Materials
Poly(D,L-lactic-co-glycolic acid) (PLGA7510; MW, 10,000) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Human cyclin B1-specific siRNA, which was modified with a thiol group at the 3ʹ end of the sense or antisense strand, was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The siRNA sequences were as follows: cyclin B1 siRNA sense: 5ʹ-GGC GAA GAU CAA CAU GGC ATT-3ʹ; and cyclin B1 siRNA antisense: 5ʹ-UGC CAU GUU GAU CUU CGC CTT-3ʹ (17). Negative control siRNA was also purchased from Thermo Fisher Scientific, Inc. (Waltham). Fetal calf serum (FCS) and Roswell Park Memorial Institute (RPMI) 1640 were purchased from Gibco (Cergy-Pontoise, France). Anti-human CD71 monoclonal antibody, phycoerythrin (PE) anti-human CD71 antibody (Clone: OKT9) and were purchased from Thermo Fisher Scientific Inc. Normal mice IgG1 kappa monoclonal, which was used as the control IgG, was purchased from Abcam (ab91353, Cambridge, UK). A Pierce IgG F(abʹ)2 preparation kit was purchased from Thermo Fisher Scientific Inc. Cell Counting Kit-8 (CCK-8) reagent was purchased from Dojindo Laboratories (Tokyo, Japan). Alexa488-labeled siRNA was purchased from Thermo Fisher Scientific, Inc. (Waltham). Cy5-labeled siRNA was purchased from Nippon Gene (Toyama, Japan). FITC-labeled PLGA was synthesized by the NARD Institute, Ltd. (Amagasaki, Japan).
Preparation of siRNA–PLGA micelles
The siRNA–PLGA hybrid block copolymer was synthesized as previously described 6. Antisense and sense 3ʹ thiol-modified siRNAs were conjugated to PLGA–3-(2-pyridyldithio) propionyl hydrazide (PDPH) via disulfide exchange reaction. The synthesized siRNA–PLGA hybrids were expected to form self-assembled micelles in aqueous solutions, thereby resulting in a substantially increased charge density of clustered siRNAs on the outer shell (Figs. 1A, 1C).
Preparation and isolation of the F(abʹ)2 fragment from the whole antibody
A F(ab)2 preparation kit was used to cleave F(abʹ)2 from the whole antibody in accordance with the manufacturer’s protocol. In this study, anti-human CD71 antibody was used as a model for targeting the Fabʹ fragment because CD71 is expressed highly in the CEMx174 cell line. The yield solution was prepared in SDS-PAGE loading dyes and analyzed using SDS-PAGE.
Preparation of Fabʹ–PLGA micelles and siRNA–PLGA/Fabʹ–PLGA mixed micelles
F(abʹ)2 fragments of the antibody in phosphate-buffered saline (PBS; 0.5 mg/mL, 400 µL) were mixed with dithiothreitol (DTT) (0.5 mM, 240 µL) and incubated at 37°C for 30 min 12 to reduce F(abʹ)2 fragments into Fabʹ fragments possessing a thiol group. The Fabʹ fragment solution was then conjugated to PLGA–PDPH via disulfide exchange reaction in the same manner that was used to prepare the siRNA–PLGA hybrid. The siRNA–PLGA and Fabʹ–PLGA hybrid solutions were mixed at a volume ratio of 4:1 in accordance with previous methods19 (Figs. 1B, 1D).
Morphology and surface charge of the siRNA–PLGA micelles and siRNA–PLGA/Fabʹ–PLGA mixed micelles
The sizes and surface charges of the siRNA–PLGA micelles and siRNA–PLGA/Fabʹ–PLGA mixed micelles were measured using a dynamic light-scattering instrument (Zetasizer; Malvern Instruments, Ltd., Malvern, UK). Both these types of micelles (300 pmol) were dissolved in 500 μL distilled water. The effective hydrodynamic diameters and zeta potentials of these micelles were measured in triplicate.
Cell culture
The human T–B hybrid cell line CEMx174 was a gift from the American Type Culture Collection (ATCC; catalog CRL-1991, Rockville, Maryland, USA). CEMx174 cells were cultured in an RPMI 1640 medium (Gibco) supplemented with 10% FCS at 37°C in an atmosphere of 95% air and 5% CO2.
Cyclin B1 gene silencing by siRNA–PLGA micelles or siRNA–PLGA/Fabʹ–PLGA mixed micelles without a transfection reagent
CEMx174 cells were seeded in a 24-well plate at a density of 5 × 105 cells per well to measure the relative cyclin B1 expression by western blotting. The siRNA–PLGA micelles or siRNA–PLGA/Fabʹ–PLGA mixed micelles (80 pmol each based on the siRNA content) were added to the well. After 48 h of incubation, the cells were lysed with 1% (w/v) Triton X-100 solution in PBS and centrifuged to remove cell debris.
Western blot analysis
To detect the cyclin B1 protein, cells were lysed in a lysis buffer. The total protein concentrations of the lysates were measured using bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, IL, USA). Cell lysates containing equal amounts of protein were separated by SDS-PAGE (Bio-Rad, Hercules, CA, USA), and protein bands were transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked with Blocking One (Nacalai Tesque, Inc., Kyoto, Japan) overnight at 4°C and then incubated for 1 h at 22°–25°C with anti-cyclin B1 antibody (ab2949; Abcam, Cambridge, UK) or an anti-glyceraldehyde 3-phosphate dehydrogenase antibody (ACR001PT; Acris Antibodies, Inc., San Diego, CA, USA) at a 1:1000 and 1:10,000 dilution, respectively, in a blocking solution. After washing three times, the membranes were incubated for 1 h at 22°C–25°C with a secondary antibody (horseradish peroxidase-conjugated species-specific antibody). Immunoreactive bands were visualized with ImmunoStar LD (Wako Pure Chemical Industries).
Cell viability assay
CyclinB1 gene knockdown effect on the proliferation of CEMx174 cells was evaluated using the CCK-8 reagent. Briefly, 1 × 104 cells in 100 µL RPMI 1640 containing 10% FCS were seeded into 96-well plates. Each well was then treated with 50 µL of micelles for 48 h or with 50 µL PBS as a control. Then, 15 µL CCK-8 reagent was added to each well, and the cells were further incubated at 37°C for 2 h. Absorbance was measured using a microplate reader at test and reference wavelengths of 450 and 655 nm, respectively, to evaluate the relative viability of the treated cells.
Flow cytometry
Evaluation of the binding activity of the antibody fragment
IgG, F(abʹ)2, or Fabʹ was added to CEMx174 cells blocked by 40% normal goat serum at a density of 5 × 105 cells per tube. After a 30-min incubation, cells were washed using a washing buffer, and PE-labeled secondary antibody against IgG was added to the cells. After a 30-min incubation, the fluorescence intensity of the cells was determined using a flow cytometer (NovoCyte, ACEA Biosciences, Inc., San Diego, CA, USA).
Evaluation of the mixing volume ratio in siRNA–PLGA/Fabʹ–PLGA mixed micelles
CEMx174 cells were seeded in a 24-well plate at a density of 5 × 105 cells per well. siRNA–PLGA mixed with Fabʹ–PLGA was added to the well at a volume ratio of 0:0, 10:0, 10:2, 10:5, 10:8, or 0:10. The 10-volume ratio of siRNA–PLGA was determined to be 80 pmol based on the siRNA content, whereas the 8-volume ratio of Fabʹ–PLGA was 5 µg based on the Fabʹ content. Cells were used to evaluate the fluorescence intensity after 24 h of incubation. The fluorescence intensity of Alexa488-siRNA was detected using a flow cytometer.
Localization of the siRNA–PLGA or Fabʹ–PLGA conjugate consisting of mixed micelles after cellular uptake into the CEMx174 cells
CEMx174 cells were seeded in a 24-well plate at a density of 5 × 105 cells per well. The four types of siRNA–PLGA/Fabʹ–PLGA mixed micelles were added to the well to evaluate whether each part of the micelle was taken up into the cells. After 24 h of incubation, the cells were used to evaluate the fluorescence intensity. In this evaluation, Cy5-labeled siRNA or FITC-labeled PLGA was used to prepare three types of a siRNA–PLGA hybrids, (Cy5-labeled siRNA)-PLGA, siRNA-(FITC-labeled PLGA), and Fabʹ-(FITC-labeled PLGA). The fluorescence intensity of Cy5 and FITC was observed using a confocal microscope (LMS710, Carl Zeiss, Oberkochen, Germany) with a 40× objective lens to evaluate the localization of the fluorescence intensity in the cells by slicing to distinguish between the surface and cell interior. Image analysis was performed using LSM Software ZEN 2009 (Carl Zeiss,).
Statistical analysis
Values were expressed as mean ± standard deviation (SD) (n = 3–5). To determine the differences among the groups, data were evaluated for statistical significance using the Bonferroni test. Overall significance was determined using a one-way repeated measures analysis of variance (ANOVA). A p-value of <0.05 was considered to be statistically significant.
Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.