Tumor-Penetrating Peptide-Functionalized Redox-Responsive

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Tumor-penetrating peptide-functionalized redox-responsive hyperbranched poly(amido amine) delivering siRNA for lung cancer therapy Zhong Guo, Sha Li, Zonghua Liu, and Wei Xue ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00971 • Publication Date (Web): 27 Jan 2018 Downloaded from http://pubs.acs.org on January 28, 2018

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ACS Biomaterials Science & Engineering

Tumor-penetrating peptide-functionalized redox-responsive hyperbranched poly(amido amine) delivering siRNA for lung cancer therapy Zhong Guo a, Sha Li a, Zonghua Liu a, *, Wei Xue a, b, *

a

Key Laboratory of Biomaterials of Guangdong Higher Education Institutes,

Department of Biomedical Engineering, Jinan University, Guangzhou, 510632, China b

Institute of Life and Health Engineering, Key Laboratory of Functional Protein

Research of Guangdong Higher Education Institutes, Jinan University, Guangzhou, 510632, China

* Corresponding authors: [email protected] (Wei Xue) [email protected] (Zonghua Liu) Tel and Fax: 86-20-85223062

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Abstract: Bio-safety and the targeting ability of gene delivery systems are critical aspects for gene therapy of cancer. In this study, we report the synthesis and use of redox-responsive poly(amido amine) (PAA) with good biocompatibility and biodegradation as a gene carrier material. A tumor-specific tissue penetration peptide, internalizing-RGD (iRGD) was then conjugated to PAA with an amidation reaction. In experiments using H1299 cells, PAA-iRGD was found to have a lower cytotoxicity and higher cellular uptake efficiency, compared to PAA. An siRNA, specific to epidermal growth factor receptor (EGFR) that is over-expressed on the lung cancer cell surface and often targeted in lung cancer treatment, was designed to silence EGFR (i.e., siEGFR) for delivery by the gene carrier PAA-iRGD. EGFR gene silencing, apoptosis, anti-proliferation, and anti-tumor effects of PAA-iRGD/siEGFR were evaluated in vitro and in vivo. PAA-iRGD/siEGFR displayed a much higher gene silencing ability compared to PAA and polyethyleneimine (25 kDa), significantly inhibited the proliferation and migration of H1299 cells, and elicited significant cell apoptosis. Moreover, intravenously injected PAA-iRGD/siEGFR inhibited lung tumor growth in vivo. These results suggest that PAA-iRGD with good biocompatibility, biodegradation, and targeting ability could be a promising gene delivery system for gene therapy of cancers. Keywords: redox-responsive, poly(amido amine), iRGD peptide, anti-tumor


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1. INTRODUCTION Gene therapy is an important tumor treatment method, involving the efficient delivery of nucleic acid therapeutics.1 Among the nucleic acid therapeutics, small interfering RNA (siRNA) has been widely used in treating various diseases due to its high specificity and low non-specific toxicity.2-3 Nevertheless, siRNA cannot be used directly since it would be degraded in the complex physiological environment in vivo, and the cellular uptake of siRNA is difficult due to its negative surface charge.4 To address these issues, a number of viral and non-viral delivery systems have been used for siRNA delivery.5 Compared to the viral vectors, non-viral vectors are widely used for gene delivery, because of their good safety, low toxicity, and abundant source.6 Among various polymer-based gene delivery systems,7-8 disulfide-linked, bio-reducible hyperbranched poly(amido amine) (PAA) has gained much attention due to its excellent properties for gene delivery, such as its biodegradability, biocompatibility and redox-sensitivity.9-10 In recent years, PAA has been widely developed by several research groups for use in gene delivery,9, 11-12 and it has shown high transfection efficiency and low toxicity. The high transfection efficiency results from its branched topological structure, chemical functionality of the terminal amino groups, and the presence of bio-reducible disulfide linkages.13-14 The incorporation of disulfide-linkages in the chemical structure of PAA can provide a redox-sensitive release of nucleic acid therapeutics into the cytoplasm, which reduces the cytotoxicity and increases the transfection efficiency and gene silencing ability.15 As a result, PAA demonstrates a higher transfection efficiency and gene silencing ability, with much



less toxicity compared to the 25 kDa “gold standard” polyethyleneimine (PEI).11 Thus, PAA shows promise for use in gene delivery. The further functionalization of PAA could significantly enhance its efficacy in gene therapy. The tumor-penetrating iRGD peptide carries a tumor-specific RGD motif and a CendR motif.16 Like conventional RGD peptides, iRGD homes to the tumor tissue by binding to αv integrins (especially αvβ3), which are highly expressed on tumor vasculature and tumor cells but poorly expressed in normal tissue cells.17-18 Differing from conventional RGD peptides, the introduction of the CendR motif promotes more efficient penetration of the iRGD peptide into tumor cells.16 These features confer tumor cell-specific penetration activity to iRGD.19 In this study, we conjugated the iRGD peptide to PAA to provide a greater targeting ability for PAA-based gene delivery systems. The iRGD-conjugated PAA (PAA-iRGD) was prepared and used in the gene delivery system for lung cancer therapy. Lung cancer has become a leading cause of cancer-related deaths with over one million deaths each year.20 Epidermal growth factor receptor (EGFR), which is overexpressed in lung cancer cells, contributes to the growth of solid lung tumor.21 A knockdown of EGFR could inhibit the proliferation and migration of lung cancer cells, and promote apoptosis of lung cancer cells, ultimately reducing the growth of solid lung tumor.22 In fact, EGFR has become a key agent in gene targeting for the treatment of several cancers.23 In this study, an siRNA was designed to silence the expression of EGFR (i.e., siEGFR), and delivered by PAA-iRGD for the treatment of lung cancer. The efficacy of gene therapy with


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PAA-iRGD was investigated in detail with lung cancer cell assays in vitro and examining animal lung tumor growth in vivo. 2. MATERIALS AND METHODS 2.1. Materials 1-(2-aminoethyl)piperazine

(AEPZ),

acryloyl

chloride,

cystamine

dihydrochloride, branched PEI with average molecular weight of 25 kDa, 1-(3-dimethylaminopropyl)-3-ethylcarbodimide

hydrochloride

(EDC),

and

N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (USA). The iRGD peptide (Ac-CRGDKGPDC) was purchased from GL Biochem Ltd (Shanghai, China). OPTI-MEM medium was purchased from Invitrogen (NY, USA). siEGFR (sense

strand:

5’-GGCUGGUUAUGUCCUCAUUdTdT-3’;

3’-dTdTCCGACCAAUACAGGAGUAA-5’),

siNC,

antisense

Cy3-labeled

strand:

siNC,

and

methylated-siRNA were synthesized by RiboBio Co. Ltd (Guangzhou, China). The H1299 cell line was obtained from American Type Culture Collection (ATCC, USA). Cells were maintained in Dulbecco’s modified eagle medium (DMEM, Gibco) with 10% fetal bovine serum (FBS, Gibco), 2 mM glutamine, 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco) at 37°C in an incubator with a 5% CO2 atmosphere. 2.2. Synthesis of PAA-iRGD Cystamine dihydrochloride (1 M) was dissolved in distilled water. NaOH (10 M) and acryloyl chloride (50%, v/v, in dichloromethane) were then added dropwise alternately under vigorous stirring at 4°C. The solution was then maintained at room



temperature

for

8

h

under

gentle

stirring.

The

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resulting

precipitate

N,N’-bis(acryloyl)cystamine (BAC) was extracted three times with 10-fold volumes of dichloromethane, and washed three times with distilled water. Finally, BAC was collected by vacuum rotary evaporation and vacuum drying. PAA was synthesized by one-pot two-step Michael addition polymerization according to our previous methods.12 Briefly, BAC (2 mmol) dissolved in 10 mL of methanol was mixed with 400 mM CaCl2. The mixture was heated to 50°C, and AEPZ (1 mmol) dissolved in 200 µL of methanol was added dropwise under vigorous stirring. The mixture was then stirred at 50°C for 36 h. After that, 2 mmol AEPZ was added dropwise, and the resulting solution was maintained at 50°C for 8 h under gentle stirring. The final product PAA was dialysed and lyophilized. The iRGD (25 µmol, pH=4-5) was dissolved in distilled water, and then EDC (30 µmol, pH=4-5) and NHS (30 µmol, pH=4-5) were added, and mixed at room temperature for 1 h to activate the carboxyl groups of iRGD. After that, 100 mg PAA (pH=4-5) was added to the mixture. The conjugation reaction was maintained at room temperature for 24 h under gentle stirring. The resulting PAA-iRGD was dialysed and lyophilized. 2.3. Characterization of PAA-iRGD The chemical structures of BAC, PAA, and PAA-iRGD were characterized at room temperature by nuclear magnetic resonance spectroscopy (1H NMR) (BRUKE, AVANCEIII 300 MHz, Germany). CDCl3 was used as the solvent for BAC, and D2O was used as the solvents for PAA and PAA-iRGD. The molecular weights of PAA and PAA-iRGD were measured with gel permeation chromatography (GPC) (Malvern,


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UK). GPC measurements were conducted with a VE 1122 solvent delivery system, a model 270 DUAL detector, and a 3580 RI detector using pullulan as the standard (molecular weights from 5.9-708 kDa). A NaNO3 solution (0.02 M) containing 0.2‰ (w/v) sodium azide was used as the eluent with a flow rate of 1 mL/min. The molecular weights of PAA and PAA-iRGD were calculated according to the calibration method (curve fit 5) attached to the GPC. The hydrodynamic diameters and zeta potentials of PAA and PAA-iRGD were measured with a Zeta Potential Analyzer instrument (Malvern, UK). 2.4. Gel Electrophoresis Assay The siRNA condensing ability of PAA-iRGD was examined by agarose gel electrophoresis. PAA-iRGD/siNC polyplexes were prepared at different weight ratios (0/1, 5/1, 10/1, 20/1, 30/1, 40/1, or 50/1). In brief, 5 µL of PAA-iRGD solution in distilled water (RNase-free) at different concentrations was added to 5 µL of siNC solution (0.2 mg/mL). After incubating for 30 min at room temperature, the solution was subjected to electrophoresis using 1% agarose gel electrophoresis in 1×tris-acetate-EDTA buffer (40 mM tris-acetate, 1 mM EDTA, pH 7.5) at 150 V for 10 min. The reduction sensitivity of PAA-iRGD was evaluated by determining the release of siNC from PAA-iRGD in vitro. The polyplexes of PAA-iRGD/siNC were prepared as mentioned above. After incubation with 10 mM glutathione for 30 min, the polyplexes were subjected to electrophoresis using 1% agarose gel electrophoresis at 150 V for 10 min.



To evaluate the siRNA protection of the PAA-iRGD carrier, the polyplexes were prepared as mentioned above and incubated with 1 µL RNase A (0.1 µg/µL) at 37°C. At different time points (0, 10, 20, 40, and 60 min), 10 µL of the incubation solution was sampled and mixed with 9 µL of stop buffer (250 mM NaCl, 25 mM EDTA, 2% SDS) on ice for 15 min. The siRNA was released by incubation with 10 mM glutathione for 30 min. Finally, the released siRNA was detected with 1% agarose gel electrophoresis at 150 V for 10 min. 2.5. In vitro cytotoxicity of PAA-iRGD H1299 cells (1×104 cells/well) were seeded with complete medium on a 96-well plate and cultured overnight. The old medium was replaced with fresh media containing PAA, PAA-iRGD or PEI for 24 h. Then, 10 µL of a cell counting kit-8 (CCK-8, Dojindo, Japan) solution was added to the media. After incubation for 2 h, the OD450nm of the medium was measured using a microplate reader (Thermo Fisher Scientific, USA). Cell viability was calculated using the formula: % cell viability = (AbsTest - AbsBlank) / (AbsControl - AbsBlank) ×100%. 2.6. Cellular uptake of PAA-iRGD H1299 cells (1×105 cells/well) were seeded with complete medium on 24-well plates and cultured overnight. Some of the cells were pretreated with iRGD (20 µM) for 1h. The old media were replaced with 500 µL of PAA/siNC-Cy3 or PAA-iRGD/siNC-Cy3 polyplexes (weight ratio, 50/1) in OPTI-MEM for 6 h. Cells were washed, trypsinized, centrifuged, resuspended with 500 µL PBS, and analyzed using a BECKMAN CytoFLEX (Beckman, USA).


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2.7. In vitro gene silencing H1299 cells (2×105 cells/well) were seeded with complete medium on a 6-well plate and cultured overnight. The old medium was replaced with fresh OPTI-MEM containing PAA/siEGFR, PAA-iRGD/siEGFR or PEI/siEGFR. After 6 h of incubation, the supernatant was removed, and the cells were incubated with fresh complete medium for 48 h. The cells were then washed with pre-cooled PBS three times, and resuspended in 100 µL SDS lysis buffer supplemented with phenylmethanesulfonyl fluoride and protease inhibitor. The cell lysates were ultrasonicated (on 5 s /off 5 s). The total protein was obtained by centrifugation at 13,200 rpm for 30 min and the concentration was measured using a BCA protein assay kit. Total proteins (40 mg) were loaded onto 10% SDS-PAGE and electrophoresized at 100 V for 90 min. The proteins were then transferred to polyvinylidene fluoride membranes and blocked with 5% skimmed milk on a horizontal shaker for 1 h. The membranes were incubated with 1:1,000 rabbit monoclonal antibody EGFR (D38B1), Akt (AP20658c), p-Akt (T308, bs-2720R), Erk1/2 (AM2189b), or p-Erk1/2 (T202/Y204, AP3607a) overnight at 4°C, and then incubated with HRP-conjugated anti-rabbit antibodies (1:2,000; ab191866, Abcam) at room temperature for 2 h. Finally, the membranes were exposed with the Bio-rad ChemiDoc XRS System. For RT-qPCR analysis, H1299 cells (2×105 cells/well) were seeded with complete medium on a 6-well plate and cultured overnight. The old medium was replaced with fresh OPTI-MEM containing PAA/siEGFR, PAA-iRGD/siEGFR, or PEI/siEGFR. After 6 h of incubation, the supernatant was removed, and the cells were



incubated with fresh complete medium for 48 h. Cells were washed with PBS and lysed with TRizol (1 mL per well) for 30 min, and then 200 µL chloroform was added. The tubes were shaken vigorously by hand for 15 s, and incubated at room temperature for 15 min. After centrifugation at 12,000 g for 15 min at 4°C, the colorless upper aqueous phase (about 600 µL) was transferred into 500 µL isopropyl alcohol in a clean tube, and incubated at -20°C for 30 min. The samples were centrifuged at 12,000 g for 10 min at 4°C, and then the supernatant was removed. The RNA pellet was washed twice with 75% ethanol, and centrifuged at 7,500 g for 5 min at 4°C. The RNA pellet was dissolved in 30 µL RNase-free H2O. The concentration of RNA was measured with a NanoDrop 2000 spectrophotometer, and the RNA quality was assessed with a SYBR Safe DNA gel stain (Invitrogen Life Technologies). Reverse transcription reactions were carried out with a TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit (Transgen, China). Real-time quantitative PCRs reactions were carried out with a TransStart® Top Green qPCR SuperMix kit (Transgen, China). The cycle threshold (Ct) value was measured, and the relative quantification of specific gene expression was determined using the 2-∆∆Ct method, with the β-actin as the reference gene. The primer sequence for RT-qPCR EGFR was 5’- TAACAAGCTCACGCAGTTGG-3’; the primer sequence for EGFR rev was 5’-ACCAAGGACCACCTCACAGTT-3’; the primer sequence for β-actin was 5’-ACGTGGACATCCGCAAAG-3’; and the primer sequence for β-actin rev was 5’-GACTCGTCATACTCCTGCTTG-3’. 2.10. In vitro proliferation


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H1299 cells (2×105 cells/well) were seeded with complete medium on a 6-well plate and cultured overnight. The old medium was replaced with fresh OPTI-MEM containing PAA/siEGFR, PAA-iRGD/siEGFR, or PEI/siEGFR. After 6 h of incubation, the supernatant was removed, and the cells were incubated with fresh complete medium for 48 h. The cells were measured with an EdU Apollo®488 in vitro flow cytometry kit. 2.11. In vitro apoptosis H1299 cells (2×105 cells/well) were seeded with complete medium on a 6-well plate and cultured overnight. The old medium was replaced with fresh OPTI-MEM containing PAA/siEGFR, PAA-iRGD/siEGFR, or PEI/siEGFR. After 6 h of incubation, the supernatant was removed, and the cells were incubated with fresh complete medium for 48 h. After trypsinization, the cells were centrifuged, washed with PBS, and measured for apoptosis with an apoptosis kit (Transgen, China). 2.12. In vitro migration H1299 cells (2×105 cells/well) were seeded with complete medium on a 6-well plate and cultured overnight. The old medium was replaced with fresh OPTI-MEM containing PAA/siEGFR, PAA-iRGD/siEGFR, or PEI/siEGFR. After 6 h of incubation, the supernatant was removed, and the cells were incubated with fresh complete medium for 48 h. The cells were then detached and resuspended in serum-free medium. The cells (2×105) were added in the upper transwell-chamber, and complete medium was added into the bottom chamber. The filtered cells were stained with 5% crystal violet. The cell migration was observed with an optical



microscope (ZEISS, Germany) and analyzed with ImageJ software (version 1.49). 2.13. In vivo tumor growth inhibition study Female BALB/c nude mice (6-8 weeks old) were purchased from HFK Bioscience Co., Ltd. (Beijing, China). The animal experiments were approved by the Laboratory Animal Ethics Committee of Jinan University and conformed to the legal mandates and national guidelines for the care and maintenance of laboratory animals. H1299 cells (4×106 cells) were inoculated subcutaneously into the right flank of each mouse. Tumor volume was estimated using the formula: volume (mm3) = length × width2/2. When the tumor volume reached approximately 50-100 mm3, the mice were randomly divided into 5 groups (5 animals per group). The mice were administered with (1) saline, (2) PAA-iRGD/siNC, (3) PAA/siEGFR, (4) PAA-iRGD/siEGFR, and (5) PEI/siEGFR via tail vein injection at an siRNA dose of 0.5 mg/kg every other day. The body weight and tumor volume of each mouse were measured daily during the whole period of treatment. Two days after the fourth administration, the mice were sacrificed and the tumors were collected for assays. 2.14. In vivo toxicity Two days after the fourth administration, major organs including heart, liver, spleen, lung, and kidney were collected, fixed in 10% formalin, processed routinely into paraffin, sectioned into 5 micron thick slices, stained with hematoxylin and eosin (H&E), and examined by light microscopy. Histological assessment was performed in terms of inflammatory cell infiltration and the severity of necrosis as previously described.2, 24 Each parameter was scored as follows: inflammatory: none (scored 0),


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rare inﬂammatory cells (1 point), increased numbers of inflammatory cells (2 points), present in approximately half of the fields (3 points) and present in most fields (4 points); necrosis: none (scored 0), mild piecemeal necrosis (1 point), moderate piecemeal necrosis (2 points), and marked piecemeal necrosis (3 points). 2.15. Statistical analysis Student’s t-test for independent means was used for the statistical analysis between two groups. The differences between any two groups of several groups were analyzed by one-way analysis of variance (ANOVA) followed by LSD multiple comparisons; p values < 0.05 were considered significant. Statistical analyses were performed using SPSS 22.0 software (SPSS Inc, Chicago). Data is shown as mean+s.d., *P < 0.05, **P < 0.01, ***P < 0.001. 3. RESULTS AND DISCUSSION 3.1. Physicochemical characterizations of PAA-iRGD In this work, PAA-iRGD was prepared via a facile amidation reaction of iRGD peptides with PAA synthesized by Michael addition polymerization, according to Scheme 1. The chemical structure of PAA-iRGD was characterized by nuclear magnetic resonance spectroscopy (1H NMR). As shown in Figure S1, typical hydrogen

signals

were

clearly

observed

in

δ5-6

ppm

in

the

N,N’-bis(acryloyl)cystamine (BAC) spectrum, while the signals disappeared and new characteristic peaks of PAA (δ2-3 ppm) appeared in the PAA spectrum, indicating that PAA had been successfully synthesized. In addition, new characteristic peaks (about δ3.7-4.5 ppm and δ1-2 ppm) of the iRGD peptides appeared in the PAA-iRGD



spectrum, suggesting that the iRGD peptides had been conjugated to the PAA. Further, the molecular weights of PAA and PAA-iRGD were measured by gel permeation chromatography (GPC) (as shown in Figure S2), and calculated to be 158 kDa and 172 kDa, respectively. The size and surface charge of gene carriers are important parameters affecting the whole gene delivery process,25-26 and gene carriers of about 150 nm are well established to be suitable for gene delivery.27-28 In this work, the size and surface charge of PAA and PAA-iRGD were measured with dynamic light scattering. The mean hydrodynamic diameter and zeta potential of PAA were 181.5±26 nm and 39.9±1.3 mV, respectively and for PAA-iRGD they were 223.7±3.1 nm and 35.2±3.2 mV, respectively. The gene condensing, release, and protection capabilities of gene carriers are critical for evaluating the gene delivery efficacy of the carrier materials, and they can be determined by the gel retardation assay.29 In this work, the gene condensing ability of PAA-iRGD by electrostatic attraction was evaluated with an agarose gel electrophoresis assay. As shown in Figure 1A, the migration of siNC (the negative control of siEGFR) was completely retarded by PAA-iRGD when the weight ratio of PAA-iRGD to siNC was higher than 30/1. Further, the gene release capacity of redox-responsive biodegradable PAA-iRGD was evaluated to predict the escape of gene from the carrier PAA-iRGD into the intracellular redox microenvironment. To this aim, PAA-iRGD/siNC polyplexes were mixed with 10 mM glutathione and subjected to the gel retardation assay. As shown in Figure 1B, the siNC was efficiently released from the polyplexes under the redox microenvironment. This indicates that


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PAA-iRGD delivered siRNA that could be efficiently released in tumor cells with high concentrations of glutathione in the cytoplasm.14 Genes also need to be protected from degradation before implementing their biological functions. Therefore, the gene protection ability of PAA-iRGD was evaluated. Naked siNC or the polyplexes (weight ratio=30/1) were incubated with RNase A for various durations, and then glutathione was added to release the siNC. From Figure 1C, the naked siNC was completely degraded, while the polyplexes protected the siNC from complete degradation. 3.2. Cytotoxicity of PAA-iRGD Ideal gene carrier materials should have high gene transfection efficiency and a low toxicity to the host.30 Bio-safety is critical for gene carrier materials used in clinical applications, With cytotoxicity being the common parameter for assessing bio-safety. In this work, the in vitro cytotoxicity of PAA and PAA-iRGD (from 0 to 1000 µg/mL) was measured with the CCK8 assay on H1299 cells. From Figure 2A, the viability of the H1299 cells that were treated with up to 250 µg/mL of PAA or PAA-iRGD was not significantly different from the control. In addition, the viability of H1299 cells was higher in the presence of 500 or 1000 µg/mL of PAA-iRGD compared to the same concentrations of PAA. The cytotoxicity of PAA and PAA-iRGD was significantly decreased in comparison to that of PEI (Figure 2B). These results suggest that PAA-iRGD has good bio-safety and biocompatibility. 3.3. Cellular uptake of PAA-iRGD and iRGD targeting For gene delivery, efficient cell uptake and targeting are prerequisites for achieving high therapy efficiency.3 In this study, polyplexes of PAA-iRGD and



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siNC-Cy3 were prepared to assess their cellular uptake into H1299 cells. As shown in Figure 3A, 61, 75, 79, and 43% Cy3 positive cells were obtained for the PAA-iRGD/siNC-Cy3 polyplexes at weight ratios of 30/1, 40/1, 50/1, and 80/1, respectively.

Moreover,

the

percentage

of

Cy-3

positive

cells

for

the

PAA-iRGD/siNC-Cy3 polyplexes was significantly higher than that for the PAA/siNC-Cy3 polyplexes. The percentage of Cy-3 positive cells for the PAA-iRGD/siNC-Cy3 polyplexes at the weight ratios of 50/1 was also significantly higher than that for the PEI/siNC-Cy3 polyplexes. From Figure 3B, pre-treatment of the cells with free iRGD peptides did not significantly affect the internalization of the PAA/siNC-Cy3 polyplexes, but it significantly decreased the internalization of the PAA-iRGD/siNC-Cy3 polyplexes. This suggests that the PAA-iRGD/siNC-Cy3 polyplexes were easily taken up by the H1299 cells, and could effectively target the H1299 cells that were overexpressing integrin αv. 3.4. In vitro gene silencing The selection of a therapeutic target gene is important in gene therapy, and for this study, EGFR was chosen as the therapeutic target. EGFR is an important transmembrane receptor tyrosine kinase that is overexpressed on lung cancer cells and associated with cell proliferation, apoptosis, angiogenesis, tissue invasion, and metastasis.21,

31

Western-blot analysis of the EGFR protein and Real-Time

Quantitative PCR (RT-qPCR) analysis of the EGFR mRNA expression level were carried out for H1299 cells incubated with various polyplexes. From the western-blot analysis (Figure 4A), the negative control and the PAA-iRGD/siNC polyplexes did


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not contribute to the downregulation of the EGFR protein level. Compared to the negative control and PAA/siEGFR, the PAA-iRGD/siEGFR treatment led to a much lower EGFR protein level with an expression level that was approximately equivalent to the positive control PEI/siEGFR. From the RT-qPCR analysis of the EGFR mRNA level (Figure 4B), PAA-iRGD/siEGFR caused a significant decrease in the EGFR mRNA level, when compared to the negative control or PAA/siEGFR, but was not significantly different from PEI/siEGFR. Thees results suggest that PAA-iRGD can efficiently deliver siRNA to silence target proteins in tumor cells. 3.5. In vitro cell proliferation and apoptosis EGFR is a transmembrane receptor tyrosine kinase with a critical role in regulating cell proliferation, differentiation, migration and apoptosis. In this study, the EdU Apollo®488 in vitro flow cytometry kit was used to examine the proliferation of H1299 cells treated with various polyplexes. EdU can label newly synthesized DNA to detect cell proliferation at the molecular level. As shown in Figure 5, PAA-iRGD/siEGFR and PEI/siEGFR significantly inhibited cell proliferation in H1299 cells, compared to the negative control. In addition, PAA-iRGD/siEGFR significantly reduced cell proliferation, compared to PEI/siEGFR. Further, the apoptosis of H1299 cells induced by various polyplexes was examined using an apoptosis detection kit. The cells were double-stained with Annexin-V-FITC (marker for early apoptosis) and PI (marker for cell death). As shown in Figure 6, the percentages of apoptotic cells (both early apoptosis and late apoptosis) was 16%, 37% and 24%, treated with PAA/siEGFR, PAA-iRGD/siEGFR,



and PEI/siEGFR, respectively. Thus, PAA-iRGD/siEGFR elicited a significantly higher degree of apoptosis in the H1299 cells, compared to PAA/siEGFR and PEI/siEGFR. 3.6. In vitro cell migration and signal pathway A transwell-chamber was used to examine migration of H1299 cells treated with various polyplexes. In the migration experiment, an increased number of transwell cells indicated a stronger cell migration capability. In the field shown in Figure 7A, 34 transwell cells were observed after treatment with PAA-iRGD/siEGFR, versus 54 or 45 transwell cells treated with PAA/siEGFR or PEI/siEGFR, respectively. The statistical results were shown in Figure 7B. Thus, PAA-iRGD/siEGFR caused a significantly reduced migration capability in the H1299 cells, compared to the negative control, PAA/siEGFR, or PEI/siEGFR. In addition, the EGFR downstream signaling cascades with Ras-MAPK and PI3K/Akt were detected to clarify the signal pathways by which PAA-iRGD/siEGFR polyplexes inhibit cell proliferation and migration, leading to cell apoptosis. As shown in Figure 8, treatment with PAA-iRGD/siEGFR downregulated the expression level of p-Akt and p-Erk1/2 proteins in the H1299 cells, compared to the negative control, PAA/siEGFR, or PEI/siEGFR. This suggests that the low-expression of the EGFR protein inhibited Akt and Erk1/2 phosphorylation, to block the signal pathways and attenuate proliferation and migration, which led to large-scale apoptosis of the H1299 cells. 3.7. In vivo systemic toxicity evaluation


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Systemic toxicity is always important when evaluating a gene vector for clinical applications. In this work, we systematically evaluated the toxicity of the PAA-iRGD/siRNA polyplexes in BALB/c nude mice. Body weight changes were recorded each day during treatment with the polyplexes, and tissue toxicity was assessed. During the whole experimental period, no deaths occurred and all animals appeared to have normal activity. As shown in Figure 9A, no significant body weight change occurred in the animals that were treated with the various polyplexes. Figure 9B shows the H&E staining to reveal tissue toxicity and histological scores for the major organs including heart, liver, spleen, lung, and kidney. Except for slight kidney toxicity found in the animals treated with PEI/siEGFR, no significant abnormalities were found in the organs of the mice treated with the polyplexes. These results demonstrate that the PAA-iRGD/siRNA polyplexes did not induce any detectable toxicity in vivo. 3.8. In vivo tumor growth inhibition To investigate the in vivo therapeutic efficiency of PAA-iRGD/siEGFR in treating BALB/c nude mice, animals were intravenously injected with the polyplexes, and the tumor volumes were recorded. As shown in Figure 10A, the mean tumor volumes were 801, 633, 511, 378, and 273 mm3, when treated with saline, PAA-iRGD/siNC, PAA/siEGFR, PAA-iRGD/siEGFR, and PEI/siEGFR, respectively. The injection with PAA-iRGD/siEGFR caused a significantly smaller tumor size compared to that of the PAA/siEGFR and PEI/siEGFR groups. These results suggest that PAA-iRGD/siEGFR targets tumor tissue and inhibits tumor growth by



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downregulating the expression of the EGFR protein.

4. CONCLUSION From these results, PAA-iRGD displayed a suitable particle size, positive surface charge, strong gene condensing and protection capabilities, efficient intracellular release,

low

cytotoxicity,

and

high

cell

uptake

efficiency.

Moreover,

PAA-iRGD/siRNA displayed a high gene silencing efficacy in vitro and significant inhibition of tumor growth in vivo. Overall, these fingdings indicate that PAA-iRGD is a promising gene carrier material for gene therapy in cancer.

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Grant No. 31271019).

ASSOCIATED CONTENT Supporting information The Supporting Information is available free of charge on the ACS Publications website at DOI: . Figure S1, 1H NMR spectra of BAC, PAA, and PAA-iRGD; Figure S2, GPC chromatograms of PAA and PAA-iRGD.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]


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*E-mail: [email protected] Notes The authors declare no competing financial interest.

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For Table of Contents Use Only Tumor-penetrating peptide-functionalized redox-responsive hyperbranched poly(amido amine) delivering siRNA for lung cancer therapy Zhong Guo a, Sha Li a, Zonghua Liu a, *, Wei Xue a, b, *

Table of Contents


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Scheme 1. Synthesis route of PAA-iRGD. 277x102mm (300 x 300 DPI)



Figure 1. Gel electrophoresis of PAA-iRGD/siNC polyplexes. A) Retardation of siNC was determined with different weight ratios between PAA-iRGD and siNC. B) siNC release from the polyplexes incubated for 30 min in 10 mM glutathione solution at 37°C. C) Gene protection ability of PAA-iRGD. The polyplexes were pre-incubated with RNase A for different times at 37°C. 122x100mm (300 x 300 DPI)


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Figure 2. Cytotoxicity of PAA, PAA-iRGD (A) and PEI (B) on H1299 cells. 107x48mm (300 x 300 DPI)



Figure 3. Cellular uptake and targeting ability of PAA-iRGD/siNC polyplexes in H1299 cells. A) The cellular uptake of different weight ratios of the polyplexes after incubated with the cells for 6 h. B) The targeting ability of PAA-iRGD. 91x41mm (300 x 300 DPI)


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Figure 4. Gene silencing of PAA-iRGD/siEGFR polyplexes in vitro. A) EGFR expression was determined by using western blotting. B) The relative EGFR mRNA level was measured by using RT-qPCR. 143x207mm (300 x 300 DPI)



Figure 5. Proliferation of H1299 cells treated with various polyplexes. 86x82mm (300 x 300 DPI)


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Figure 6. Apoptosis of H1299 cells treated with various polyplexes. A) The flow cytometry data of H1299 cells stained with Annexin-V-FITC and PI after pre-treated with various polyplexes. B) The statistical analysis of the flow cytometry data. 133x66mm (300 x 300 DPI)



Figure 7. Migration of H1299 cells treated with various polyplexes. A) The microscopy images of transwell H1299 cells. B) The statistical analysis of the transwell H1299 cells. 219x93mm (300 x 300 DPI)


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Figure 8. Expression levels of Akt, p-Akt, Erk1/2, and p-Erk1/2 in the H1299 cells pre-treated with various polyplexes. 63x49mm (300 x 300 DPI)



Figure 9. In vivo toxicity of various polyplexes in mice. A) The body weight changes of the mice during the treatment process. B) H&E stained sections and histological scores of the heart, liver, spleen, lung, and kidney harvested from the mice (20×). a: Saline; b: PAA-iRGD/siNC; c: PAA/siEGFR; d: PAA-iRGD/siEGFR; e: PEI/siEGFR. 289x132mm (300 x 300 DPI)


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Figure 10. In vivo anti-tumor effect of various polyplexes. A) Tumor growth curve of the mice treated with various polyplexes (The red arrows indicate the injection time points). B) All the tumors harvested from the mice at 23 d post all the injections. 284x113mm (300 x 300 DPI)


Tumor-Penetrating Peptide-Functionalized Redox-Responsive

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