Journal of Drug Targeting
ISSN: 1061-186X (Print) 1029-2330 (Online) Journal homepage: http://www.tandfonline.com/loi/idrt20
Application of biodegradable dendrigraft poly-Llysine to a small interfering RNA delivery system Yukinobu Kodama, Haruka Kuramoto, Yukari Mieda, Takahiro Muro, Hiroo Nakagawa, Tomoaki Kurosaki, Miako Sakaguchi, Tadahiro Nakamura, Takashi Kitahara & Hitoshi Sasaki To cite this article: Yukinobu Kodama, Haruka Kuramoto, Yukari Mieda, Takahiro Muro, Hiroo Nakagawa, Tomoaki Kurosaki, Miako Sakaguchi, Tadahiro Nakamura, Takashi Kitahara & Hitoshi Sasaki (2016): Application of biodegradable dendrigraft polyL-lysine to a small interfering RNA delivery system, Journal of Drug Targeting, DOI: 10.1080/1061186X.2016.1184670 To link to this article: http://dx.doi.org/10.1080/1061186X.2016.1184670
Accepted author version posted online: 29 Apr 2016.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=idrt20 Download by: [Orta Dogu Teknik Universitesi]
Date: 03 May 2016, At: 01:42
Journal of Drug Targeting, original paper
Application of biodegradable dendrigraft poly-L-lysine to a small interfering RNA delivery system
Nakagawaa, Tomoaki Kurosakia, Miako Sakaguchib, Tadahiro Nakamuraa, Takashi
Department of Hospital Pharmacy, Nagasaki University Hospital, 1-7-1 Sakamoto,
TE
a
D
Kitaharaa, Hitoshi Sasakia,*
AC C
Sakamoto, Nagasaki 852-8523, Japan
EP
Nagasaki 852-8501, Japan; bInstitute of Tropical Medicine, Nagasaki University, 1-12-4
*Corresponding author: Department of Hospital Pharmacy, Nagasaki University Hospital, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan +81-95-819-7245
Fax:
+81-95-819-7251
ST
Tel.:
JU
Downloaded by [Orta Dogu Teknik Universitesi] at 01:42 03 May 2016
Yukinobu Kodamaa, Haruka Kuramotoa, Yukari Miedaa, Takahiro Muroa, Hiroo
E-mail:
[email protected]
Abstract Dendrigraft poly-L-lysine (DGL), including its central core, consists entirely of lysine, hence it is completely biodegradable. We applied DGL in a small interfering RNA (siRNA) delivery system. Binary complexes with siRNA and DGL had particle
D
significant silencing effects in a mouse colon carcinoma cell line expressing luciferase
TE
(Colon26/Luc cells). The siRNA-DGL complexes induced slight cytotoxicity and hematological toxicity at a high charge ratio of DGL to siRNA, probably because of
EP
their cationic charges. Therefore, we recharged the siRNA-DGL complexes with
AC C
γ-polyglutamic acid (γ-PGA), a biodegradable anionic compound, which was reported to reduce the cytotoxicity of cationic complexes. The ternary complexes showed particle sizes of 35–47 nm at a charge ratio of greater than 14 to siRNA with negative charges.
ST
Strong silencing effects of the ternary complexes were observed in Colon26/Luc cells without cytotoxicity or hematological toxicity. The cellular uptake and degradation of
JU
Downloaded by [Orta Dogu Teknik Universitesi] at 01:42 03 May 2016
sizes of 23–73 nm and ζ-potentials of 34–42 mV. The siRNA-DGL complexes showed
the binary and ternary complexes were confirmed by fluorescence microscopy. The ternary complexes suppressed luciferase activity in the tumor after direct injection into the tumors of mice bearing Colon26/Luc cells. Thus, a potentially important siRNA delivery system was constructed using biodegradable DGL.
Keywords: dendrigraft poly-L-lysine; biodegradable; siRNA delivery; γ-polyglutamic
interference
(RNAi),
which
involves
highly
specific-sequence
TE
RNA
D
1. Introduction
post-transcriptional gene silencing, results from the formation of double-stranded RNA
EP
and plays an important role in the regulation of protein expression. Double-stranded
AC C
small interfering RNAs (siRNAs), consisting of 21–23 nucleotides, can induce RNAi and inhibit the expression of target proteins without affecting genomic DNA.1 Gene silencing using siRNA has potential applications in the treatment of diseases including
ST
cancer, viral infection, and genetic disorders.2-4 However, the therapeutic use of siRNA requires a drug-delivery system because unmodified naked siRNA is immediately
JU
Downloaded by [Orta Dogu Teknik Universitesi] at 01:42 03 May 2016
acid; nanoparticles
degraded by nucleases, it penetrates poorly through the plasma membrane, and it induces interferon responses after systemic injection.5 Dendrimers are regular, highly branched monodisperse and usually highly symmetric spherical synthetic macromolecules with a tunable structure, molecular size,
and surface charge. Dendrimers are characterized by the presence of numerous ionizable terminal groups, which means that they can efficiently bind to a large amount of genetic material.6 Polycationic forms such as polyamidoamine dendrimers have been studied extensively as carriers for plasmid DNA (pDNA), antisense oligonucleotides, and
D
strong cytotoxicity.10,11 On the other hand, dendrigraft poly-L-lysine (DGL), including
TE
its central core, consists entirely of lysine and is completely biodegradable. In addition, DGL is usually synthesized by “protect/deprotect”, activation schemes, or aqueous and it is water-soluble, thermally stable,
AC C
and non-immunogenic.
13
EP
N-carboxyanhydride polycondensation,12,
We focused on biodegradable DGL, which comprises a recently discovered subset of dendritic polymers. Hofman et al. already reported that the complex of siRNA and
ST
DGL showed a high silencing effect using Hep2 cells in vitro.14 We also found a high silencing effect of the binary complexes of siRNA and DGL using a mouse colon
JU
Downloaded by [Orta Dogu Teknik Universitesi] at 01:42 03 May 2016
siRNA.7-9 However, polyamidoamine dendrimers are not biodegradable and exhibit
carcinoma cell line, Colon26, expressing luciferase (Colon26/Luc cells) in the preliminary experiment, although a slight cytotoxicity of the complexes was observed. In order to reduce the cytotoxicity of DGL, Tang et al developed a PEGylated DGL, which cleaved in tumor relevant glutathione conditions.15 Another promising approach
is to prepare the ternary complexes coated with biodegradable γ-polyglutamic acid (γ-PGA), which reduced cytotoxicity of the complexes without a decrease of transgene efficiency.16 In the present study, we investigated the usefulness of ternary complexes, which were constructed of binary complexes and γ-PGA.
D
2.1. Chemicals
TE
Fifth-generation DGL compounds (MW: 172,300 Da, 963 lysine groups, dimeter (pH=7): 16 nm) and DGL-Fluo inside (1-fluorescein isothiocyanate labeled at the core
EP
of DGL; DGLin) were purchased from COLCOM S.A.S. (Montpellier, France). γ-PGA
AC C
(average MW: 55,000 Da) was provided by Yakult Pharmaceutical Industry Co., Ltd. (Tokyo, Japan). Lipofectamine RNAiMAX and Alexa Fluor 555-labeled siRNA (BLOCK-iT Alexa Fluor Red Fluorescent Oligo) were purchased from Invitrogen CA,
USA).
ST
(Carlsbad,
Firefly
luciferase
siRNA
5-CUUACGCUGAGUACUUCGAdTdT-3,
JU
Downloaded by [Orta Dogu Teknik Universitesi] at 01:42 03 May 2016
2. Materials and methods
5-UCGAAGUACUCAGCGUAAGdTdT-3) 5-CUUACGCUGUCAUGAUCGAdTdT-3,
(sense: antisense:
and
scrambled
siRNA
(sense: antisense:
5-UCGAUCAUGACAGCGUAAGdTdT-3) were obtained from GeneDesign, Inc. (Osaka, Japan). Bovine serum albumin was purchased from Sigma Aldrich (St. Louis,
MO, USA), and fetal bovine serum was purchased from Biological Industries Ltd. (Kibbutz Beit Haemek, Israel). RPMI 1640 medium, Opti-MEM I, antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin), and other culture reagents were obtained from GIBCO BRL (Grand Island, NY, USA). Antibiotic G418 solution was obtained Roche
Diagnostics
(Indianapolis,
IN,
USA).
The
D
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium
TE
salt (WST-1), and 1-methoxy-5-methylphenazinium methyl sulfate (1-methoxy PMS) were purchased from Dojindo Laboratories (Kumamoto, Japan). All other chemicals
AC C
EP
were of reagent grade.
2.2. Preparation of complexes
In this study, we constructed complexes using theoretical charge ratios based on
ST
phosphate in siRNA, nitrogen in DGL, and carboxylate in γ-PGA. To prepare binary complexes, an appropriate amount of stock DGL solution (5
JU
Downloaded by [Orta Dogu Teknik Universitesi] at 01:42 03 May 2016
from
mg/ml, pH 7.4) was mixed with siRNA solution (1 mg/ml) dissolved in diethylpyrocarbonate-treated water (Invitrogen) by thorough pipetting, before being left for 30 min at room temperature. The siRNA and DGL were mixed at charge ratios of 1:2, 1:4, 1:6, 1:8, 1:10, and 1:12 (siRNA-DGL2, 4, 6, 8, 10, and 12 complexes) to
produce binary complexes (siRNA-DGL complexes). For example, in the case of siRNA-DGL10, 10 μL of siRNA solution (1 mg/mL) and 11.3 μL of DGL solution (5 mg/mL) were mixed. γ-PGA solution (10 mg/ml) was added to the siRNA-DGL10 complex by pipetting to produce complexes with charge ratios of 1:10:4, 1:10:8, 1:10:12,
For example, in the case of
TE
for another 30 min at room temperature.
D
construct ternary complexes (siRNA-DGL10-γ-PGA complexes), which were then left
siRNA-DGL10-γ-PGA14, 10 μL of siRNA solution (1 mg/mL), 11.3 μL of DGL
EP
solution (5 mg/mL), and 5.7 μL of γ-PGA solution (10 mg/mL) were mixed.
AC C
The siRNA was mixed with Lipofectamine RNAiMAX in accordance with the standard protocol to make a binary complex (siRNA-Lipofectamine complexes) as a
ST
positive control.
2.3. Physicochemical properties of the complexes
JU
Downloaded by [Orta Dogu Teknik Universitesi] at 01:42 03 May 2016
1:10:14, and 1:10:16 (siRNA-DGL10-γ-PGA4, 8, 12, 14, and 16 complexes) to
The particle sizes and ζ-potentials of each complex were measured using a
Zetasizer Nano ZS (Malvern Instruments, Ltd., Malvern, UK). Particle size is shown as the number-weighted mean diameter To determine the complex formation, 20 µL aliquots of each complex solution
containing 1 µg siRNA were mixed with 4 µL loading buffer (30% glycerol and 0.2% bromophenol blue) and loaded onto 2% agarose gels. Electrophoresis (i-Mupid J; Cosmo Bio, Tokyo, Japan) was carried out at 100 V in running buffer solution (40 mM Tris/HCl, 40 mM acetic acid, and 1 mM ethylenediaminetetraacetic acid [EDTA]) for 20
D
a Gel Doc EZ System (Bio-Rad Laboratories, Inc., Tokyo, Japan). The electrophoresis
TE
of the complexes was also carried out after their incubations with 10% serum for 60min
EP
at 37 °C.
AC C
2.4. Cell culture
Colon26/Luc cells was prepared in our laboratory. Briefly, to establish Colon26/Luc cells, Colon26 cells were transfected with pDNA encoding a luciferase
ST
reporter gene (pCMV-Luc) and selected using antibiotic G418 solution. pCMV-Luc was constructed by subcloning the HindIII/XbaI firefly luciferase cDNA fragment from
JU
Downloaded by [Orta Dogu Teknik Universitesi] at 01:42 03 May 2016
min, and the retardation of siRNA was visualized with ethidium bromide staining using
pGL3-control vector (Promega, Madison, WI, USA) into the polylinker of the pcDNA vector (Invitrogen).
2.5. In vitro gene silencing and cellular uptake experiments
Colon26/Luc cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotics (culture medium) in a humidified atmosphere of 5% CO2 in air at 37°C, before being plated into 24-well plates (Becton-Dickinson, Franklin Lakes, NJ, USA) at a density of 1.0 × 104 cells/well and cultivated in 500 µL culture
D
Opti-MEM I medium (transfection medium) after a 24 h pre-incubation, and then the
TE
cells were treated with each complex containing 1 µg siRNA and incubated for 2 h. In preliminary experiment, we examined the transfection efficiency of the complexes in
EP
various dosages and chose the proper dosage. After the cells had been transfected, the
AC C
transfection medium was replaced with culture medium, and the cells were cultured for a further 22 h in a humidified atmosphere of 5% CO2 in air at 37°C. After 22 h incubation, the cells were washed with phosphate-buffered saline (PBS) and then lysed
ST
in 100 µL lysis buffer (pH 7.8; 0.1 M Tris/HCl buffer containing 0.05% Triton X-100 and 2 mM EDTA). Lysate samples (10 µL) were mixed with 50 µL luciferase assay
JU
Downloaded by [Orta Dogu Teknik Universitesi] at 01:42 03 May 2016
medium. In the transfection experiment, the medium was replaced with 500 µL
buffer (PicaGene; Toyo Ink, Tokyo, Japan), and the amount of fluorescence produced was immediately measured using a luminometer (Lumat LB 9507; EG & G Berthold, Bad Wildbad, Germany). The protein content of the lysate was determined using a Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA), in which bovine
serum albumin was used as a standard, and absorbance was measured using a microplate reader (Sunrise RC-R; Tecan Japan Co., Ltd., Kanagawa, Japan) at 595 nm. Luciferase activity is shown as relative light units (RLU) per mg protein, and the results are shown as a percentage of untreated cells (control).
D
incubated with complexes composed of Alexa Fluor 555-labeled siRNA and DGLin, as
TE
described above. After 22 h incubation, the relative levels of Alexa Fluor 555-labeled siRNA and DGLin in the cells were characterized using fluorescence microscopy (200 ×
EP
magnification). DGLin had DGL-labeled fluorescein isothiocyanate at its core, which
2.6. Cytotoxicity
AC C
produced strong green fluorescence after DGL degradation.
ST
The cytotoxic effects of the various complexes on Colon26/Luc cells were measured using a WST-1-based commercially available cell proliferation reagent. The
JU
Downloaded by [Orta Dogu Teknik Universitesi] at 01:42 03 May 2016
To visualize the uptake and degradation of the complexes, Colon26/Luc cells were
reagent was prepared (5 mM WST-1 and 0.2 mM 1-methoxy PMS in PBS) and filtered through a 0.22 µm filter (Millex-GP; Millipore Co, Bedford, MA, USA) just before the experiments. Colon26/Luc cells were plated on 96-well plates (Becton-Dickinson) at a density of 5.0 × 103 cells/well in culture medium. Each complex containing 0.25 µg
siRNA in 100 µL Opti-MEM I medium was added to each well and incubated for 2 h. In preliminary experiment, we examined the cytotoxicity of the complexes in various dosages and chose the proper dosage. Then, the medium was replaced with 100 µL culture medium and incubated for another 22 h at 37°C. The medium was then
D
well. The cells were incubated for an additional 2 h at 37°C, and the absorbance of each
TE
well was measured at a wavelength of 450 nm with a reference wavelength of 630 nm, using a microplate reader. The results are shown as percentages of the value for the
AC C
2.7. Agglutination study
EP
untreated cells (control).
Mouse erythrocytes were subjected to three rounds of centrifugation at 2,430 ×g
prepare
a
ST
at 4°C (Kubota 3500; Kubota, Tokyo, Japan) for 5 min and then resuspended in PBS to 2%
(v/v)
stock
suspension.
The
siRNA-DGL10
complexes
or
JU
Downloaded by [Orta Dogu Teknik Universitesi] at 01:42 03 May 2016
substituted for 100 µL culture medium, and 10 µL of WST-1 reagent was added to each
siRNA-DGL10-γ-PGA14 complexes were added to the erythrocyte suspension (complex: stock suspension = 1:1), which were then incubated for 30 min at room temperature. Then, 10 µL of each sample was placed on a glass plate, and the extent of agglutination was observed by microscopy (200 × magnification).
2.8. Transmission electron microscopy The siRNA-DGL10 complexes or siRNA-DGL10-γ-PGA14 complexes (5 μL) were loaded on a 200-mesh copper grid with carbon-coated plastic film (Nisshin EM,
D
10 s. The morphology of the complexes was observed using a JEM-1230 (JEOL Ltd.,
TE
Tokyo, Japan) with 80 kV acceleration voltage, and imaged using a 2k × 2k Veleta CCD
EP
camera (Olympus Soft Imaging Solutions, Lakewood, CO, USA).
AC C
2.9. Animals
Animal care and experimental procedures were performed in accordance with the Guidelines for Animal Experimentation of Nagasaki University with approval from the
ST
Institutional Animal Care and Use Committee. Female BALB/c mice (5 weeks old) were purchased from Japan SLC (Shizuoka, Japan). After shipping, mice were
JU
Downloaded by [Orta Dogu Teknik Universitesi] at 01:42 03 May 2016
Tokyo, Japan), and negatively stained with 10 μL uranyl acetate solution (1%, w/v) for
acclimatized to the environment for at least 1 day before experiments.
2.10. In vivo gene silencing experiment The transfected Colon26/Luc cells (5 × 105 cells per mouse) suspended in 100 μL
PBS were injected intracutaneously into the flank of BALB/c mice. To evaluate the in vivo silencing effect of the ternary complex, when the volume of the tumors reached 100 mm3, each complex containing 25 μg siRNA and naked siRNA that targeted luciferase was injected directly into the tumors. After 24 h, mice
D
lysis buffer, and the homogenates were centrifuged at 15,000 rpm for 5 min (Kubota
TE
3500; Kubota, Tokyo, Japan). The supernatants were used for the luciferase assay, as described above. Luciferase activity was described as RLU per g of tissue and the
2.11. Statistical analysis
AC C
EP
results are shown as a percentage of untreated cells (control).
The statistical significance of differences between two groups was assessed using
ST
Student’s t test. Multiple comparisons among the groups were performed using Dunnett’s pairwise multiple comparisons t test.
JU
Downloaded by [Orta Dogu Teknik Universitesi] at 01:42 03 May 2016
were sacrificed and the tumors were dissected. The tumor tissues were homogenized in
3. Results 3.1. Physicochemical properties and electrophoretic assay of siRNA-DGL complexes The particle sizes and ζ-potentials of siRNA-DGL complexes are summarized in Table 1. The siRNA-DGL2 complexes aggregated and the size could not be determined.
D
34–42 mV.
TE
During a gel retardation assay, naked siRNA was detected as bands on the agarose gel. The siRNA-DGL2 complexes showed a slight release of siRNA. No such bands of
EP
naked siRNA were detected in the lanes for other siRNA-DGL complexes (Fig. 1A).
AC C
The siRNA-Lipofectamine complexes, siRNA-DGL2, 4, and 6 complexes showed a slight release of siRNA in the presence of the 10% serum (Fig. 1B).
ST
3.2. In vitro silencing efficiency of siRNA-DGL complexes The in vitro silencing efficiency of siRNA-DGL complexes was evaluated by
JU
Downloaded by [Orta Dogu Teknik Universitesi] at 01:42 03 May 2016
The other siRNA-DGL complexes had particle sizes of 23–73 nm and ζ-potentials of
measurement of luciferase activity expressed in Colon26/Luc cells after incubation for 22 h and is shown as a percentage of the control in Fig. 2A. A commercial vector, siRNA-Lipofectamine complexes, showed a strong silencing effect of siRNA. siRNA-DGL6, 10, and 12 complexes showed high silencing efficiency to a significant
level. On the other hand, no silencing effect was confirmed for siRNA-DGL10 complexes using scrambled siRNA (Fig. 2B).
3.3. Cytotoxicity of siRNA-DGL complexes
D
determined using WST-1 assays (Fig. 2C). The siRNA-Lipofectamine complex
TE
displayed significant cytotoxicity compared with the control (P
