ARTICLE IN PRESS Cancer Letters ■■ (2015) ■■–■■
Contents lists available at ScienceDirect
Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Original Articles
A prostate cancer-targeted polyarginine-disulfide linked PEI nanocarrier for delivery of microRNA Q1 Tingting Zhang a,b,*, Xiang Xue a, Dalin He b,c, Jer-Tsong Hsieh d a
Department of Gynecology, The Second Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an, China Oncology Research Lab, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an, China Department of Urology, The First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an, China d Department of Urology, University of Texas Southwestern Medical Center, Dallas, TX, USA b c
A R T I C L E
I N F O
Article history: Received 25 February 2015 Received in revised form 30 April 2015 Accepted 4 May 2015 Keywords: MicroRNA R11 peptide Prostate cancer Polyplex
A B S T R A C T
Recent advances in efficient microRNA (miRNA) delivery techniques using prostate cancer-targeted nanoparticles offer critical information for understanding the functional role of miRNAs in vivo, and for supporting targeted gene therapy in terms of treating miRNA-associated prostate cancer. Here, we report the polyarginine peptide (R11)-labeled non-toxic SSPEI nanomaterials capable of prostate cancer-specific miR-145 delivery to prostate cancer in vivo where they display full bioactivity at completely nontoxic concentrations. The R11-labeled BPEI-SS (R11-SSPEI) nanocarrier showed less toxicity in prostate cancer, and electrostatic interaction of R11-SSPEI with miR-145 exhibited optimal transfection efficacy. The R11-SSPEI/ miR-145 polymer could be specifically uptaken in prostate cancer using FAM-miR-145 mixed with R11-SSPEI. The functional action of miR-145 oligomers released from polyplexes was evaluated by a reporter vector containing a miR-145-binding sequence, and showed a significantly reduced reporter signal in a dosedependent manner. More importantly, in a peritoneal mouse tumor model, the systemic administration of the R11-SSPEI/FAM-miR-145 complex leads to the delivery of miR-145 into the tumors, dramatically inhibiting tumor growth and prolonged survival time. Hence, we establish a novel and prostate cancerspecific targeting system for the systemic in vivo application of microRNAs through R11-SSPEI complexation as a powerful tool for future therapeutic use. © 2015 Elsevier Ireland Ltd. All rights reserved.
41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Introduction Prostate cancer (PCa) is a commonly diagnosed cancer and the second leading cause of cancer-related deaths in US men. Common treatments for prostate cancer such as surgery, hormone therapy, radiation therapy, and chemotherapy have been commonly used for PCa treatments [1,2]. Alternative therapeutic modalities such as targeted therapy with cancer specificity would be a better treatment to enhance therapeutic efficacy in prostate cancer patients. The development of an efficient nanocarrier for successful delivery of therapeutic genes into the tumor provides strong support for effective treatment of genetically based tumors in terms of gene therapy. MicroRNA (miRNA), an endogenously expressed non-coding RNA molecule, has emerged as an important regulator for various developmental, physiological, and pathological conditions. And numerous miRNAs have been considered as potential therapeutic targets [3,4]. However, the development of an efficient in vivo miRNA delivery system has been challenging due to many limitations including rapid degradation in serum conditions and the lack of a
61 62 63 64
* Corresponding author. Tel.: +86 29 8532 3661; fax: +86 29 8532 3203. E-mail address: [email protected] (T. Zhang).
reliable delivery system that is capable of trapping miRNA in intracellular space [4]. Therefore, the development of the miRNA delivery vehicle that can deliver miRNA into the right region in vivo would clearly offer advantages. Of these nonviral carriers, cationic polymers such as polyethyleneimine (PEI) could form noncovalent interpolyelectrolyte complexes with DNA and RNA [5,6]. And PEIs with relatively low immune response, degrees of branching, and other modifications have been used as transfection reagent in a variety of cell lines and live animals to establish their efficacy for efficient small interfering RNA (siRNA) delivery for therapeutic purposes [7–10]. However, a major concern of using a PEI polymer as a nanocarrier is showing its unfavorable adverse effects such as high cellular toxicity and nontargeted uptake in tumors [9]. Previous studies have proven that the introduction of disulfide linkage in the branched PEI (SSPEI) containing multiple amine backbone is able to increase cell biocompatibility, and introduced a biodegradable capability by inducing degradation of the SSPEI polymer by endogenous enzymes such as glutathione reductase [11]. Cell permeable peptide (CPP) or protein transduction domain (PTD), as a delivery vehicle to introduce a functional molecule into the cell, has received increasing attention because of its novel biologic properties and capabilities [12–14]. Our previous study
http://dx.doi.org/10.1016/j.canlet.2015.05.003 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Tingting Zhang, Xiang Xue, Dalin He, Jer-Tsong Hsieh, A prostate cancer-targeted polyarginine-disulfide linked PEI nanocarrier for delivery of microRNA, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.05.003
65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87
ARTICLE IN PRESS T. Zhang et al./Cancer Letters ■■ (2015) ■■–■■
2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
indicated that poly-arginine (R11) compared with other four CPPs exhibited the highest uptake by different prostate cancer cell lines [15]. Moreover, in vivo evaluation of the tissue distribution of R11 in nude mice showed that R11 exhibited an organ-specific uptake in prostate cancer and prostate tissues 24 h after intravenously delivery. Thus R11 could be used as a potential delivery vehicle in prostate cancer therapy. Fusion CPP, such as RVG, with the cationic polyarginine chain was used to label negatively charged microRNA with electrostatic interaction [16]. However, this approach is susceptible to causing the problem of microRNA stability and has the limitation of not being able to specifically carry the targeting agents to tumor. Therefore, to not only facilitate the stability of oligonucleotides such as microRNA in vitro and in vivo environment but also to improve the organ-specific uptake, the use of condensed R11-SSPEI polymer is beneficial for efficient microRNA delivery in vivo. In this regard, we introduced an R11-labeled SSPEI polymer to deliver miR-145 to the prostate cancer by introducing a polyethylene glycol (PEG) chain linker that can help enhance biocompatibility and extend circulation time in the bloodstream. We targeted miR-145, a well-known tumor suppressive miRNA which inhibits cell growth, invasion and migration in cancer cells [17–19]. From the therapeutic aspect, restoration of miR-145 has the potential function of suppressing cell proliferation, migration and invasion in prostate cancer [19]. In the current study, we evaluated R11-synthesized SSPEI polyplexes as an efficient polymer carrier for delivery of miR-145 to the prostate cancer region by examining the targeted pattern of FAMlabeled miR-145 coated with R11-SSPEI to the prostate cancer.
31
Synthesis of the R11-SSPEI nanocarrier
32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67
Synthesis of thiolated BPEI (BPEI-SH) and disulfide-crosslinked BPEI (BPEI-SS) was performed as previously described with some modifications [11]. The green solid obtained was dissolved in methanol (30 mL) in a bottlenecked flask (100 mL), and the sample was purged with nitrogen and kept under a vacuum for 10–20 min. A calculated amount of propylene sulfide (5 the molar excess to BPEI1.2 K) was added using a syringe. This solution was stirred at 60 °C for 24 h. The reaction mixture was evaporated to dryness under reduced pressure and was taken up in methanol, followed by precipitation in cold diethyl ether twice. The product’s thiol group content was determined using Ellman’s method. The BPEI-SH product exhibited a similar kind of proton NMR except for the integral value of protons at d 1. 5e1.2 compared with BPEI1.2 K; 1 H NMR of BPEI-SH (300 MHz, D2O) was d 3. 15e2.55 (m, NCH 2CH 2N, NCH2 CHS) vs. 1.5e1.2 (m, CH 3). BPEI-SH (0.5 g) was dissolved in anhydrous DMSO (50 mL) and the solution was stirred for 48 h at room temperature (r.t.) for crosslinking via oxidation of the thiol group. The product was purified by dialysis against D.I. water (MWCO 3500) and extensively lyophilized, and the chemical structure was confirmed by proton NMR. The extent of disulfide crosslinking was determined using Ellman’s method. BPEI-SS (12 equiv) and MAL-PEG-NHS were dissolved in 50 mL of anhydrous methanol and DMSO in the proportion of 1:5 (v/v), and this was stirred for 4 h at r.t., followed by the addition of RVG peptide (1.2 equiv to MAL-PEG-NHS). The reaction mixture was stirred for another 48 h at r.t. to afford BPEI-SS-PEG-RVG. Other experimental and characterization conditions were the same as described above. For more precise evaluation of R11-mediated BBB targeting, methoxy PEG-conjugated BPEI-SS (BPEI-SS-MPEG) was synthesized as a control. MPEG 5 K (1 g, 7 mmol), NPC (400 mg, 20 mmol), and triethylamine (2.7 mL, 7 mmol) were dissolved in 50 mL of methylene chloride (MC) and reacted for 2 h in an ice bath. The reaction mixture was then stirred at r.t. for another 16 h. The reactant was evaporated to dryness under reduced pressure and dissolved in MC, followed by precipitation in cold diethyl ether twice. For the conjugation of BPEI-SS to NPC activated MPEG, BPEI-SS (193 mg, 0.16 mmol), and NPC activated MPEG (40 mg, 6 mmol) were dissolved in 20 mL of anhydrous methanol and DMSO in the proportion of 1:3 (v/v), and this mixture was stirred for 24 h at r.t. The product was extensively purified by dialysis against D.I. water (MWCO 10,000) and lyophilized, and its chemical structure was confirmed by proton NMR. The R11 peptides (RRRRRRRRRRR) corresponding to protamine were prepared in mass quantities using a peptide synthesizer (APEX 396, AAPP TEC, Louisville, KY, USA) based on standard fluoren-9-ylmethoxycarbonyl (F-moc) chemistry.
68 69 70
ethidium bromide for 20 min and analyzed on a UV illuminator to identify the locations of the miRNA. The dispersity of the R11-SSPEI/miR-145 was assessed by transmitting electron micrograph (TEM). The complexes were stained with uranylacetate to examine their morphology. The polymer/miRNA complexes were prepared by addition of the polymer solution to the miRNA oligomer solution in PBS buffer (pH 7.4, 140 mM NaCl). The mixtures were then incubated for 30 min at room temperature. The particle size of each sample was measured using a particle size analyzer using Zetasizer Nano S (Malvern Instruments, Malvern, UK). The surface charge was measured by determination of zeta potential using a Zetasizer Nano Z (Malvern Instruments) in PBS buffer.
71 72 73 74 75 76 77 78 79 80
Cell culture
81
The human PCa cell lines, PC3 and LNCAP, were obtained from the American Type Culture Collection (Manassas, VA, USA). The PC cell lines were cultured in monolayer in T-medium (Invitrogen) supplemented with 5% fetal bovine serum, and maintained in an incubator with a humidified atmosphere of 95% air and 5% CO2 at 37 °C.
82 83 84 85 86
Cell toxicity test
87
The cytotoxic effects of free R11-SSPEI peptides were studied on prostate cancer cells. The cells were seeded at a density of 5000 cells/well in 96-well plates. After 24 h of seeding, the culture medium was replaced with medium containing R11-SSPEI peptides (0, 50, 100, 200, 500, and 1000 μg/mL). The cells were incubated for 8 h and 48 h, followed by the addition of MTS reagent, and the cell viability was determined following the manufacturer’s instructions (Promega, Madison, WI).
88 89 90 91 92 93
Confocal microscopy analysis
94
PC3 cells (1 × 105) that were pre-incubated with the fluorescence cell tracker CM-DiI (Invitrogen) for 20 min were seeded on glass coverslips in 12-well plates and grown for 24 h at 37 °C. After the complexation reaction of FAM-labeled miR-145 oligomer with SSPEI or R11-SSPEI for 30 min, this polymer complex was treated into each well and incubated for 1 h. Several washing steps were performed using PBS and the cells were fixed using 4% paraformaldehyde solution (Wako Pure Chem., Osaka, Japan) under gentle shaking for 20 min. Cells were then rinsed twice with PBS for 10 min, and the cells attached to the coverslip were transferred onto a slide that was pretreated with mounting solution containing 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution (Vector Laboratories, Inc, Burlingame, CA, USA). The fluorescence images were acquired using a confocal laser scanning microscope (LSM 510; Carl Zeiss, Inc., Thornwood, NY, USA).
95 96 97 98 99 100 101 102 103 104 105 106
In vitro fluorescence or luciferase assay
107
The harvested PC cells were seeded into a 24-well plate and incubated with R11SSPEI/FAM-miR-145 complex for 1 h. After several washing steps using PBS, the cells were lysated using lysis buffered solution containing Triton X-100. Collected PC cells were transferred into a black 96-well microplate and the fluorescence signals were analyzed using an Infinite M200 (Tecan, GmbH, SZ, Austria). CMV promoter driven Gaussia luciferase vector containing 3 copy sequences of perfect target of miR-145 (CMV/Gluc/3 × PT_miR-145) and synthetic miR-145 oligomer (Bioneer, Inc., Korea) modified with phosphorothioate (10 nM, 50 nM) were mixed with the R11-SSPEI polymer for 15 min at r.t. The mixed polyplexes were added to PBS-washed PC cells, which were then incubated for 1 h in OptiMEM medium. The incubated cells were washed twice with PBS and maintained for 24 h in a serum-containing medium. Luciferase analyses were conducted using Luciferase Assay System Kits (Promega, MI, USA). Washed cells were lysed with the supplied lysis solution (100 mL per 24well plate), and the cell lysates were transferred to white 96-well plates. In vitro luciferase signals were measured using a 96-well microplate luminometer (TR717; Applied Biosystems, CA, USA) at 20 s acquisition time.
108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123
In vivo fluorescence study
124
For the in vivo study, FAM-labeled miR-145 was incubated with R11-SSPEI or SSPEI for 15 min. The polyplexes were administered into 6-week-old male BALB/c mice (n = 3) via the tail vein using an insulin syringe (BD Biosciences Inc.). Four hours after administration of the R11-SSPEI/miR-145 complex, the mice were sacrificed and several organs were isolated. Fluorescence images were detected using an IVIS-100 imaging system (Caliper Life Sciences, Inc.) equipped with a cooled CCD camera at a 1 s exposure time. The average photon counts per pixel (cm2) were recorded for each organ. Relative mean fluorescence photon value was divided by tissue weight (the mean value of ten tissue organs). All of the experimental animals were housed under specific pathogen-free conditions and handled according to the Institutional Animal Care and Use Committee of The Second Hospital of Xi’an Jiao Tong University.
125 126 127 128 129 130 131 132 133 134 135
Characterization of R11-SSPEI/miR-145 complexes
Statistical analysis
136
The complex formation was monitored by 2% agarose gel electrophoresis using molecular markers. Following electrophoresis, the gels were stained with 0.5 mg/mL
Data are represented as mean ± standard error of mean (SEM) and were calculated using Student’s t-test. Statistical significance was set at P values
Contents lists available at ScienceDirect
Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Original Articles
A prostate cancer-targeted polyarginine-disulfide linked PEI nanocarrier for delivery of microRNA Q1 Tingting Zhang a,b,*, Xiang Xue a, Dalin He b,c, Jer-Tsong Hsieh d a
Department of Gynecology, The Second Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an, China Oncology Research Lab, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an, China Department of Urology, The First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an, China d Department of Urology, University of Texas Southwestern Medical Center, Dallas, TX, USA b c
A R T I C L E
I N F O
Article history: Received 25 February 2015 Received in revised form 30 April 2015 Accepted 4 May 2015 Keywords: MicroRNA R11 peptide Prostate cancer Polyplex
A B S T R A C T
Recent advances in efficient microRNA (miRNA) delivery techniques using prostate cancer-targeted nanoparticles offer critical information for understanding the functional role of miRNAs in vivo, and for supporting targeted gene therapy in terms of treating miRNA-associated prostate cancer. Here, we report the polyarginine peptide (R11)-labeled non-toxic SSPEI nanomaterials capable of prostate cancer-specific miR-145 delivery to prostate cancer in vivo where they display full bioactivity at completely nontoxic concentrations. The R11-labeled BPEI-SS (R11-SSPEI) nanocarrier showed less toxicity in prostate cancer, and electrostatic interaction of R11-SSPEI with miR-145 exhibited optimal transfection efficacy. The R11-SSPEI/ miR-145 polymer could be specifically uptaken in prostate cancer using FAM-miR-145 mixed with R11-SSPEI. The functional action of miR-145 oligomers released from polyplexes was evaluated by a reporter vector containing a miR-145-binding sequence, and showed a significantly reduced reporter signal in a dosedependent manner. More importantly, in a peritoneal mouse tumor model, the systemic administration of the R11-SSPEI/FAM-miR-145 complex leads to the delivery of miR-145 into the tumors, dramatically inhibiting tumor growth and prolonged survival time. Hence, we establish a novel and prostate cancerspecific targeting system for the systemic in vivo application of microRNAs through R11-SSPEI complexation as a powerful tool for future therapeutic use. © 2015 Elsevier Ireland Ltd. All rights reserved.
41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Introduction Prostate cancer (PCa) is a commonly diagnosed cancer and the second leading cause of cancer-related deaths in US men. Common treatments for prostate cancer such as surgery, hormone therapy, radiation therapy, and chemotherapy have been commonly used for PCa treatments [1,2]. Alternative therapeutic modalities such as targeted therapy with cancer specificity would be a better treatment to enhance therapeutic efficacy in prostate cancer patients. The development of an efficient nanocarrier for successful delivery of therapeutic genes into the tumor provides strong support for effective treatment of genetically based tumors in terms of gene therapy. MicroRNA (miRNA), an endogenously expressed non-coding RNA molecule, has emerged as an important regulator for various developmental, physiological, and pathological conditions. And numerous miRNAs have been considered as potential therapeutic targets [3,4]. However, the development of an efficient in vivo miRNA delivery system has been challenging due to many limitations including rapid degradation in serum conditions and the lack of a
61 62 63 64
* Corresponding author. Tel.: +86 29 8532 3661; fax: +86 29 8532 3203. E-mail address: [email protected] (T. Zhang).
reliable delivery system that is capable of trapping miRNA in intracellular space [4]. Therefore, the development of the miRNA delivery vehicle that can deliver miRNA into the right region in vivo would clearly offer advantages. Of these nonviral carriers, cationic polymers such as polyethyleneimine (PEI) could form noncovalent interpolyelectrolyte complexes with DNA and RNA [5,6]. And PEIs with relatively low immune response, degrees of branching, and other modifications have been used as transfection reagent in a variety of cell lines and live animals to establish their efficacy for efficient small interfering RNA (siRNA) delivery for therapeutic purposes [7–10]. However, a major concern of using a PEI polymer as a nanocarrier is showing its unfavorable adverse effects such as high cellular toxicity and nontargeted uptake in tumors [9]. Previous studies have proven that the introduction of disulfide linkage in the branched PEI (SSPEI) containing multiple amine backbone is able to increase cell biocompatibility, and introduced a biodegradable capability by inducing degradation of the SSPEI polymer by endogenous enzymes such as glutathione reductase [11]. Cell permeable peptide (CPP) or protein transduction domain (PTD), as a delivery vehicle to introduce a functional molecule into the cell, has received increasing attention because of its novel biologic properties and capabilities [12–14]. Our previous study
http://dx.doi.org/10.1016/j.canlet.2015.05.003 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Tingting Zhang, Xiang Xue, Dalin He, Jer-Tsong Hsieh, A prostate cancer-targeted polyarginine-disulfide linked PEI nanocarrier for delivery of microRNA, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.05.003
65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87
ARTICLE IN PRESS T. Zhang et al./Cancer Letters ■■ (2015) ■■–■■
2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
indicated that poly-arginine (R11) compared with other four CPPs exhibited the highest uptake by different prostate cancer cell lines [15]. Moreover, in vivo evaluation of the tissue distribution of R11 in nude mice showed that R11 exhibited an organ-specific uptake in prostate cancer and prostate tissues 24 h after intravenously delivery. Thus R11 could be used as a potential delivery vehicle in prostate cancer therapy. Fusion CPP, such as RVG, with the cationic polyarginine chain was used to label negatively charged microRNA with electrostatic interaction [16]. However, this approach is susceptible to causing the problem of microRNA stability and has the limitation of not being able to specifically carry the targeting agents to tumor. Therefore, to not only facilitate the stability of oligonucleotides such as microRNA in vitro and in vivo environment but also to improve the organ-specific uptake, the use of condensed R11-SSPEI polymer is beneficial for efficient microRNA delivery in vivo. In this regard, we introduced an R11-labeled SSPEI polymer to deliver miR-145 to the prostate cancer by introducing a polyethylene glycol (PEG) chain linker that can help enhance biocompatibility and extend circulation time in the bloodstream. We targeted miR-145, a well-known tumor suppressive miRNA which inhibits cell growth, invasion and migration in cancer cells [17–19]. From the therapeutic aspect, restoration of miR-145 has the potential function of suppressing cell proliferation, migration and invasion in prostate cancer [19]. In the current study, we evaluated R11-synthesized SSPEI polyplexes as an efficient polymer carrier for delivery of miR-145 to the prostate cancer region by examining the targeted pattern of FAMlabeled miR-145 coated with R11-SSPEI to the prostate cancer.
31
Synthesis of the R11-SSPEI nanocarrier
32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67
Synthesis of thiolated BPEI (BPEI-SH) and disulfide-crosslinked BPEI (BPEI-SS) was performed as previously described with some modifications [11]. The green solid obtained was dissolved in methanol (30 mL) in a bottlenecked flask (100 mL), and the sample was purged with nitrogen and kept under a vacuum for 10–20 min. A calculated amount of propylene sulfide (5 the molar excess to BPEI1.2 K) was added using a syringe. This solution was stirred at 60 °C for 24 h. The reaction mixture was evaporated to dryness under reduced pressure and was taken up in methanol, followed by precipitation in cold diethyl ether twice. The product’s thiol group content was determined using Ellman’s method. The BPEI-SH product exhibited a similar kind of proton NMR except for the integral value of protons at d 1. 5e1.2 compared with BPEI1.2 K; 1 H NMR of BPEI-SH (300 MHz, D2O) was d 3. 15e2.55 (m, NCH 2CH 2N, NCH2 CHS) vs. 1.5e1.2 (m, CH 3). BPEI-SH (0.5 g) was dissolved in anhydrous DMSO (50 mL) and the solution was stirred for 48 h at room temperature (r.t.) for crosslinking via oxidation of the thiol group. The product was purified by dialysis against D.I. water (MWCO 3500) and extensively lyophilized, and the chemical structure was confirmed by proton NMR. The extent of disulfide crosslinking was determined using Ellman’s method. BPEI-SS (12 equiv) and MAL-PEG-NHS were dissolved in 50 mL of anhydrous methanol and DMSO in the proportion of 1:5 (v/v), and this was stirred for 4 h at r.t., followed by the addition of RVG peptide (1.2 equiv to MAL-PEG-NHS). The reaction mixture was stirred for another 48 h at r.t. to afford BPEI-SS-PEG-RVG. Other experimental and characterization conditions were the same as described above. For more precise evaluation of R11-mediated BBB targeting, methoxy PEG-conjugated BPEI-SS (BPEI-SS-MPEG) was synthesized as a control. MPEG 5 K (1 g, 7 mmol), NPC (400 mg, 20 mmol), and triethylamine (2.7 mL, 7 mmol) were dissolved in 50 mL of methylene chloride (MC) and reacted for 2 h in an ice bath. The reaction mixture was then stirred at r.t. for another 16 h. The reactant was evaporated to dryness under reduced pressure and dissolved in MC, followed by precipitation in cold diethyl ether twice. For the conjugation of BPEI-SS to NPC activated MPEG, BPEI-SS (193 mg, 0.16 mmol), and NPC activated MPEG (40 mg, 6 mmol) were dissolved in 20 mL of anhydrous methanol and DMSO in the proportion of 1:3 (v/v), and this mixture was stirred for 24 h at r.t. The product was extensively purified by dialysis against D.I. water (MWCO 10,000) and lyophilized, and its chemical structure was confirmed by proton NMR. The R11 peptides (RRRRRRRRRRR) corresponding to protamine were prepared in mass quantities using a peptide synthesizer (APEX 396, AAPP TEC, Louisville, KY, USA) based on standard fluoren-9-ylmethoxycarbonyl (F-moc) chemistry.
68 69 70
ethidium bromide for 20 min and analyzed on a UV illuminator to identify the locations of the miRNA. The dispersity of the R11-SSPEI/miR-145 was assessed by transmitting electron micrograph (TEM). The complexes were stained with uranylacetate to examine their morphology. The polymer/miRNA complexes were prepared by addition of the polymer solution to the miRNA oligomer solution in PBS buffer (pH 7.4, 140 mM NaCl). The mixtures were then incubated for 30 min at room temperature. The particle size of each sample was measured using a particle size analyzer using Zetasizer Nano S (Malvern Instruments, Malvern, UK). The surface charge was measured by determination of zeta potential using a Zetasizer Nano Z (Malvern Instruments) in PBS buffer.
71 72 73 74 75 76 77 78 79 80
Cell culture
81
The human PCa cell lines, PC3 and LNCAP, were obtained from the American Type Culture Collection (Manassas, VA, USA). The PC cell lines were cultured in monolayer in T-medium (Invitrogen) supplemented with 5% fetal bovine serum, and maintained in an incubator with a humidified atmosphere of 95% air and 5% CO2 at 37 °C.
82 83 84 85 86
Cell toxicity test
87
The cytotoxic effects of free R11-SSPEI peptides were studied on prostate cancer cells. The cells were seeded at a density of 5000 cells/well in 96-well plates. After 24 h of seeding, the culture medium was replaced with medium containing R11-SSPEI peptides (0, 50, 100, 200, 500, and 1000 μg/mL). The cells were incubated for 8 h and 48 h, followed by the addition of MTS reagent, and the cell viability was determined following the manufacturer’s instructions (Promega, Madison, WI).
88 89 90 91 92 93
Confocal microscopy analysis
94
PC3 cells (1 × 105) that were pre-incubated with the fluorescence cell tracker CM-DiI (Invitrogen) for 20 min were seeded on glass coverslips in 12-well plates and grown for 24 h at 37 °C. After the complexation reaction of FAM-labeled miR-145 oligomer with SSPEI or R11-SSPEI for 30 min, this polymer complex was treated into each well and incubated for 1 h. Several washing steps were performed using PBS and the cells were fixed using 4% paraformaldehyde solution (Wako Pure Chem., Osaka, Japan) under gentle shaking for 20 min. Cells were then rinsed twice with PBS for 10 min, and the cells attached to the coverslip were transferred onto a slide that was pretreated with mounting solution containing 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution (Vector Laboratories, Inc, Burlingame, CA, USA). The fluorescence images were acquired using a confocal laser scanning microscope (LSM 510; Carl Zeiss, Inc., Thornwood, NY, USA).
95 96 97 98 99 100 101 102 103 104 105 106
In vitro fluorescence or luciferase assay
107
The harvested PC cells were seeded into a 24-well plate and incubated with R11SSPEI/FAM-miR-145 complex for 1 h. After several washing steps using PBS, the cells were lysated using lysis buffered solution containing Triton X-100. Collected PC cells were transferred into a black 96-well microplate and the fluorescence signals were analyzed using an Infinite M200 (Tecan, GmbH, SZ, Austria). CMV promoter driven Gaussia luciferase vector containing 3 copy sequences of perfect target of miR-145 (CMV/Gluc/3 × PT_miR-145) and synthetic miR-145 oligomer (Bioneer, Inc., Korea) modified with phosphorothioate (10 nM, 50 nM) were mixed with the R11-SSPEI polymer for 15 min at r.t. The mixed polyplexes were added to PBS-washed PC cells, which were then incubated for 1 h in OptiMEM medium. The incubated cells were washed twice with PBS and maintained for 24 h in a serum-containing medium. Luciferase analyses were conducted using Luciferase Assay System Kits (Promega, MI, USA). Washed cells were lysed with the supplied lysis solution (100 mL per 24well plate), and the cell lysates were transferred to white 96-well plates. In vitro luciferase signals were measured using a 96-well microplate luminometer (TR717; Applied Biosystems, CA, USA) at 20 s acquisition time.
108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123
In vivo fluorescence study
124
For the in vivo study, FAM-labeled miR-145 was incubated with R11-SSPEI or SSPEI for 15 min. The polyplexes were administered into 6-week-old male BALB/c mice (n = 3) via the tail vein using an insulin syringe (BD Biosciences Inc.). Four hours after administration of the R11-SSPEI/miR-145 complex, the mice were sacrificed and several organs were isolated. Fluorescence images were detected using an IVIS-100 imaging system (Caliper Life Sciences, Inc.) equipped with a cooled CCD camera at a 1 s exposure time. The average photon counts per pixel (cm2) were recorded for each organ. Relative mean fluorescence photon value was divided by tissue weight (the mean value of ten tissue organs). All of the experimental animals were housed under specific pathogen-free conditions and handled according to the Institutional Animal Care and Use Committee of The Second Hospital of Xi’an Jiao Tong University.
125 126 127 128 129 130 131 132 133 134 135
Characterization of R11-SSPEI/miR-145 complexes
Statistical analysis
136
The complex formation was monitored by 2% agarose gel electrophoresis using molecular markers. Following electrophoresis, the gels were stained with 0.5 mg/mL
Data are represented as mean ± standard error of mean (SEM) and were calculated using Student’s t-test. Statistical significance was set at P values
