410 nm (released p-nitroaniline) was recorded for 10 min. The change in absorption over 10 min was ∆A ) 0.488. The amount of degradation of the oligonucleotides was measured by HPLC [Dynamax, Varian Inc., Palo Alto, CA, C18 column (Varian), mobile phase: acetonitrile and 50 mM triethylamine acetate buffer (pH 7)] after digestion with the tritosomes. The oligonucleotide and oligonucleotide-HPMA copolymer conjugate, P1 (both at 1 µM final concentration of oligonucleotide), were digested in 250 µL of the citrate-phosphate cocktail (above) with 50 µL of tritosomes at 37 °C. The eluting oligonucleotide was assayed for fluorescence using a fluorescent detector [Waters (Milford, MA), excitation 495 nm, emission 520 nm], and the peak area of the respective peaks was used
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to determine the amount of oligonucleotide degradation at various time points in a 8 h of incubation to see staining for a majority of the cells. Such variability was not observed in this work as all cells that were incubated with oligonucleotides (free or conjugated) exhibited similar amounts and distribution of staining. The difference is likely due to the presence of the oligonucleotides on the polymers, but more studies are needed to determine the effect of conjugated moieties on the polymer’s subcellular fate. The subcellular distribution of the oligonucleotide conjugated to the polymer by the lysosomally degradable GFLG spacer (P2) differed significantly from that attached by the nondegradable spacer. Labeling the polymer backbone with LR and the oligonucleotide with
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fluorescein allowed separate tracking of each component. At incubation times shorter than 2 h, the fluorescence from both dyes was predominantly found together in small vesicles in the cytoplasm (Supporting Information Figure 2). The green (oligonucleotide) and red (polymer) signals began to separate between 2 and 4 h and increase slightly after 24 h of incubation (Supporting Information Figure 2). The staining of the cytosol, nuclei, and nuclear structures by the oligonucleotide conjugated via the cleavable spacer (polymer P2) appeared to be a combination of the staining of the free oligonucleotide and the oligonucleotide-HPMA copolymer conjugate P1. At incubation times of 4 h or greater, the oligonucleotide of polymer P2 was observed in the cytoplasm and nucleus (Figures 5A and 6A) similar to that of the free oligonucleotide (Figure 3A). There was only minimal staining of the cytoplasm by the polymer (Figures 5C and 6C) similar to that observed for the oligonucleotide-HPMA copolymer P1 with the nondegradable spacer (Figure 4A). There was increased staining of nuclear structures by the oligonucleotide in polymer P2 (Figure 6A) similar to that seen when incubating with the free oligonucleotide (Figure 3A). These results suggest that the oligonucleotide conjugated to the polymer by the degradable spacer was released from the polymer and escaped from lysosomes in a fashion similar to the free oligonucleotide. The oligonucleotide appeared to be able to escape from lysosomes faster than the polymer as more (qualitatively) of the oligonucleotide entered the cytoplasm and nucleus than the polymer (compare Figures 5A vs 5C and 6A vs 6C). The increased uptake of the oligonucleotide-HPMA copolymer conjugate compared to the LR-labeled polymer (Supporting Information Figure 3) supported previous findings (36) that Hep G2 cells actively internalize phosphorothioate oligonucleotides. The antiviral assay was needed to determine if the antisense oligonucleotide was still active after passing through the lysosome. The confocal microscopy supplied evidence that oligonucleotide delivered to the lysosome escaped into the cytoplasm and nucleus. The positive antiviral activity of the polymer-oligonucleotide conjugate P2 (Table 2) demonstrated that a fraction of the antisense oligonucleotide had not been degraded and was still active. The relatively high concentration of the oligonucleotide-HPMA copolymer conjugate P2 required for antiviral activity was likely due to a combination of three causes: inefficient delivery of the oligonucleotide to its target, inactivation of the antisense oligonucleotide, and a reduced potency antisense oligonucleotide (fluorescein bound to the oligonucleotide and an unoptimized sequence). A significant amount of the fluorescence from the oligonucleotide appeared to remain in vesicles, indicating that only a fraction of oligonucleotide escaped into the cytoplasm and nucleus of the cells. The inactivity of the free oligonucleotide, at the concentrations studied, suggested that some of the fluorescence observed outside of the lysosomes was due to inactivated oligonucleotide. Degradation was the likely causesas supported by the stability assay and by the fact that protein binding or modification should have been similar for free and polymer-liberated oligonucleotides. After observing the free oligonucleotide in the cytoplasm and nuclei of the cells, we expected it to have antiviral activity also. This inactivity was most likely due to degradation of the oligonucleotide in the cell media or endosome/lysosome as some stability was afforded to the oligonucleotide by conjugation to the polymer (Table 1).
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Using a similar antisense oligonucleotide, Wu and Wu (7) also found that the free oligonucleotide had lower activity than the oligonucleotide complexed to a targeted poly-lysine carrier. They attributed the difference in activity to the increased amount of oligonucleotide delivered with the targeted polymeric carrier, increased stability due to the carrier, and potential help from the carrier in escaping the lysosome. In this work, the polymeric carrier likely increased the activity of the oligonucleotide by increasing its stability as the HPMA copolymer was not targeted nor was it likely to help the oligonucleotide escape the lysosomesit was uncharged, had no useful buffering capacity, and was less able to escape the lysosome than the oligonucleotide (Figure 6). The initial rapid degradation of a small fraction of the conjugated oligonucleotide followed by the slow degradation phase suggested that some of the oligonucleotide was easily accessible for degradation, but that the majority of the conjugated oligonucleotide was not. The slow degradation phase of the polymer-conjugated oligonucleotide was probably not due to inactivation of the lysosomal enzymes as the free oligonucleotide was continuously degraded over the course of the experiment. It is unlikely that the uncharged, hydrophilic polymer inactivated the enzymes as it was present from the beginning of the experiment. Cleavage of the degradable tetrapeptide bond and protection of the oligonucleotide, both on the polymer, may appear an apparent contradiction. Both are likely afforded limited protection from degradation. Cleavage of the tetrapeptide is particularly efficient as its sequence was chosen as a good substrate for lysosomal thiol proteinases, especially cathepsin B (37, 38), whereas the phosphorothioate oligonucleotide is designed to be resistant to nucleases. CONCLUSIONS
• Phosphorothioate oligonucleotides internalized by endocytosis were able to escape into the cytoplasm and nucleus of Hep G2 cells. • Binding the oligonucleotides to the HPMA copolymers via nondegradable spacers restricted the oligonucleotides from entering the cytoplasm and nucleus of the cells. • Conjugation of the oligonucleotides to the HPMA copolymers via degradable spacers afforded them with increased stability and prevented their inactivation before escaping the lysosome and actively inhibiting the hepatitis B virus. To better deliver antisense oligonucleotides to their targets in the cytoplasm and nucleus, knowledge of their subcellular distribution and ability to escape lysosomes is needed. The many positive examples of antisense oligonucleotides in the literature suggests that despite their large size and multiple negative charges, oligonucleotides are able to escape from lysosomes and actively inhibit synthesis of the target protein (1). To study this hypothesis, we delivered phosphorothioate oligonucleotides to the lysosome using HPMA copolymers. Conjugation via lysosomally cleavable spacers assured release of the oligonucleotide only in the lysosome. Confocal microscopy and an antiviral assay provided evidence that the oligonucleotide delivered to the lysosome was able to escape into the cytoplasm and nucleus of Hep G2 cells and actively inhibit its target. The high concentration of antisense oligonucleotides required for antiviral activity, the short half-life of the oligonucleotide, and the high fluorescence of vesicles suggest directions for improving antisense therapies. Increasing the activity and stability of the antisense oligonucleotides, while
Oligonucleotides Delivered to the Lysosome Escape
maintaining high specificity, will provide potent drugs. Better delivery methods are also needed to ensure interaction of the antisense oligonucleotide with its intended target. ACKNOWLEDGMENT
The antiviral activities of the antisense oligonucleotides were determined by Dr. Brent Korba (Georgetown University, Rockville, MD) under NIAID Contract N01 AI85349. This research was supported in part by National Institutes of Health Grants CA51578 and CA88047 from the National Cancer Institute. Partial support of K.D.J. was provided by NIH Training Grant GM08537 and by a fellowship from the American Foundation of Pharmaceutical Education. Supporting Information Available: Three figures showing Hep G2 cells incubated with P1, P2, and polymer P-LR. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Chirila, T. V., Rakoczy, P. E., Garrett, K. L., Lou, X., and Constable, I. J. (2002) The use of synthetic polymers for delivery of therapeutic antisense oligodeoxynucleotides. Biomaterials 23, 321-342. (2) Lebedeva, I., Benimetskaya, L., Stein, C. A., and Vilenchik, M. (2000) Cellular delivery of antisense oligonucleotides. Eur. J. Pharm. Biopharm. 50, 101-119. (3) Crooke, S. T. (1998) Basic principles of antisense therapeutics. Antisense research and application (Crooke, S. T., Ed.) pp 1-50, Springer, Berlin. (4) de Smet, M. D., Meenken, C. J., and van den Horn, G. J. (1999) Fomivirsensa phosphorothioate oligonucleotide for the treatment of CMV retinitis. Ocul. Immunol. Inflammation 7, 189-198. (5) Tamm, I., Dorken, B., and Hartmann, G. (2001) Antisense therapy in oncology: new hope for an old idea? Lancet 358, 489-497. (6) Knowles, B. B., and Aden, D. P. (1983) Human hepatoma derived cell line, process for preparation thereof, and uses therefore. U.S. Patent 4,393,133. (7) Wu, G. Y., and Wu, C. H. (1992) Specific inhibition of hepatitis B viral gene expression in vitro by targeted antisense oligonucleotides. J. Biol. Chem. 267, 12436-12439. (8) Kopecˇek, J., and Bazˇilova´, H. (1973) Poly[N-(2-hydroxypropyl)methacrylamide]. 1. Radical polymerization and copolymerization. Eur. Polym. J. 9, 7-14. (9) Rathi, R. C., Kopecˇkova´, P., R ˇ ´ıhova´, B., and Kopecˇek, J. (1991) N-(2-hydroxypropyl)methacrylamide copolymers containing pendant saccharide moieties. Synthesis and bioadhesive properties. J. Polym. Sci., Part A: Polym. Chem. 29, 1895-1902. (10) Rejmanova´, P., Labsky´, J., and Kopecˇek, J. (1977) Aminolyses of monomeric and polymeric 4-nitrophenyl esters of methacryloylated amino acids. Makromol. Chem. 178, 21592168. (11) Ulbrich, K., Sˇ ubr, V., Strohalm, J., Plocova´, D., Jelı´nkova´, M., and R ˇ ´ıhova´, B. (2000) Polymeric drugs based on conjugates of synthetic and natural macromolecules. I. Synthesis and physicochemical characterization. J. Controlled Release 64, 63-79. (12) Jensen, K. D., Kopecˇkova´, P., Bridge, J. H. B., and Kopecˇek, J. (2001) The cytoplasmic escape and nuclear accumulation of endocytosed and microinjected HPMA copolymers and a basic kinetic study in Hep G2 cells. AAPS PharmSci. [serial online] 3, Article 32. (13) Trouet, A. (1974) Isolation of modified liver lysosomes. Methods Enzymol. 31 (Pt. A), 323-329.
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