Rod‐Shaped Active Drug Particles Enable Efficient and Safe Gene Delivery

Abstract Efficient microRNAs (miRNA) delivery into cells is a promising strategy for disease therapy, but is a major challenge because the available conventional nonviral vectors have significant drawbacks. In particular, after these vectors are entrapped in lysosomes, the escape efficiency of genes from lysosomes into the cytosol is less than 2%. Here, a novel approach for lethal‐7a (let‐7a) replacement therapy using rod‐shaped active pure drug nanoparticles (≈130 nm in length, PNPs) with a dramatically high drug‐loading of ≈300% as vectors is reported. Importantly, unlike other vectors, the developed PNPs/let‐7a complexes (≈178 nm, CNPs) can enter cells and bypass the lysosomal route to localize to the cytosol, achieving efficient intracellular delivery of let‐7a and a 50% reduction in expression of the target protein (KRAS). Also, CNPs prolong the t 1/2 of blood circulation by ≈threefold and increase tumor accumulation by ≈1.5–2‐fold, resulting in significantly improved antitumor efficacies. Additionally, no damage to normal organs is observed following systemic injection of CNPs. In conclusion, rod‐shaped active PNPs enable efficient and safe delivery of miRNA with synergistic treatment for disease. This nanoplatform would also offer a viable strategy for the potent delivery of proteins and peptides in vitro and in vivo.

were incubated with 5× loading buffer containing GelRed (Generay Biotechnology, China) for 30 min. and then 10% glycerine and 5 µL of 2% sodium dodecyl sulfate (SDS) were added to the mixture. Gel electrophoresis was performed at 110 V for 30 min. Finally, the gel was photographed using Bio-Rad high-sensitivity chemiluminescence imaging system (Chemidoc XRS+, USA).
To assess the protection for let-7a in CNPs against degradation by RNase A, naked let-7a (0.5 µg) or CNPs were cultured with 10 µg/mL RNase A at 37 °C. At predetermined time points, the resultant samples were stopped by incubation with 1% SDS for 5 min at 60 °C followed by addition of 2% SDS to displace miRNAs from CNPs. The samples were assayed by agarose gel electrophoresis as described above.
To evaluate the serum stability of let-7a in CNPs, CNPs were cultured in 10% FBS solution at 37 °C for different times. Then, the samples were removed, processed according to the procedure described above and finally assayed by agarose gel electrophoresis.

In vitro drug release
The drug release from Taxol, PNPs and CNPs was tested using a dialysis method performed in an incubator (SHA-C, Jintan, China) with a shaking speed of 100 rpm/min at 37 ± 1 °C. The molecular weight cutoff of the dialysis bag was 3500 Da, and the release medium was phosphate-buffered saline (PBS) with three different pH values (pH 7.4, 6.8 and 5.0). At predetermined intervals, samples were withdrawn from outside the dialysis bag and replaced with an equal volume of corresponding fresh PBS. After filtration through a 0.2-µm membrane filter, the PTX content in these samples was detected by high performance liquid chromatography (HPLC) as depicted in a previous report. [3]

Caveolae-mediated cellular uptake
The 4T1 and Caco-2 cells were seeded on 12-well plates at a density of 2×10 5 cells/well and cultured for 48 h at 37 °C, respectively. After pretreatment with caveolae inhibitors, nystatin (10 µM) or methyl-β-cyclodextrin (M-CD, 2.5 mM), for 0.5 h at 37 °C, the cells were incubated with dual-labeled CNPs in serum-free culture medium at 37 °C for 4 h. At the end of the experiment, the cells were washed three times with PBS and collected by trypsinization to assess the fluorescence intensity by flow cytometry (FCM, BD FACSCalibur, USA).

Co-localization of CNPs with caveolae
For the location detection of CNPs in caveolae, Caco-2 cells were incubated with the CNPs at 100 nM Cy5-let-7a in DMEM for 4 h at 37 °C. Subsequently, the cells were sequentially washed, fixed with 4% (w/w) paraformaldehyde for 10 min, permeabilized in 0.1% Triton X-100 for 5 min, and blocked in blocking buffer (1% bovine serum albumin/10% normal goat serum/0.3 M glycine in 0.1% PBS-Tween) for 15 min. The cells were then incubated with Caveolae Marker (Alexa Fluor ® 488) at a working dilution of 1 in 50 for 2 h at room temperature. After washing, the co-localization was examined by confocal laser scanning microscopy (CLSM, LSM700, Carl Zeiss, Germany).
For the co-localization of CNPs with actin, a similar procedure was conducted as described above. Briefly, the cells were cultured with Cy5-CNPs at 100 nM Cy5-let-7a for 4 h at 37 °C. The cells were then rinsed and fixed with 4% (w/w) paraformaldehyde (10 min), permeabilized in 0.5% Triton X-100 for 5 min, and incubated with 200 nM FITC phalloidine in PBS containing 1% bovine serum albumin for 30 min at room temperature.
To study the co-localization of PTX-Ns with cholera toxin subunit B (CTB), the cells were co-incubated with Alexa Fluor ® 488-CTB at 20 µg/mL and Cy5-CNPs at 100 nM Cy5-let-7a in DMEM for 3 h at 37 °C. The cells were then rinsed and observed by CLSM (LSM700, Carl Zeiss, Germany).

Cellular trafficking and time lapse fluorescence imaging
The 4T1 and Caco-2 cells were seeded on 30-mm glass-bottomed cell culture dishes at a density of 1×10 5 cells/dish and cultured for 48 h. The cells were then cultured with FITC-CNPs at 100 µg/mL FITC or Cy5-CNPs at 100 nM Cy5-let-7a in serum-free culture medium for 4 h at 37 °C, following by addition of 1 mL of Lyso-tracker red or Lyso-tracker green and incubation for 1 h at 37 °C. The cells were examined with CLSM after three rinses with PBS.
To confirm the co delivery of PNPs and let-7a to cells, cellular uptake of dual-labeled CNPs was performed using a method similar to that described above, except that the lysosomes were not marked by staining with Lyso-tracker red or green.
For time-lapse live cell imaging, 4T1 cells at a density of 1×10 5 were cultured in the dish for 48 h prior to use. After incubation with Cy5-CNPs at 100 nM Cy5-let-7a, the cells were placed in the live cell imaging chamber with 5% CO 2 at 37 °C on the CLSM (LSM700, Carl Zeiss, Germany). A series of images were obtained at 30-s intervals over a period of 30 min.

Transcytosis
The in vitro transcellular model was prepared as described in a previous report. [4] In brief, Caco-2 cells at a density of 2×10 5 cells /well were cultured on a 12-well polycarbonate Transwell filter with a pore size of 3 mm and diameter of 12 mm (Costar, Corning, NY) for 7 days. Culture medium in both apical and basolateral chambers was renewed every 2 days during the culture period. The integrity of the cell monolayer was confirmed by determining the transepithelial electrical resistance (TEER), and the cell monolayer with a TEER value greater than 500 Ω cm 2 was chosen for further analysis.

The cell monolayers on porous Transwell insert membranes were incubated with
Cy5-CNPs at 100 nM Cy5-let-7a and dual-labeled CNPs at FITC 10 µg/mL at 37 °C for a predetermined duration. Subsequently, the polycarbonate membranes covered with the cell monolayers were isolated from the Transwell insert and washed with cold PBS, fixed with 4% paraformaldehyde for 10 min, and stained to detect F-actin and nuclei with FITC-phalloidine and 4',6-diamidino-2-phenylindole (DAPI) for 30 min, respectively. Finally, the membranes were examined by CLSM (LSM700, Carl Zeiss, Germany) in X-Y-Z scanning mode after placement and sealing on glass slides.
At designated time points, the media in both apical and basolateral chambers were collected and subjected to size-distribution analysis by DLS.

Penetration into multicellular spheroids
A multicellular spheroid model was employed to determine the ability of CNPs to penetrate the tumor tissue. [3] In brief, 4T1 cells with a density of 2×10 3 cells/well were cultured in a 96-well plate for 7 days at 37 °C. Then, the spheroids were incubated with dual-labeled CNPs with 10 µg/mL FITC or 100 nM Cy5-let-7a at 37 °C. At predetermined time intervals, the location of CNPs in the tumor spheroids was observed using CLSM (LSM700, Carl Zeiss, Germany). The penetration ratio (%) was also calculated using the ratio of the penetration depth from the surface to center of the spheroid diameter.

Migration of cancer cell inhibition
The migration assay was performed on transwell insert chambers (Corning, USA).
The 4T1 cells were cultured at a density of 6 × 10 5 cells/well in 6-well plates for 48 h prior to use. The cells were incubated with Taxol, naked let-7a, CLG, PNPs and CNPs in 2 mL of culture media without serum for 4 h at 10 µg/mL PTX or 100 nM let-7a, followed by two washes with PBS and incubation in RPMI-1640 medium with 10% For the western blot assay, the treated cells were lysed in cold lysis buffer followed by centrifugation at 10, 000 g for 10 min, and then, the supernatants were collected for protein determination with the BCA protein assay kit (KeyGEN Biotech., China) and protein separation by SDS-PAGE. Subsequently, the separated proteins were transferred onto a PVDF membrane (Millipore, USA), incubated with blocking solution (5% fat-free dried milk) at room temperature for 1 h, treated with a monoclonal antibody against KRAS overnight at 4 °C, cultured with goat anti-rabbit secondary antibody for 1 h, and stained with an ECL chemiluminescence kit (KeyGEN Biotech., China). Finally, blotting images were obtained from a G: Box ChemiXR5 (Syngene, Cambridge, UK) using GAPDH as an internal reference for normalization.

Blood circulation study
The animals used in the experiments received care in compliance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals.
All the animal experiments were performed in accordance with the protocol approved by the China Pharmaceutical University Institutional Animal Care and Use Committee.
The in vivo pharmacokinetics of Taxol (free drug), PNPs and CNPs were investigated in rats. The rats were randomly divided into three groups (4 rats per group) and intravenously injected with Taxol, PNPs or CNPs, respectively, at a PTX dose of 10 mg/kg, according to the animal's body weight. At specific time intervals, 0.5-mL blood samples were collected from the orbit and then immediately centrifuged for 10 min at 5,000 g (Anke TGL-16G, Shanghai, China) to separate the plasma. The extraction of PTX from plasma and determination of the plasma concentration were performed as described in a previous report. [5] The pharmacokinetic parameters were calculated based on a statistical moment theory using the Microsoft Excel 2007 program.

Ex vivo imaging and biodistribution
The qRT-PCR and western blot, respectively. The other mice were subjected to the survival study until day 30, and the endpoint was detected as depicted in a previous report. [6] 1.

Safety study
Female BALB/c mice were randomly divided into groups (n = 6 for each group) that received an intravenous injection of saline, CLG, PEI, Taxol, naked let-7a, PEI/let-7a complexes, PNPs and CNPs via the tail vein at 1, 4, 7, 10 and 13 days. The administered doses of active or inactive ingredients in these formulations were the same as described for the study of antitumor efficacy. At 15 days post-administration, the mice were sacrificed to harvest the major tissues, following by preparation of 5-µm-thick tissue sections and H&E and CD68 immunohistochemical analysis.

Statistical analysis
One-way analysis of variance was performed to assess the statistical significance of the differences between samples. The results are expressed as the means ± standard deviation. Significant differences were set as P < 0.05.