Enzyme-Responsive Charge-Reversal Polymer Mediated Effective

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Enzyme-Responsive Charge-Reversal Polymer Mediated Effective Gene Therapy for Intraperitoneal Tumors Nasha Qiu, Jianqing Gao, Qi Liu, Jinqiang Wang, and Youqing Shen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00440 • Publication Date (Web): 08 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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Enzyme-Responsive Charge-Reversal Polymer-Mediated Effective Gene Therapy for Intraperitoneal Tumors Nasha Qiu,1,2* Jianqing Gao,1 Qi Liu,3 Jinqiang Wang,2 Youqing Shen2* 1

Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University,

Hangzhou 310058, China. 2Center for Bionanoengineering and Key Laboratory of Biomass Chemical Engineering of the Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China. 3Division of Pharmacoengineering and Molecular Pharmaceutics and Center for Nanotechnology in Drug Delivery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

E-mail: [email protected] or [email protected] Abstract:

Gene therapy has demonstrated effectiveness in many genetic diseases as evidenced by recent clinical applications. Viral vectors have been extensively tested in clinical gene-therapy trials, but nonviral vectors such as cationic polymers or lipids are much less used due to their lower gene-transfection efficiencies. However, the advantages of nonviral vectors, such as easily tailored structures, nonimmunogenetics, and relatively low cost, still drive great efforts to improve their transfection efficiencies. A reverse question asks if nonviral vectors with


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current gene transfection efficiency can find application niches. Herein, we synthesized a cationic

polymer,

poly{N-[2-(acryloyloxy)ethyl]-N-[p-acetyloxyphenyl]-N,N-diethylammonium

chloride}

(PQDEA) as a gene-delivery carrier and compared it side by side with chemotherapy drugs for cancer treatment. PQDEA is rapidly hydrolyzed by intracellular esterases into anionic poly(acrylic acid) to give low cytotoxicity and fast release of DNA for expression. PQDEA formed stable complexes with DNA (PQDEA/DNA polyplexes), which were further coated with a lipid layer to make serum-stable lipidic polyplexes, LPQDEA/DNAs, for in vivo use. In an intraperitoneal tumor xenograft model mimicking late-stage metastatic cervical cancer, the LPQDEA/DNA vector with TRAIL suicide gene exerted strong tumor inhibition as effective as paclitaxel, the first-line anticancer drug, but gave much less tumor relapse and much longer survival than the clinical chemotherapy drugs, irinotecan and paclitaxel. Equally important, the gene therapy showed much fewer adverse effects than the chemotherapy drugs. This work shows that nonviral vectors with current transfection efficiencies may produce therapeutic advantages and may be safe and worthy of clinical translation in, for example, intraperitoneal cancer therapy.

Keywords: Cancer gene therapy; Esterase-responsive; Intraperitoneal tumor; Charge-reversal polymer



INTRODUCTION Gene therapy has proven very effective against various genetic diseases. In particular, the recent first-ever FDA approval of Luxturna by Spark Therapeutics for treatment of hereditary retinal disease reactivates the widespread interest in the technology.1-3 Gene therapy is avidly sought for cancer treatment because it has fewer side effects than most anticancer chemotherapeutic drugs2, 4. However, the availability of safe and effective gene-delivery vectors has been the bottleneck to its wide clinical applications.5 Viral gene vectors such as adenovirus, adeno-associated virus, and lentivirus are characteristic in their high gene transfection efficiencies6 and thus have been the main players in gene therapy clinical trials, but are concerned with high cost (e.g., Luxturna) and safety issues1, 7. Nonviral gene vectors including synthetic polymers and liposomes are readily available and can be tailor-made nonimmunogenic and safe. However, they are much less efficient than viral vectors8-10. Recently, vectors for siRNA delivery have made great advances4, 11-15. For instance, the combinatorial library approach has screened out many structures with superior capability for RNA delivery16-20 and codelivery21. One example is lipopeptides shown as very potent siRNA carriers with efficient silencing effects in mice (ED50 ∼ 0.002 mg/kg), rats (ED50 < 0.01 mg/kg), and nonhuman primates (over 95% silencing at 0.3 mg/kg) 22. However, for DNA-based gene delivery, given great efforts on increasing nonviral vectors’ DNA transfection efficiency,23-25 optimizing carrier structures,26-32 and studying the underlying mechanisms33, 34, the transfection efficiencies of the systems are still similar to or slightly better than that of the gold standard, 25 KDa polyethyleneimine (PEI 25 KDa).


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While most efforts on nonviral DNA vectors should remain focused on increasing DNA-transcription efficiency, an alternative is to ask whether a nonviral system must have a viral-vector-equivalent transfection efficiency to achieve sufficient therapeutic advantages to merit clinical translation. A clear answer to this question would be critical and directional for this area as it indicates if we should direct some efforts to clinical translation of current nonviral DNA-delivery systems. Some nonviral DNA-delivery systems have shown impressive in vivo activity and therapeutic effects alone.35, 36

However, we lack side-by-side

comparison with chemotherapy to draw this conclusion.

Scheme 1. Illustration of esterase-responsive charge-reversal polymer PQDEA and its lipid-coated esterase-responsive polyplexes (LPQDEA/DNA) with TRAIL plasmid



(pTRAIL) for intraperitoneal (i.p.) cancer-gene therapy. a) PQDEA is a cationic polyacrylate composed of p-acetyloxybenzyl ammonium chloride. The hydrolysis of the acetyloxybenzyl ester triggers elimination of p-hydroxymethylphenol and consequent conversion of the polymer from cationic to anionic polyacrylic acid. b) PQDEA complexes with DNA into the PQDEA/DNA polyplexes, which are then coated with DC-Chol/DOPE lipids, forming lipidic polyplexes (LPQDEA/DNA). 1) The tumor cells internalize the LPQDEA/DNA into the cytosol, whose esterases trigger the PQDEA charge reversal to release the DNA plasmids. The delivered TRAIL plasmids (pTRAIL) effectively produce TRAIL inducing cancer cell apoptosis. 2) The intraperitoneally injected LPQDEA/pTRAIL transfect abdominal disseminated HeLa tumors in nude mice, effectively suppress tumor growth, and prolong survival compared with clinical anticancer drugs irinotecan and paclitaxel. Herein, we show a side-by-side comparison of the potency of a cancer suicide-gene therapy system with first-line clinical drugs, paclitaxel (PTX) and irinotecan (CPT11) (Scheme 1). The cationic polymer contains quaternary amines with p-acetyloxybenzyl groups, which are easily hydrolyzed by esterase to trigger an elimination reaction converting the polymer to tertiary amine-based PDEA. PDEA is unstable and undergoes self-catalyzed hydrolysis into polyacrylic acid, realizing a cationic to anionic charge reversal. Thus, the cationic polymer condenses DNA into stable polymer/DNA complexes (polyplexes), but once in cancer cells rich in esterases, the polyplexes quickly dissociate and release the carried DNA for efficient transcription. So, polyplexes carrying a cancer suicide gene, tumor necrosis factor (TNF)-related apoptosis-inducing ligand gene (pTRAIL), efficiently kill cancer cells.


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I.p. tumors were used as the animal model because they are common metastasis disseminations of advanced cancers very resistant to systemic chemotherapy.37 Thus, their treatment remains a pressing unmet medical need. Furthermore, i.p. administration of treatments can bypass the complicated in vivo delivery barriers and produce high drug concentrations surrounding the tumors,38-40 leading to improved efficacy.41 Therefore, i.p. administration is now recommended by the National Cancer Institute (NCI) for treatments of peritoneal tumors.42 This character makes i.p. gene therapy a very promising treatment strategy.41,43,44 Furthermore, given the fact that even viral vectors demonstrated their effectiveness in local or ex vivo gene therapy,1, 45 nonviral vectors may first find their niche in clinical applications in local i.p. gene therapy. MATERIALS AND METHODS Materials 2-(N,N-Diethylamino)ethyl acrylate (DEA), branched polyethylenimine with the molecular weight

of

25

KDa

(PEI

25

KDa),

4-(chloromethyl)phenyl

acetate

and

3β-N-(dimethylaminoethyl)-carbamate hydrochloride (DC-Chol) were purchased from Sigma-Aldrich (Shanghai, China). Azodiisobutyronitrile (AIBN) was purchased from Aladdin (Shanghai, China). 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and Lipofectamine 2000 were purchased from Avanti Polar Lipids Company (Alabaster, AL, USA). pGL4.13 Luciferase plasmid and luciferase assay system were purchased from Promega (Madison, WI, USA). EGFP and TRAIL plasmids were kindly provided by Zhejiang University School of Medicine and Shanghai Institute of Materia Medica, Chinese Academy of Sciences. The plasmids were propagated in Escherichia coli DH5α and extracted using Endo-Free Plasmid



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Kit (Qiagen, Hilden, Germany). LabelIT® Nucleic Acid Labeling Kit Cy5TM was purchased from Mirus Bio (Madison, WI, USA). LysoTracker Green and Hoechst 33342 were purchased from Invitrogen (Carlsbad, CA, USA). Cell culture medium RPMI 1640, fetal bovine serum, and Trypsin were purchased from GibcoTM (Thermo Fisher Scientific, Shanghai, China). Polymer Synthesis 2-(N,N-Diethylamino)ethyl

acrylate

(DEA)

was

polymerized

to

prepare

poly[2-(N,N-Diethylamino)ethyl acrylate] (PDEA) with molecular weight of 5 KDa, 9 KDa or 14 KDa as we have reported46. PDEA (0.2 g, 1.2 mmol tertiary amines) was reacted with p-(chloromethyl)phenyl acetate (0.34 g, 1.8 mmol) in 1 mL DMF at 60 °C for 24 h. The polymer was then precipitated in THF three times and then dried in vacuum, giving a white powder, PQDEA, at an 85% yield. 1H-NMR (400 MHz, CDCl3, δ): 7.70, 7.12 (4H, phenyl), 4.5-5.25(4H, COOCH2, NCH2-phenyl), 3.0-4.0 (6H, NCH2), 2,29 (3H, O=CCH3), 1.0-1.9 (9H, CHCH2, NCH2CH3). Polyplex preparation PQDEA was dissolved in DMSO to 50 mg/mL as a stock solution. The stock solution (50 µL) was diluted with pH7.4 HEPES buffer solution (10 mM, pH 7.4) to the needed concentrations according to the designated N/P ratios defined as the molar ratio of the nitrogen atoms in the polymer to the phosphate atoms of the plasmid DNA. The DNA plasmid solution was mixed with the polymer solution, vortexed for 10 seconds, and then incubated for 30 min at room temperature to prepare the polyplex solutions.


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Sizes and zeta potentials of polyplexes in HEPES buffer were measured at 25 °C using Zetasizer Nano-ZS (Malvern Instruments, UK). Data were presented as the means ± SD (n = 3). Each measurement was repeated at least three times. Esterase responsiveness and charge reversal of PQDEA PQDEA solution in DMSO (20 µL, 50 mg/mL) was added to 100 µL porcine liver esterase solution (120 units/mL, 100 mM NaCl, 100 mM Tris buffer) and the mixture was incubated at 37 °C. At timed intervals, 1 mL acetonitrile was added to the solution to deactivate the esterase; the mixture was then centrifuged at 6,000 rpm for 5 min. The supernatant was filtered through a 220 nm PTFE syringe filter and 20 µL of the filtrate was subjected to HPLC analysis (Waters, 1525 binary HPLC Pump, 2475 multi-λ-Fluorescence Detector, 2998 Photodiode Array Detector, SunFireTM C18 (4.6×250 mm, 5 µm column) to monitor the 4-hydroxybenzyl alcohol (HBA) release using a water/methanol gradient elution at a flow rate of 0.8 mL/min. Pure HMP was used as a standard. Cell culture and gene transfection HeLa (human cervix carcinoma), A549 (human lung carcinoma), SW480 (human colorectal adenocarcinoma) and SKOV3 (human ovarian carcinoma) were purchased from the American Type Culture Collection and cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Gibco), 100 units/mL penicillin and 100 units/mL streptomycin. Cells were cultured in a humidified atmosphere with 5% CO2 at 37 °C. Plasmid DNA encoding luciferase (pLUCI, Promega), enhanced green fluorescence protein (EGFP; pEGFP) or tumor-related apoptosis-inducing ligand (TRAIL; pTRAIL) was amplified in LB culture medium and extracted using Qiagen plasmid extraction kits. Quantity and



quality of the extracted plasmids were determined by the OD260/OD280 value. The plasmids were diluted with HEPES buffer to 40 µg/mL (for in vitro transfection) or 100 µg/mL (for in vivo transfection). For luciferase transfection, cells were seeded in 48-well plates at a density of 3.0×104 cells per well with 0.4 mL of 10% FBS-containing cell-culture medium and incubated overnight. The medium was then replaced with 0.4 mL fresh medium containing 0%, 10%, or 100% FBS. Polyplex solutions (50 µL) were then added to each well to reach a DNA concentration of 2.5 µg/mL and incubated for 4 h. Then medium was replaced with 0.4 mL of fresh RPMI1640 medium supplemented with 10% FBS. The cells were incubated for an additional 44 h. The medium was removed and the cells were rinsed with PBS. Cell lysis buffer (100

µL) was added to each well and incubated for 3 min on ice and then 20 µL of the cell lysate was mixed with 5 µL of luciferin for chemiluminescence measurement according to the manufacture’s protocol (Promega). The protein content of each sample was determined by Bradford protein assay kits (Sangon Biotech, Shanghai). Luciferase expression was defined as relative luciferase light units per milligram protein (RLU/mg). Data were presented as means ± SD (n = 3) and each measurement was performed in triplicate and repeated at least twice. Transfection in medium containing 0, 25, 50, and 100 µM chloroquine was also conducted to evaluate the effect of lysosomal trapping on the transfection of PQDEA polyplexes. For the EGFP transfection, cells were seeded in six-well plates at a density of 1.5×105 cells per well in 2 mL of 10% FBS-containing cell-culture medium and incubated overnight. The medium was then replaced with 2 mL of fresh medium containing 0%, 10%, or 100% FBS. Polyplex solutions were added to the wells to reach a DNA concentration of 6 µg/mL and


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incubated for 4 h. The medium was replaced with 2 mL of fresh RPMI-1640 medium supplemented with 10% FBS. The cells were incubated for another 44 h. The medium was then removed and the cells were rinsed with PBS and detached with Trypsin. The cells were collected, washed twice with PBS and resuspended in 1 mL of PBS. The expression efficiency of the EGFP polyplexes was quantitatively measured by flow cytometry (BD FACS Calibur TM, 10,000 cells counted per treatment) in terms of the percentage of GFP-positive cells. For imaging EGFP expression, cells were seeded on glass-bottom petri dishes at a density of 150,000 cells per dish in 1.5 mL of 10% FBS-containing cell-culture medium and transfected similarly. The images were acquired using a confocal laser-scanning microscope (Nikon-A1 system, Japan). The imaging parameters were kept constant for different groups. For in vitro TRAIL transfection, the procedures were the same as for EGFP transfection except that the pTRAIL concentration was 1 µg/mL in each well. The cells were imaged at 12 h and 24 h posttransfection and each sample was lysed for Western blot analysis of TRAIL expression. Cellular uptake inhibition HeLa cells were seeded in six-well culture plates at a density of 2×105 cells per well and incubated for 24 h. Inhibitors (wortmannin, 5 µM; cytochalasin D, 5 µM; chlorpromazine, 50 µM) were separately added to the cell-culture medium at the indicated concentrations and incubated with cells for 0.5 h. The cells without any inhibitors served as the control. Luciferase plasmid DNA was labeled with Cy5 (Mirus Bio) (Cy5pLUCI) according to the manufacturer’s instructions. PQDEA/Cy5pLUCI (0.3 µg) was added to each well and



incubated for another 2 h. The medium was then removed and the cells were rinsed with PBS, detached with Trypsin and collected. The cells were resuspended in PBS, washed twice, and subjected to flow cytometry for analysis of Cy5-positive cells (1×104 cells counted). Data were presented as means ± SD (n = 3) and each measurement was performed in triplicate and repeated at least twice. Subcellular distribution HeLa cells were plated onto glass-bottom petri dishes at 105 cells per dish in 1.5 mL of 10% FBS-containing cell culture medium and incubated for 24 h before use. The medium was replaced with 1.5 mL of fresh medium (containing 0% or 10% FBS). Polyplexes (50 µL) of the Cy5pLUCI were then added at a dose of 0.5 µg DNA per dish. After timed incubation, the medium was replaced with fresh medium. The lysosomes were labeled with LysoTracker Green at a concentration of 200 nM 0.5 h prior to imaging; the nuclei were stained with two drops of Hoechst 33342 (Molecular Probes, Carlsbad, CA) per mL medium at 15 min prior to imaging. The cells were washed three times with PBS before imaging with a confocal laser scanning microscope (Nikon A1). Fabrication of the lipid-coated PQDEA/DNA polyplexes (LPQDEA/DNA) The polyplexes at an optimized N/P ratio of 15 were prepared as indicated above. DOPE (22.32 mg, 0.03 mM) and DC-Chol (5.37 mg, 0.01 mM) were dissolved in 1 mL of chloroform with stirring and then evaporated to obtain a thin lipid film. HEPES buffer was added to make the final DOPE concentration at 1 mg/mL (for in vitro transfection) or 5 mg/mL (for in vivo transfection). The obtained lipid solution was stirred overnight at room temperature and sonicated for 5 min before use. The PQDEA/DNA polyplex solution (N/P =


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15) was then added dropwise to the lipid solution at a molar ratio of PQDEA repeating units/total lipids at 3:4. The lipid/PQDEA/DNA (LPQDEA/DNA) solution was stirred for 4 h at room temperature before characterization and use.

In vivo luciferase and TRAIL gene transfection, bioluminescence imaging, and tissue luciferase and TRAIL expression All animal studies were approved by the Animal Care and Use Committee of Zhejiang University in accordance with the guidelines for the care and use of laboratory animals. Female BALB/c athymic mice (6–8 weeks old) were purchased from the Animal Center of Zhejiang University and maintained under the standard conditions. The animals were housed in sterile cages within laminar airflow hoods in a pathogen-free room with a 12-h light/12-h dark schedule and fed autoclaved chow and water ad libitum. The HeLa i.p. tumor model was established via i.p. inoculation of 4 × 106 HeLa cells in 0.2 mL of PBS into the right flank belly cavities of the nude mice. For luciferase and TRAIL gene transfection, the mice were grouped randomly (n = 5) six days after the i.p. tumor inoculation. The LPQDEA/pLUCI or LPQDEA/pTRAIL solution (0.35 mL) at a dose of 0.5 mg DNA/kg was injected into the intraperitoneal cavity. PEI 25K/pLUCI and Lipo2000/pLUCI polyplexes served as positive controls. After 48 h, the mice were i.p injected with D-luciferin sodium salt (Gold Biotechnology, St. Louis, MO, USA) at 150 mg/kg body weight in 0.2 mL of PBS. After 10 minutes, the mice were imaged under anesthesia with 2.5% isofluorane using a Xenogen IVIS Lumina system (Caliper Life Sciences, Waltham, MA, USA; 30 s exposure per image). Acquired images were obtained by



superimposing the emitted light over the grayscale photographs of the animals. Quantitative analysis was performed using the Lumina II Living Image 4.2 software. For tissue luciferase expression, the mice were sacrificed 48 h after transfection and the tumor tissues were dissected and lysed in lysis buffer (Promega). Protein concentrations of the lysates were determined with Bradford protein assay kits (Sangon Biotech). The tissue lysis sample (20 µl) was added with 5 µL of the luciferase substrate (Promega) and its relative luciferase light unit (RLU) was measured by a luminometer (Birthold, Bad Wildbad, Germany). The value of relative luciferase light units per milligram protein was obtained by the measured RLU value divided by the protein concentration. All data are presented as the means of at least five independent measurements. For tumor TRAIL expression, tumor samples were collected three days, six days, and nine days posttransfection and fixed in 4% neutral-buffered paraformaldehyde and embedded in paraffin for further analysis of TRAIL expression and TdT-mediated dUTP nick end labeling (TUNEL) assay for apoptosis measurement, with PEI 25K/pTRAIL as a positive control. Western blot analysis of TRAIL expression in vitro HeLa cells were seeded in six-well plates at a density of 2×105 cells per well in 2 mL of cell-culture medium and incubated overnight. The LPQDEA/pTRAIL solution was then added to the medium at a DNA concentration of 1.0 µg/mL. After 4 h, the medium was replaced with fresh medium containing 10% FBS and the cells were further cultured for another 8 or 20 h. The cells were then rinsed with cold PBS, lysed in 200 µL of RIPA lysis buffer (Beyotime, Jiangsu, China) per well for 30 min on ice, and centrifuged at 12,000 rpm for 5 min. Protein contents were measured using the BCA protein assay (Beyotime). For each


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sample, 40 µg of total proteins were loaded and separated on 10% sodium dodecyl sulfate polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA). The membrane was incubated first with 5% BSA in tris-buffered saline with Tween-20 (TBST) to block nonspecific binding sites and then incubated with primary antibodies (anti-TRAIL 1:1,000, Cell Signaling Technology, Danvers, MA, USA). The membrane was rinsed in TBST and incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (Beyotime) at a 1:1,000 dilution. Finally, the membrane was rinsed and visualized with an electrochemiluminescence-detection reagent (Beyotime). β-Actin was used as internal reference. The grayscale images of TRAIL and β-Actin bands were quantified using ImageJ 1.47v (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).

In vivo anticancer experiments An abdominally disseminated HeLa tumor model was established via i.p. inoculation of 4 × 106 HeLa cells into the right flank belly cavities of nude mice as described above. For Tumor Dissection Study 1, the mice were grouped randomly six days after inoculation and the treatments were initiated as follows (a) PBS control, (b) PEI 25K/pTRAIL at a TRAIL dose of 0.5 mg/kg, (c) LPQDEA/pTRAIL at a TRAIL dose of 0.5 mg/kg, (d) CPT11 at a dose of 10 mg/kg, and (e) PTX at a dose of 10 mg/kg. The treatments were administered as four i.p. injections, once every three days. Three days after the fourth injection, the animals were sacrificed according to institutional guidelines and the tumors in the peritoneal cavity and other organs were carefully examined and dissected. Tumors in each mouse were weighted and the tumor number was counted.



For Dissection Study 2, the groups of mice were treated as in Dissection Study 1 except that after the fourth treatment, the mice were maintained without further treatment for another 15 days. The mice were then sacrificed and tumor nodules were dissected as described above. For long-term survival evaluation, groups of mice were treated as described in Dissection Study 1. After the fourth treatment, the mice were maintained without additional treatments and observed carefully. Mouse body weights were measured every day for body condition scores in accordance with standard rodent tumor-monitoring guidelines. The animal with severe weight loss, signs of dullness or closing eyes, or abdominal distention as evidence for accumulation of hemorrhagic ascites or large internal tumors were defined as the endpoint and were euthanized. Histology and immunohistochemical studies Tumor tissue and organ specimens were fixed in 4% neutral-buffered paraformaldehyde and embedded in paraffin. Tumor sections of 5 µm thickness were mounted on glass slides and stained with haematoxylin/eosin (H&E, Beyotime), or MASSON Trichrome Stain Kit (Abcam, Cambridge, England) for immunohistochemical studies. For immunohistochemistry, the sections were deparaffinized, rehydrated, and subjected to epitope retrieval and stained with primary antibodies for 24 h at 4 °C. They were then reacted with HRP-conjugated secondary antibody for 1 h, and diaminobenzidine (DAB) was used as the substrate to produce an observable brown color. Three primary antibodies were used: rabbit anti-TRAIL (Cell Signaling Technology), Caspase-3 (Beyotime), and α-SMA (Santa Cruz Biotechnology, Dallas, TX, USA). Apoptotic events were determined by the TUNEL assay. Sections were subjected to TUNEL staining using an in situ Cell Death Detections Kit, POD, according to


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the manufacturer’s protocol (Roche, Mannheim, Germany). Apoptotic cells were identiﬁed by positive TUNEL staining. Negative controls were carried out by omitting the primary antibodies and substituting the primary antibody with antibody diluent. Sections were examined by light microscopy (BX51, OLYMPUS, Tokyo, Japan). Serum chemistry ICR mice (6~8 weeks, average body weight: 20 g) were randomly divided into five groups (12 mice in each group; the blood of four mice was collected as one sample for analysis) and i.p. injected with PBS, LPQDEA/pTRAIL at a DNA dose of 0.5 mg/kg, CPT11 at a dose of 10 mg/kg or PTX at a dose of 10 mg/kg every two days for two cycles, respectively. Serum samples were collected for serum chemistry analysis. Serum alanine transaminase (ALT), aspartate transaminase (AST), creatine kinase (CK), creatine kinase mb isoenzyme (CKMB), lactate dehydrogenase (LDH), blood urea nitrogen (UREA), creatinine (CREA), total bilirubin (TBIL), albumin (ALB) and C-reaction (CRP) in each serum sample were measured using the Johnson & Johnson Vitros® 5600 Integrated System (Ortho-Clinical Diagnostics, Raritan, NJ, USA). Data were presented as the means ± SD (n = 3). Statistical analysis Experiments were repeated at least three times and each measurement was performed in triplicate. Data were presented as the means ± S.D. Assignments and selections of microscopic inspection fields were made at random. The two-tailed, unpaired Student's t test served for individual comparisons. P < 0.05 was regarded as statistically significant. 3. RESULTS AND DISCUSSION



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Design and synthesis of charge-reversal PQDEA, and characterizations and luciferase transfection of polyplexes This esterase-responsive polymer poly{N-[2-(acryloyloxy)ethyl]-N-[p-acetyloxyphenyl] -diethylammonium

chloride}

(PQDEA)

was

prepared

by

quaternarization

of

poly[2-(N,N-diethylamino)ethyl acrylate] (PDEA) with 4-(chloromethyl)phenyl acetate. The reaction was straightforward to complete in DMF, but not in such low-polar solvents as THF because the quaternarized polymer would precipitate in these solvents. The PQDEA molecular weight was controlled by its precursor PDEA46 and three samples of PDEA with molecular weights of 5 KDa, 9 KDa and 14 KDa were prepared. The PQDEA structure was confirmed by NMR spectra. In HEPES buffer, PQDEA had a zeta potential of about +18 mV. The hydrolysis of PQDEA phenolic acetate triggered the elimination of p-quinone methide, which quickly converted into 4-hydroxybenzyl alcohol (HBA) in water, and turned the quaternary amine into a tertiary amine, which subsequently catalyzed PDEA hydrolysis into poly(acrylic acid)46 (Scheme 1 a). This esterase-catalyzed hydrolysis was tracked by the HBA release of PQDEA incubated with 100 units/mL esterase (Figure 1a). After only 15 min incubation, the HBA signal appeared in the HPLC trace and the esters were completely hydrolysed within an hour. Correspondingly, the PQDEA zeta potential in pH 7.4 HEPES buffer shifted from + 18 mV to about -12 mV within

an

hour

(Figure

p-acetyloxyphenylammonium-based

1b).

These

PQDEA

results

validated

undergoes

an

positive-to-negative charge reversal.


our

design:

esterase-triggered

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We then characterized the complexation of PQDEA with DNA to form PQDEA/DNA polyplexes (Figure 1c, d). Due to the strong positive charges of quaternarized amines, PQDEA efficiently condensed negatively charged DNA, forming uniform and stable nanoparticles. The polyplex sizes were around 40 nm at N/P ratios greater than 14 (Figure 1c, d). Such small sizes are preferable for endocytosis and tumor penetration. The zeta potentials of these PQDEA polyplexes were about +10 mV to +20 mV. The polyplex nanoparticles were very stable and their sizes and zeta potentials were unchanged after a week (data not shown). Polymer molecular weight is an important factor affecting gene transfection efficiency. We thus first tested transfection efficiencies of three PQDEA samples made from PDEA with molecular weights 5 KDa, 9 KDa, and 14 KDa. Clearly, the PQDEA from the 14 KDa PDEA achieved the highest transfection at different N/P ratios, particularly at the N/P ratio of 15. (Figure 1e), and thus this PQDEA was used in the following studies.

Figure 1. PDEA charge-reversal (a, b), polyplex formation (c, d), and luciferase transfection assay (e). (a) HPLC traces of 4-hydroxybenzyl alcohol (HBA) in PQDEA solution incubated with porcine liver esterase for different times. HBA was used as the



standard. Polymer concentration: 30 mg/mL; porcine liver esterase (100 units/mL, 100 mM NaCl, 100 mM Tris buffer). (b) Hydrolysis and charge-reversal of PQDEA at room temperature (5 mg/mL polymer; 100 units/mL porcine liver esterase, 100 mM NaCl, 100 mM Tris buffer). (c) Sizes and zeta potentials of PQDEA/pLUCI polyplexes formed at different N/P ratios in pH7.4 HEPES buffer and (d) dynamic laser scattering pattern of the polyplexes at N/P = 15; The PQDEA was made from PDEA with molecular weight of 14 KDa. (e) Luciferase expression of HeLa cells transfected with pLUCI polyplexes at different N/P ratios of PQDEA made from PDEA with the molecular weight of 5 KDa, 9 KDa, and 14 KDa. Four hour transfection in serum-free medium followed by 44 h incubation in fresh culture medium containing 10% FBS; pLUCI vector concentration, 2.5 µg/ mL. The luciferase gene expression is presented as relative luciferase light unit (RLU) per protein milligram. Error bars represent the SD (n = 3).

Polyplex internalization and intracellular trafficking Generally, polyplex nanoparticles are endocytosed and mostly further trafficked into lysosomes, causing DNA degradation

47

. We tested the effects of three different

cellular-uptake inhibitors on the internalization of PQDEA/pLUCI polyplexes (Figure 2a). Cytochalasin D and wortmannin had no obvious inhibitory effect, and chlorpromazine showed some. Chlorpromazine is an inhibitor of clathrin-mediated endocytosis, while wortmannin, an inhibitor of PIK3 (phosphoinositide 3-kinase)48 and cytochalasin D, an inhibitor of actin polymerization49, are both macropinocytosis-related inhibitors. These results may suggest that the cellular uptake of PQDEA/pLUCI by the tumor cells was via the clathrin-related pathway.


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Confocal microscopy further imaged intracellular trafficking of the PQDEA/Cy5pLUCI polyplexes whose plasmids were labeled with Cy5 (Figure 2b). Compared with PEI 25K/Cy5pLUCI polyplexes, the PQDEA/Cy5pLUCI polyplexes entered cells very quickly; in as little as 0.5 h most cells already had polyplexes (red signals) associated with their membranes. After 1 h, almost all cells took up PQDEA/Cy5pLUCI, while only a small portion of the cells incubated with PEI 25K/Cy5pLUCI had

Cy5

DNA signals (Figure 2b). Furthermore, close

observation found that some PQDEA/Cy5pLUCI polyplexes were associated with lysosomes labeled in green and appeared yellow, but many appeared red and, thus, were not associated with lysosomes. At 4 h, some plasmids were already in the nuclei appearing pink. This may suggest that PQDEA possibly had lysosome-rupture ability. This was further proven by our observation capturing the osmotic swelling and burst of some lysosomes (Figure 2c). PEI containing a large number of secondary and tertiary amines is known to have a “proton-sponge” ability once in lysosomes and can rupture them to release the contents 50. It was surprising that PQDEA, whose amines were quaternarized, had such an efficient lysosome rupture. We further validated this by adding chloroquine—known to accumulate in the acidic vesicles and buffer their pH to induce lysosomal rupture and thus enhance gene transfection of some polymers unable to escape from lysosomes51—did not improve the PQDEA/pLUCI transfection efficiency (data not shown), indicating that PQDEA itself had the ability to escape from lysosomes. As quaternary amines of PQDEA had no buffer capacity, its strong buffer capacity and lysosomal-escape ability may result from its intermediate, PDEA, whose tertiary amines induced the proton-sponge effect. Importantly, in lysosomes the resulting protonated PDEA complexed with and thus protected DNA; once released in the



cytosol (pH 7.4) the PDEA quickly deprotonated and hydrolyzed to poly(acrylic acid), realizing charge reversal and rapid DNA release.

Figure 2. Cellular uptake and intracellular trafficking of PQDEA/pLUCI polyplexes. (a) Flow cytometry measurement of the cellular uptake of PQDEA/Cy5pLUCI polyplexes in the absence or presence of cellular uptake inhibitors. (b) Cellular uptake, lysosome escape and nuclear entry of PQDEA/pLUCI polyplexes compared with PEI 25K/pLUCI polyplexes in HeLa cells after incubation for 0.5 h, 1 h, 2 h, and 4 h observed by confocal microscopy. (c)


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Osmotic swelling and lysosome rupture in a HeLa cell cultured 2 h with PQDEA/Cy5pLUCI or PEI 25K/pLUCI polyplexes captured by confocal microscopy; similar phenomena were observed at other times. In the imaging, DNA was labeled with Cy5 shown in red, the cell nuclei were stained with Hoechst 33342 shown in blue and lysosomes were stained with LysoTracker Green shown in green, using overlapping images taken from the three channels. The Luciferase plasmid DNA dose was 0.35 µg/ml. All scale bars represent 20 µm.

Lipid-coated PQDEA/DNA polyplex (LPQDEA/DNA) fabrication for in vitro and in

vivo transfection PQDEA/pLUCI polyplexes were very sensitive to serum. Their transfection efficiency decreased sharply in the presence of serum because serum contains abundant esterases, which triggered PQDEA charge reversal before the polyplexes entered cells, leading to premature disassembly of the polyplexes. We, therefore, coated the PQDEA/DNA polyplexes with a DC-Chol/DOPE layer for in vivo transfection. The relatively hydrophobic structure of PQDEA rendered its polyplexes easily coated with DC-Chol/DOPE. The optimized composition of DC-Chol/DOPE/PQDEA (repeating units) was 1:3:3 in molar ratios, which formed stable and uniform lipidic PQDEA/DNA polyplexes (LPQDEAs) with a size around 90 nm and a zeta potential of 20 mV; this layered structure was further confirmed by transmission electron microscopy (Figure 3a). The luciferase expression of LPQDEA/pLUCI was essentially the same with and without FBS. Even in the FBS solution mimicking the blood serum or ascites, their luciferase expression still remained above the 107 RLU/mg protein level, while the noncoated PQDEA/pLUCI lost transfection ability once with serum (Figure 3b). We also loaded



LPQDEA with EGFP plasmids to directly observe and measure the GFP-positive cells (Figure 3c, d). In contrast to PEI 25K/pEGFP polyplexes, whose transfection efficiency decreased sharply in FBS-containing medium and only a few cells were GFP-positive, about 47% of the LPQDEA/EGFP-transfected cells were GFP positive when transfected in 10% FBS medium. This figure further increased to 63% when in 100% FBS (Figure 3c, d). This is advantageous for intraperitoneal transfection because the biological fluids (e.g., ascites) may favor gene transfection. Furthermore, efficient transfection of most cells is also important for cancer suicide gene therapy to induce apoptosis. LPQDEA/pLUCI transfection was also tested in SKOV3, SW480, and A549 cell lines. Transfection was relatively low transfection in SKOV3 cells, but LPQDEA/pLUCI performed well in SW480 cells and especially well in A549 cells; the luciferase expression was above 108 RLU/ mg protein in 10% FBS medium.


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Figure 3. Characterization of lipid-coated PQDEA/DNA polyplexes (LPQDEA/DNA) and their cell transfection. (a) Size distribution determined by DLS and the TEM image (inserted, bar, 100 nm) of LPQDEA/pLUCI made from the polyplexes (N/P = 15) shown in Fig. 1d. Lipid/polymer composition: DC-Chol/DOPE/PQDEA (repeating units) = 1:3:3 (molar). (b) Luciferase expression of LPQDEA/pLUCI compared with PEI 25K polyplexes in the absence and presence of serum; 4 h transfection followed by 44 h culture; Luciferase plasmid concentration, 2.5 µg/mL; error bars, SD (n = 3). (c) Confocal microscopic imaging and (d) flow cytometry quantitation of GFP expression of LPQDEA/pEGFP and PEI 25K/pEGFP polyplexes in HeLa cells. The cells were transfected in medium containing 10%



and 100% FBS for 4 h followed by 44 h in fresh cell-culture medium; EGFP plasmid dose, 6 µg/mL; all scale bars represent 200 µm. (e) Luciferase expression of LPQDEA/pLUCI in three other cell lines in 10% FBS-containing medium. Transfection conditions were the same as those in b. The in vivo transfection with LPQDEA/pLUCI was then evaluated using the i.p. tumor model with HeLa cells and compared with Lipo2000/pLUCI and PEI 25k/pLUCI as controls (Figure 4). The mice receiving a DNA dose of 0.5 mg/kg of LPQDEA/pLUCI all had strong chemiluminescence after transfection for 48 h, up to 107 RLU/mg protein level as quantified in homogenized tumors, while only one out of the five mice transfected by PEI 25K/pLUCI showed very weak chemiluminescence. Three of the five mice transfected by Lipo2000/pLUCI had observable luciferase chemiluminescence, but the averaged luciferase expression of the group was on hundredth that of the LPQDEA/pLUCI group. There was negligible luciferase expression in the main organs of the mice transfected with these three gene-delivery vectors (Figure 4b).


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Figure 4. Chemiluminescence imaging (a) and quantitation in main organs and HeLa i.p. tumors (b). 48 h post i.p. injection of the LPQDEA/pLUCI, PEI 25K/pLUCI or Lipo2000/pLUCI; plasmid dose, 0.5 mg/kg; Error bars, SD (n = 5).

In vitro and in vivo suicide gene transfection by LPQDEA/pTRAIL After validation of the reporter gene transfection by LPQDEA/DNA, we chose a functional gene, tumor suicide gene TRAIL plasmid (pTRAIL), to test its efficacy in cancer gene therapy. Similar to the reporter gene luciferase and EGFP expression, TRAIL expression in HeLa cells after LPQDEA/pTRAIL transfection for 24 h (Figure 5a, b) was nearly four times higher that in PEI 25K/pTRAIL-transfected cells. Accordingly, LPQDEA/pTRAIL effectively induced HeLa cell apoptosis; all cells shrank after 12 h and lysed after 24 h. For comparison, cells transfected by PEI 25K/pTRAIL still looked healthy in the same timeframe (Figure 5c). This effect was further confirmed in vivo in i.p. tumors. After administration of LPQDEA/pTRAIL for three days, the HeLa i.p. tumors heavily expressed TRAIL, with more thereafter (six days and nine days). Therefore, as shown by TUNEL staining, the tumors contained many fewer tumor cells in six or nine days, and the tumor sections became cracked and disrupted (six days) and finally developed necrosis in the center (nine days). For comparison, the tumors transfected with PEI 25K/pTRAIL had much less TRAIL expression in the same timeframe, and thus the tumors still had a comparable tumor cell density to the PBS control group (Figure 5d, e).



Figure 5. In vitro and in vivo TRAIL expression and cell apoptosis. (a) Western blot analysis and (b) quantitative calculation of TRAIL expression normalized to that of the corresponding PBS control by ImageJ software. HeLa cells (2×105) were transfected with LPQDEA/pTRAIL or PEI 25K/pTRAIL polyplexes at 1 µg/mL of TRAIL plasmid DNA in 10% FBS-containing medium for 4 h and then cultured in fresh medium for 20 h. (c) HeLa cells morphologies after transfection with LPQDEA/pTRAIL or PEI 25K/pTRAIL polyplexes at a DNA dose of 2 µg/mL for 4 h in 10% FBS-containing medium and then in fresh medium for 8 (12 h in total) or 20 h (24 h in total). Scale bar = 100 µm. (d) Immunohistological analysis of TRAIL expression and (e) TUNEL assay for measurement of apoptosis in HeLa i.p. tumor treated with LPQDEA/pTRAIL or PEI 25K/pTRAIL at a TRAIL dose of 0.5 mg/kg for three days, six days, or nine days; i.p. injection. Tissue paraffin sections were 5 µm thick.

In vivo anticancer study ACS Paragon Plus Environment

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HeLa i.p. tumors, a model mimicking feminine cervical metastatic cancer, were further used to evaluate the therapeutic efficacy of LPQDEA/pTRAIL and compared side-by-side with two clinical first-line chemotherapy drugs, paclitaxel (PTX) and irinotecan (CPT11). The treatment regime is as follows (Figure 6a): (1) Tumor suppression study (Dissection Study 1): four treatments (both chemo and gene therapy) were given in a three-day schedule initiated on the sixth day post-tumor-inoculation. The mice were sacrificed three days after the last injection and abdominal tumor nodules were dissected, counted, and weighed. (2) Tumor relapse study (Dissection Study 2): Another group of tumor-bearing mice received the same treatments as in (1), but they were maintained without any treatment after the first four for 15 days and then sacrificed to analyze their tumor burdens. (3) 90-day survival for prognosis evaluation: Another group of the mice received the same treatments as in (1) and were then maintained without any treatment for 90 days (Figure 6). Dissection Study 1 illustrates the immediate anticancer activities of the treatments. After four treatments, the average tumor weights in the mice treated with LPQDEA/pTRAIL, PTX or CPT11 were all significantly lower than that of the PBS control group (p < 0.01). The tumor burdens in the LPQDEA/pTRAIL and PTX treated groups were similar, and much lower than in the CPT11 and PEI 25K/pTRAIL groups in terms of total tumor weight and number of tumor nodules per mouse (Fig 6b, c). An important phenomenon is that the averaged tumor number in the LPQDEA/pTRAIL treated mice was significantly less than that in the PTX group (p < 0.05) although the two groups had very similar total tumor weight per mouse.



Figure 6. In vivo anticancer study of LPQDEA/pTRAIL compared with PEI 25K/pTRAIL and two clinical chemotherapy drugs. (a) Treatment regime. HeLa i.p. tumor model was established via i.p. injection of 4×105 HeLa cells into each mouse abdominal cavity. Six days postinoculation, mice were divided for three experiments. Dissection Study 1: The mice (n = 5 mice) received four treatments, one every three days, either PBS control, LPQDEA/pTRAIL at TRAIL-vector dose of 0.5 mg/kg, PEI 25K/pTRAIL at TRAIL vector dose of 0.5 mg/kg, CPT11 at 10 mg/kg, or PTX at 10 mg/kg. Three days after the last shots, the mice were sacrificed and tumor nodules were dissected, weighed, and counted. Dissection Study 2: Groups of mice (n = 5) were first treated the same as in Dissection Study 1 and then


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maintained for another 15 days without any treatment. Then mice were sacrificed and tumor nodules were dissected, weighed, and counted (Dissection Study 2). One mouse in the PBS group died before the end of Dissection Study 2. Survival Study: Groups of mice (n = 8 mice) were treated as in Dissection Study 1 and then the mice were maintained without any treatment until 90 days postinoculation. Mouse survival in each group was measured throughout. Mouse body condition score was evaluated every day according to the rodent tumor-monitoring guideline. The end point of each mouse was defined as severe weight loss, dullness or closing eyes, or abdominal distention as the evidence for accumulation of hemorrhagic ascites or large internal tumors. The mice were then euthanized. (b) Dissected tumor nodules in Dissection Studies 1 and 2. (c) Comparison of the averaged tumor weight and number in each group in Dissection study 1 (blue bar) and Dissection Study 2 (orange bar) (***p < 0.001; **p < 0.01; *p < 0.05). (d) Long-term 90-day survival curves mice receiving the above treatments. Tumor resistance and relapse were cancer’s main lethal modes and the targets of cancer chemotherapy, so we further tested the tumor relapse in each group after a 15-day treatment-free period (Dissection Study 2). The mice in the PBS-treated group developed ascites from around Day 20. At the end of Dissection Study 2, 30 days postinoculation, three in five of the CPT11-treated mice developed ascites. The averaged tumor burden of the group increased by six times in weight and by three times in tumor number compared to that in Dissection Study 1. Surprisingly, although the PTX-treated mice only had only a few tumors in Dissection Study 1, their tumor burden had sharply increased after the 15-day treatment-free period, 70-fold in tumor weight and fourfold in tumor number (Dissection



Study 2). In contrast, in the LPQDEA/pTRAIL treated group, the averaged tumor weight increased only slightly (threefold) and the tumor number remained essentially unchanged in that timeframe. This indicates that the gene-therapy-treated tumor only grew larger and did not disseminate new tumors. Furthermore, after the LPQDEA/pTRAIL therapy, few tumor nodules were found around the cervical area, where the hormone–organ microenvironment induced tumor homing and supported tumor survival, whereas, in the chemotherapy and PBS-treated mice, tumors spread all over abdominal cavity, leading to peritoneal fluid and blood leaking to form ascites. This is consistent with our previous finding that the esterase-responsive gene therapy selectively induced cancer cell death without damaging tumor fibroblasts, which consequently were not stimulated to express WNT16B30, the signal promoting tumor cell survival and metastasis.52 In contrast, cytotoxin-based anticancer drugs can also intoxicate fibroblasts, stimulate WNT16B secretion, and promote tumor-cell survival and metastasis.52 The long-term survival of tumor-bearing mice is a direct measure of therapeutic efficacy (Figure 6d). The time of 50% mice survival (t0.5) was 34 days for the PBS-treated mice, 40 days for the CPT11-treated mice, and 60-days for the PTX-treated mice. The LPQDEA/pTRAIL-gene-therapy group had a much-prolonged survival, 62.5% of mice were still alive at the 90-day end point with sound body condition. Tumor samples were dissected, fixed in 4% paraformaldehyde, and sectioned to analyze each treatment’s mechanism (Figure 7a). HE and Masson staining together with α-SMA immunology showed that the untreated tumor (PBS control) was characterized by tumor cells associated with abundant collagen and fibroblasts. Chemotherapy-treated tumors had much


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less stroma but higher tumor-cell density, especially the PTX-treated tumors. The LPQDEA/pTRAIL-treated tumors, however, showed lower tumor density with large acellular cracks and fragmentation. Both the Caspase-3 and the TUNEL assays showed all treatments induced tumor-cell apoptosis, but LPQDEA/pTRAIL showed much more intense Caspase-3 and induced more tumor-cell apoptosis. As an Apo2 ligand, TRAIL binds to death receptors and forms death-inducing signaling complex (DISC), activating the Caspase-3 dependent apoptosis pathway53. As normal cells have TRAIL decoy receptors, which can bind with TRAIL without initiating the subsequent apoptotic cascade, the TRAIL suicide gene delivery may selectively kill cancer cells with few side effects54. Furthermore, complete in vivo PQDEA hydrolysis produces inert and water-soluble poly(acrylic acid), whose molecular weight was designed to be much lower than the renal filtration threshold (~40 KDa) 55. Thus, these end products can be completely excreted from the body. The effects of the LPQDEA/pTRAIL gene therapy and chemotherapy treatments on the mice were first checked by monitoring the body weights of the mice in the survival experiment (Figure 7b). There was no obvious weight loss after any of the therapies, but the PBS group at 27 days, CPT11 group at 39 days and PTX group at 63 days had a sharp increase in body weight as the mice started to develop ascites, but the body weight of the gene therapy group increased slowly as expected in normal mice during this period. The CPT11 and PTX treatments also caused abnormal spleen hypertrophy and a significant increase in spleen weight compared with the control, but the LPQDEA/pTRAIL-treated mice had normal spleens (Figure 7c) and serum-chemistry analysis showed no significant differences (Figure 7d).



Figure 7. (a) Histological and immunohistochemical analysis of HeLa i.p. tumors after 4 treatments. Tissue paraffin sections were 5 µm thick. (b) Body-weight profiles of mice in the survival experiment; n = 8 in each group. (c) Spleens weights of HeLa i.p. tumor-bearing mice after four treatments. (d) Serum biochemistry analysis of the mouse blood after treatments. ICR mice (6~8 weeks, average body weight of 20 g) were randomly grouped (n = 12) and PBS, LPQDEA/pTRAIL (DNA dose, 0.5 mg/kg), CPT11 (10 mg/kg), or PTX (10 mg/kg) was i.p. injected every two days for two treatments. Whole-blood samples were collected 24 h after the last injection for serum chemistry analysis of serum alanine


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transaminase (ALT), aspartate transaminase (AST), creatine kinase (CK), creatine kinase mb isoenzyme (CKMB) and lactate dehydrogenase (LDH). All data are presented as mean ± SD.

4. CONCLUSIONS We report an esterase-responsive cationic polymer, PQDEA, for intraperitoneal gene therapy compared with chemotherapeutic drugs. PQDEA can be hydrolyzed by intracellular esterases and turn into negatively charged poly(acrylic acid). Thus, in the cytosol its polyplexes can efficiently dissociate and release free DNA for transcription. Particularly, the lipidic-PQDEA/DNA polyplexes, LPQDEA/DNA, can resist serum and other biological fluids and achieve efficient gene delivery, and so do in i.p. tumors even with ascites. The vector delivers a suicide gene, pTRAIL, and effectively induces tumor-cell apoptosis. In the HeLa i.p. tumor model, LPQDEA/pTRAIL treatment effectively suppresses tumor growth, and particularly has minimal tumor relapse and much-prolonged survival during the treatment-free period compared with PEI 25K/pTRAIL and two first-line anticancer drugs, CPT11 and PTX. Furthermore, the gene therapy shows much-reduced adverse effects. This work shows that, with current gene-delivery efficiency, nonviral gene vectors may find their niche in some local gene-therapy applications.

Acknowledgement We thank National Basic Research Program (2014CB931900), National Natural Science Foundation (21704090 and 51390481), and Fundamental Research Funds for the Central Universities 2018QNA7022.



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TOC


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Scheme 1 155x130mm (220 x 220 DPI)



Fig1 145x75mm (220 x 220 DPI)


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Fig 2 139x162mm (220 x 220 DPI)



Fig 3 225x186mm (150 x 150 DPI)


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Fig 4 143x58mm (220 x 220 DPI)



Fig 5 160x96mm (220 x 220 DPI)


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Fig 6 217x194mm (150 x 150 DPI)



Fig 7a 144x103mm (220 x 220 DPI)


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Fig 7b 137x95mm (220 x 220 DPI)


Enzyme-Responsive Charge-Reversal Polymer Mediated Effective

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