Efficient and Tumor Targeted siRNA Delivery by Polyethylenimine

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Efficient and tumor targeted siRNA delivery mediated by polyethyleniminegraft-polycaprolactone-block-poly (ethylene glycol)-folate (PEI- PCL-PEG-Fol) Li Liu, Mengyao Zheng, Damiano Librizzi, Thomas Renette, Olivia Merkel, and Thomas Kissel Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00575 • Publication Date (Web): 07 Dec 2015 Downloaded from http://pubs.acs.org on December 11, 2015

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Molecular Pharmaceutics

Efficient and tumor targeted siRNA delivery by polyethylenimine-graft-polycaprolactone-block-poly (ethylene glycol)-folate (PEI- PCL-PEG-Fol) Li Liua,b ‡, Mengyao Zheng a,‡, Damiano Librizzic, Thomas Renettea, Olivia Merkela,d*, Thomas Kissela a

Department of Pharmaceutics and Biopharmacy, Philipps-University Marburg, Ketzerbach 63,

35032 Marburg, Germany b

School of Pharmacy, Shanghai Jiao Tong University, 200240 Shanghai, China

c

Department of Nuclear Medicine, University of Hospital Giessen and Marburg GmbH,

Baldingerstrasse, 35043 Marburg, Germany d

Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health

Sciences, Wayne State University, 259 Mack Ave, Detroit, MI 48201, USA

KEYWORDS Folate targeting, siRNA delivery, prolonged circulation, SKOV-3 tumor xenograft, SPECT imaging, tumor targeting

ACS Paragon Plus Environment

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ABSTRACT

Efficient delivery of functional nucleic acids into specific cells or tissues is still a challenge for gene therapy and largely depends on targeted delivery strategies. The folate receptor (FR) is known to be over-expressed extracellularly on a variety of human cancers and is therefore an outstanding gate for tumor-targeted Trojan horse like delivery of therapeutics. In this study, an amphiphilic and biodegradable ternary copolymer conjugated with folate as ligand, polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol)-folate (PEI-PCL-PEG-Fol) was synthesized and evaluated for targeted siRNA delivery via folate-FR recognition. The amphiphilic character of similar polymers was shown previously to support endosomal release of endocytosed nanocarriers and to promote formation of long circulating micelles. The obtained PEI-PCL-PEG-Fol exhibited less cytotoxicity in comparison with the corresponding ternary copolymer without folate (PEI-PCL-PEG) and with unmodified PEI25kDa. Stable micelle-like polyplexes with hydrodynamic diameters about 100 nm were found to have a zeta potential of +8.6 mV which was lower than that of micelleplexes without folate-conjugation (+13-16 mV). Nonetheless, increased cellular uptake and in vitro gene knockdown of PEI-PCL-PEGFol/siRNA micelleplexes were observed in SKOV-3 cells, an FR over-expressing cell line, in comparison with the non-folate-conjugated ones. Moreover, PEI-PCL-PEG-Fol/siRNA micelleplexes exhibited excellent stability in vivo during the analysis of 120 min and a longer circulation half live than hyPEI25kDa/siRNA polyplexes. Most interestingly, the targeted delivery system yielded 17% deposition of the i.v. injected siRNA per g in the tumor after 24 h of due to the effective folate targeting and the prolonged circulation.


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INTRODUCTION RNA interference (RNAi) by short interfering RNA (siRNA) has emerged as a means of gene silencing, which holds promise as a new class of gene therapy for various diseases such as cancer, viral infections and inflammatory diseases.1,

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Successful applications of RNAi in

mammalian cells depend upon efficient intracellular delivery of siRNA and effective knockdown of targeted transcripts. Although siRNA needs to be delivered to the cytosol only,3 compared to plasmid DNA, safe and effective systemic delivery of siRNA with specificity to target cells remains a challenge.4, 5 Problems include the instability and rapid degradation of siRNA as well as their poor cellular uptake. To address these problems, a large number of polymeric carriers6-9 have been designed for intracellular delivery of siRNA via polyelectrolyte complex formation. The resulting polyplexes can provide excellent protection of siRNA from degradation and allow facile cellular uptake through an endocytic pathway. Moreover, polymeric siRNA delivery carriers have gained great interest due to their advantages such as low immunogenicity, convenience of handling, and functionalization to provide the carriers with biodegradability, prolonged circulation and targeted delivery properties. Polyethylenimine (PEI) is one of the most successful and efficient cationic polymers employed for siRNA delivery both in vitro10, 11 and in vivo.12-14 It features the unique ‘proton sponge effect’ for endosomal release of the polyplexes into the cytosol. To address the cytotoxicity issue of high molecular weight PEI and targeted delivery of siRNA, a wide array of chemical modifications of PEI including the introduction of hydrophilic and hydrophobic segments to PEI molecules as well as the coupling of cell/tissue-specific ligands has been reported.14-17


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The folate receptor (FR) has been identified as a target molecular biomarker over-expressed in several carcinoma cells.18-20 Folic acid, a variant of Vitamin B9, exhibits particularly high affinity to the FR. Therefore, the conjugation of folate to a variety of polymeric and lipid carriers has been successfully applied as a strategy for target specific delivery of siRNA to FR-bearing tumor cells.15, 21-23 In our previous study, an amphiphilic and biodegradable ternary copolymer conjugated with folate as ligand, polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol)-folate (PEI-PCL-PEG-Fol) was synthesized through a modular procedure as targeted gene delivery carrier.24 In this strategy, the folate-conjugated copolymers of PEI-PCL-PEG-Fol contained (1) PEI as a polycation to condense nucleic acids, (2) hydrophobic PCL to provide biodegradability and/or encapsulate drugs, (3) hydrophilic PEG to offer excellent biocompatibility and prolonged circulation, and (4) folate conjugated via PEG spacer to target FR-positive tumor cells. These triblock copolymers are expected to form micelle-like polyplexes with DNA/siRNA, exhibiting not only a core-corona structure with colloidal stability but also bearing folate ligands located on the micelle surface for efficient target recognition (Figure 1). Previous results concerning their ability to deliver pDNA demonstrated decreased cytotoxicity, enhanced cellular uptake and increased transfection efficiency compared to the non-folate conjugated PEI-PCL-PEG system.24 Additionally, PEI-PCL-PEG-based siRNA delivery systems have shown enhanced in vivo circulation times and improved cytoplasmatic delivery of siRNA.25 These copolymers therefore seem promising for targeted siRNA delivery. Hence, similar ternary copolymers of PEI-PCL-PEG-Fol were synthesized to explore their applications for siRNA delivery in this study. The physico-chemical properties such as siRNA complexation, complex stability, size, and zeta potentials were determined, and in vitro evaluations of cellular uptake and gene knockdown in FR-overexpressing SKOV-3 were


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performed. Furthermore, in vivo pharmacokinetics and biodistribution experiments of siRNA polyplexes were carried out by SPECT imaging and gamma scintillation counting in xenografted mice bearing FR-overexpressing tumors to demonstrate the targeted siRNA delivery by PEIPCL-PEG-Fol.

Figure 1. Chemical structure of PEI-PCL-PEG-Fol (A) and schematic illustration of the micellelike polyplex formation (B).

EXPERIMENTAL SECTION Materials


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Hyper branched polyethylenimine (hyPEI 25k Da) was provided by BASF. ε-Caprolactone was purchased from Fluka and purified by distillation under vacuum over CaH2 before use. Bifunctional poly(ethylene glycol) (HO-PEG-COOH, 3 kDa) was purchased from Rapp Polymere GmbH (Germany). Folic acid was obtained from Acros. Solvents and other reagents were provided by Sigma-Aldrich and used without further purification. AlexaFluor488 fluorescently labeled firefly luciferase (FLuc) dicer substrate interfering RNA (DsiRNA), aminemodified DsiRNA, hGAPDH and negative control (NegCon) DsiRNA was obtained from IDT (Leuven, Belgium). SYBR™ Gold was bought from Invitrogen. The chelator 2-(4isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (p-SCN-Bn-DTPA) was purchased from Macrocyclics (Dallas, TX, USA), and

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InCl3 from Covidien Deutschland GmbH

(Neustadt a.d. Donau, Germany). All solutions used for siRNA-coupling were treated with 0.1% diethyl-pyrocarbonate (DEPC). Synthesis and characterization PEI-PCL-PEG-Fol

was

synthesized

by

conjugating

azido-activated

folate

with

heterobifunctional diblock copolymer acrylated-PCL-b-PEG-alkyne, followed by coupling acrylated-PCL-b-PEG-Fol to PEI, as described in our previous study.24 All intermediate compounds and the resulting folate-conjugated ternary copolymer were characterized by 1H NMR and UV spectroscopy, which verified their structures and enabled calculation of their compositions. Preparation of micelleplexes A stock polymer solution of PEI-PCL-PEG-Fol was obtained by dissolving the copolymer in sterile water at a concentration of 1 mg/mL PEI25k and filtrated using a 0.22 µm syringe filter to


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maintain sterility. The stock solution was then diluted to precalculated concentrations with sterile water to prepare micelleplexes/polyplexes at different N/P ratios. Briefly, 50 µL of polymer dilution at a calculated concentration was mixed with an equal volume of 2 µM siRNA by vigorous pipetting, followed by incubation at room temperature for 20 min. Micelleplexes with the corresponding non-targeted copolymer PEI-PCL-PEG and polyplexes with PEI25k were prepared accordingly for comparison. SYBR Gold Assay SYBR Gold quenching assays were performed to evaluate the complexation between copolymer and siRNA as previously reported.26 Briefly, micelleplexes/polyplexes were prepared at different N/P ratios and incubated at room temperature for 20 min. Then, 100 µL of micelleplexes/polyplexes containing 0.2 nmol GAPDH-siRNA were added in opaque FluoroNunc 96-well plates, followed by the addition of 20 µL 4×SYBR™ Gold solution and incubation for another 10 minutes in the dark. The fluorescence was detected at 495 nm excitation and 537 nm emission using a fluorescence plate reader (BMG Labtech, Offenburg, Germany). The evaluation was carried out with triplicate samples and graphed as normalized average fluorescence percentage values ± the standard deviation (SD). The fluorescence of intercalating SYBR Gold with free siRNA represents 100%, while background fluorescence of SYBR Gold without siRNA represents 0%. Size and zeta-potential analysis The size of the siRNA-free micelles, that of micelleplexes and PEI polyplexes as well as the zeta potential of the micelleplexes and polyplexes were monitored by dynamic light scattering (DLS) and Laser Doppler Anemometry (LDA) using a Zetasizer Nano ZS (Malvern Instruments,


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Worcestershire, UK) equipped with a He-Ne laser source at a wavelength of 633 nm at 25 ºC. First, the hydrodynamic diameters of the sample were measured in a low volume cuvette of 100 µL. Then the micelleplex/polyplex solution (100 µL) was diluted with additional 600 µL of pure water to a final volume of 700 µL and transferred into a transparent zeta cuvette for zetapotential measurements at 25 ºC. Triplicate measurements were performed on each sample. Results are given as mean values ± SD.

Cell culture SKOV-3 cells (human ovarian carcinoma) were obtained from ATCC (LG Promochem, Wesel, Germany) and were cultured in folate-free RPMI-1640 medium supplemented with 10 % fetal calf serum (FCS) at 37 ºC in a humidified atmosphere containing 5 % CO2. Cytotoxicity assay SKOV-3 cells were seeded at a density of 8×103 cells/well in 96-well plates 24 h before the experiments. Predetermined concentrations of the polymer were prepared from the stock solution by serial dilution with cell culture medium containing FCS. Afterwards, the used cell culture medium was replaced by 200 µL of a polymer/medium mixture, and the cells were cultured for another 24 hours at 37 °C. Then, the medium was aspirated followed by the addition of medium without serum containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The plate was incubated at 37 °C in the dark for 4 h. After the medium was removed, 200 µL of DMSO was added into the wells to dissolve the formazan crystals formed by proliferating cells. The absorbance of the DMSO solution was measured using an ELISA reader (Titertek Plus MS 212, ICN, Eschwege, Germany) at wavelengths of 570 nm and 690 nm (for


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background correction), respectively. Relative viability was calculated compared with untreated cells as 100 % viability control. Data are presented as mean values (±SD) of four experiments. The IC50 values were calculated with Origin 8 using logarithmic fit. Cellular Uptake by Flow Cytometry SKOV-3 cells were seeded at a density of 8×104 cells/well in 24 well plates and cultured for 24 h. AlexaFluor488-labeled siRNA was used to prepare micelleplexes/polyplexes at N/P=5 as described above. The medium within the wells was exchanged for fresh, serum containing medium, and 100 µL of micelleplexes/polyplexes containing 50 pmol siRNA were added per well. In the folate competition studies, the cells were preincubated with normal RPMI-1640 medium (containing 1 mg/L folic acid) 1 h before micelleplexes/polyplexes were added. Cells were incubated at 37 °C for 4 h, followed by washing with PBS once, incubation with 0.4 % trypan blue solution for 5 min (to quench extracellular fluorescence) and were washed again. Afterwards, the cells were treated with 100 µL of trypsin per well. Subsequently, 900 µL of PBS solution containing 10 % FCS was added, and the cells were collected by centrifugation. Cell suspensions were fixed in 300 µL of Cellfix solution (BD Biosciences, San Jose, CA), and measured on a FACS Canto™ II (BD Biosciences, San Jose, CA) with excitation at 488 nm and the emission filter set to 530/30 bandpass. In each experiment, ten thousand viable cells were investigated. Results are presented as the mean value of triplicate samples. Confocal Laser Scanning Microscopy SKOV-3 cells were seeded at a density of 3×104 cells/well in 8 well chamber slides (Nunc, Wiesbaden, Germany) and cultured for 24 h. Micelleplexes/polyplexes at N/P ratio of 5 were


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prepared with AlexaFluor488 labeled siRNA as described above. Subsequently, cells were incubated with micelleplexes/polyplexes containing 50 pmol siRNA per well at 37 °C for 4 h. Cells were washed with PBS pH 7.4 and then incubated with 4% paraformaldehyde PBS solution for fixation. After the fixed cells were nucleus-stained with DAPI and membrane-stained with TRITC-conjugated wheat germ agglutinin (WGA) lectin (Invitrogen, Karlsruhe, Germany), they were embedded in FluorSave Reagent (Calbiochem, San Diego, CA) for confocal microscopy measurement with a Zeiss Axiovert 100 M microscope coupled to a Zeiss LSM 510 scanning device (Zeiss, Oberkochen, Germany). AlexaFluor488, DAPI and TRITC were excited with excitation wavelengths of 488 nm, 364 nm and 543 nm, respectively. The fluorescence of AlexaFluor488, DAPI and TRITC and their overlay were recorded seperately. RT-PCR The knockdown efficiency of micelleplexes/polyplexes containing siRNA against the housekeeping gene GAPDH was determined by RT-PCR. Lipofectamine™ 2000 (Invitrogen, Karlsruhe, Germany) (LF) was used as positive control, and all formulations containing NegCon siRNA were used as negative controls. SKOV-3 cells were seeded in 6-well-plates, respectively, (Nunc, Wiesbaden, Germany) at the density of 500,000 cells per well with 3 mL medium (containing 10% serum) 24 h before transfection. On the day of transfection, the old medium was changed with 1 mL fresh serum containing medium, and 100 µL micelleplexes/polyplexes prepared at N/P-ratio 5 containing 100 pmol siRNA (GAPDH or siNegCon) were added in each well. After 4 h of incubation at 37 ºC, the medium was replaced again with fresh medium. Cells were incubated for another 24 h before they were washed with PBS buffer and lysed with lysis buffer (PureLinkTM RNA Mini Kit, Invitrogen, Karlsruhe, Germany). The mRNA was isolated from culture cells according to the protocol of the PureLinkTM RNA Mini Kit (Invitrogen,


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Karsruhe, Germany) with additional DNase I (18U) digestion (Qiagen, Hilden, Germany). Finally, the concentration and purity of the extracted mRNA was measured using a spectrophotometer (Ultrospect 3000, Amersham Pharmacia Biotech Europe, Freiburg, Germany). Total RNA (1 µg) from each sample was reverse transcribed to cDNA in a 20 µL reaction with the First Strand cDNA Synthesis Kit (FERMENTAS, St. Leon-Rot, Germany) on a TGradient thermocycler (Biometra, Goettingen, Germany), and Real-Time PCR was performed using the QuantiFastTM SYBR™ Green PCR Kit (Qiagen, Germany) and the Rotor-Gene 3000 real-time PCR thermal cycler (Corbett Research, Sydney, Australia) as described before.25 QuantiTect Primer Assays (Qiagen, Germany) for human GAPDH and human β-actin for normalization were used respectively for every sample. Radiolabeling and purification of siRNA To investigate in vivo pharmacokinetics and biodistribution, micelleplexes/polyplexes were formed using radiolabeled siRNA. Amine-modiﬁed DsiRNA was labeled with p-SCN-Bn-DTPA and

111

InCl3 by a previously described method.13, 14, 27, 28 The radiolabeling was followed by size

exclusion chromatography (SEC) on PD-10 Sephadex G25 (GE Healthcare, Freiburg, Germany) to find the fractions, which showed both a high radioactive signal and the strongest UV absorption at 260 nm but did not contain any free p-SCN-Bn-DTPA or indium. Fractions were combined and further purified as well as concentrated using RNeasy columns.27 In vivo pharmacokinetics, biodistribution and SPECT imaging For in vivo experiments, 8-week-old female balb/c nu/nu mice were maintained on a folatedeficient diet (Altromin C 1027, Altromin, Lage, Germany) in order to reduce their serum folate to a level near that of human serum.29 All animal experiments were carried out according to the


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German law of protection of animal life and were approved by an external review committee for laboratory animal care. After an acclimation period of 21 days, the mice were inoculated subcutaneously with 0.1 ml of cell suspensions containing 1×106 SKOV-3 cells. Tumor growth was observed for 14 days, and animals were randomly distributed in groups of five. On the day of experiments, mice were anesthetized with xylazine and ketamine and injected i.v. with micelleplexes/polyplexes at N/P 5 containing 35 µg of 111In-labeled siRNA via the tail vein. The radiolabeled dose of 35 µg siRNA was equivalent to an average of 4.61 MBq per animal. For pharmacokinetic studies, 25 µL blood samples were drawn retro-orbitally at 1 min, 3 min, 5 min, 15 min, 30 min, 60 min, and 120 min after the injection and measured by gamma scintillation counting on a Packard 5005 Gamma Counter (Packard Instruments, Meriden, CT). Biodistribution was recorded non-invasively in anesthetized mice using three-dimensional Single photon emission computed tomography (SPECT) and planar gamma camera imaging (Siemens E.cam with custom made pinhole collimator, Siemens AG, Erlangen, Germany) 2 h, 5 h, and 24 h after treatment. For competition studies, 300 µg FA (30 µl, 10 mg/ml in PBS) was injected 30 min prior to administration of FA-conjugated polyplex. Finally, one set of animals were sacrificed 5 h after the injections, another set of animals was sacrificed 24 h after the treatment; the tumor and selected organs were dissected, weighed and analyzed for the deposition of radioactive siRNA using a Gamma Counter Packard 5005. The counts per minute (CPM) measured within excised organs was compared to a standard curve made of dilutions of 35 µg of 111

In-labeled siRNA and normalized to the injected dose. All biodistribution results are given as

percent of the injected dose (%ID) per gram of organ weight (%ID/g). Statistics


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In all analytical assays, results are presented as mean values ± SD. Significance between the means was tested by two way ANOVA. Statistical evaluation, calculation of the AUC and two way ANOVA were performed using Graph Pad Prism 4.03 (Graph Pad Software, La Jolla, CA).

RESULTS AND DISCUSSION Polymer synthesis The folate-conjugated ternary copolymer of PEI-PCL-PEG-Fol was synthesized as shown in Figure 1 through a modular procedure described previously. It contains an amphiphilic structure as hydrophilic PEG chains are coupled with hydrophobic PCL segments that are grafted onto branched PEI molecules. The folate moiety is conjugated to the distal PEG end. In our previous studies on PEI-PCL-mPEG for siRNA delivery, we found that the hydrophobic PCL segment and graft density exhibited a great impact on the siRNA binding affinity, polyplex stability, blood circulation and siRNA transfection efficacy, and PEI25k-(PCL570-PEG5k)5 with high graft density of PCL-PEG was more promising for gene silencing in SKOV-3 cells than PEI25k(PCL570-PEG5k)3 or PEI25k-(PCL570-PEG5k)1.25 PEI-PCL-PEG-Fol copolymers with a PCL molecular weight of 1000 Da, PEG spacer of 3000 Da, and graft density of five were accordingly designed here for targeted siRNA delivery and were synthesized successfully. The structure of the resulting copolymers was characterized and confirmed by 1H NMR spectroscopy (Fig. S1), and information on their composition is listed in Table S1 (See Supporting information). The folate content in PEI-PCL-PEG-Fol was found to be 6.7 × 10-8 mol/mg, obtained by UV absorbance at 360 nm, which accounts for a conjugate that can be described as PEI25k(PCL1000-PEG3k)5-Fol3. A PEI-PCL-mPEG copolymer with similar structure of five branches


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but without folate conjugation was also synthesized as control in the following physicochemical and biological assays.

Cytotoxicity Cytotoxicity studies were performed by incubating FR-positive SKOV-3 cells with PEI-PCLPEG-Fol and PEI-PCL-PEG copolymers at different concentrations and detection of the cell viability after 24 h. Results of cell viability of both polymers along with PEI25k are shown in Fig. S-2 and the calculated IC50 values are listed in Table 1. The IC50 value of PEI-PCL-PEG-Fol was found to be 0.0207 mg/mL in SKOV-3 cells, which was about 1.5-fold higher than that of PEI-PCL-PEG and 3-fold higher than the IC50 value of PEI 25kDa (pPDI>0.40 than PEI-PCLPEG micelles of around 180 nm with 0.55>PDI>0.41, most likely due to the rigid folate ligands.


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After condensation with siRNA at N/P=5, micelleplexes showed a significant decrease in hydrodynamic diameters compared to the micelles for both PEI-PCL-PEG-Fol and PEI-PCLPEG. The size distributions also became narrower (0.38>PDI>0.26). The decrease of hydrodynamic diameter could be explained by the strong interaction between the siRNA and cationic PEI, as reported by Mao et. al.31 Generally, the micelleplexes of PEI-PCL-PEG-Fol and PEI-PCL-PEG at N/P=5 were within a size range of 92-100 nm, which was comparable to the size of PEI25k polyplexes at around 108 nm. Our FR-targeted siRNA delivery system described here is therefore considerably smaller in size than previously reported examples based on chitosan or low molecular weight PEI that ranged between 220 nm32 and 250 nm.33 The sizes we observed were similar, however, to previously described PAMAM polyplexes coupled with cyclodextrin, PEG, and folic acid, which were 120 nm small.34 The zeta-potentials of PEI-PCLPEG-Fol micelleplexes were positive (+7-9 mV) and slightly lower than those of non-folateconjugated PEI-PCL-PEG micelleplexes (+15 mV) and PEI25k polyplexes (+14 mV). The decreased zeta potentials of the targeted vs. non-targeted micelleplexes probably may result from the shielding effect of folate ligands, which could also be a reason for the reduced cytotoxicity of the folate conjugated copolymer.


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Figure 3. Hydrodynamic diameters and zeta-potentials of copolymer micelles and the corresponding micelleplexes/polyplexes at N/P=5.

Cellular uptake Cellular uptake of micelleplexes/polyplexes was determined by flow cytometry to evaluate their folate receptor targeting efficiency. Fluorescently labeled siRNA was used to prepare micelleplexes/polyplexes from PEI-PCL-PEG-Fol, PEI-PCL-PEG and PEI 25k. The samples were incubated with FR-positive SKOV-3 cells. Figure 4 shows that the amount of the internalized siRNA, as determined from the mean fluorescence intensity of the cells, increased by more than 10% for the folate-conjugated micelleplexes (PEI-PCL-PEG-Fol) in comparison to folate-negative PEI-PCL-PEG micelleplexes (p < 0.05) in SKOV-3 cells. This finding is in line with Benoit et al. who also described a 10-20% increase of delivering fluorescent siRNA, in HeLa cells, with their targeted diblock nanocarrier in comparison to the non-targeted formulation.35 Taking into consideration that the targeted micelleplexes have a lower zeta potential than the non-targeted ones or the PEI polyplexes, which usually results in decreased uptake, these results demonstrate the targeting effect of the PEI-PCL-PEG-Fol micelleplexes. It should be noted that the amount of the internalized PEI-PCL-PEG-Fol/siRNA micelleplexes decreased to 81% of the mean fluorescence detected in cells incubated with PEI-PCL-PEGFol/siRNA in presence of free folate (p < 0.05) in the competition experiments. Benoit et al. also found very similar results in presence of 10 µg/ml free folic acid,35 whereas others reported more efficient competition at 40 µg/ml32 or 2-4 mM free folic acid,34 which is equivalent to about 9001800 µg/ml. Due to the higher valency of folate bearing micelleplexes compared to free folate,


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the competition with free folate is not expected to inhibit uptake completely as reported by others.35 In the ternary copolymers used here, a hydrophobic PCL segment was used as a linker between PEI and PEG to increase the biodegradability of the copolymers, which is also advantageous to affect the hydrophilic–hydrophobic balance of the polymer and thus to enhance the uptake of the complexes through cell membranes.17, 30 Therefore, delivery of siRNA is not exclusively mediated by the FR and can therefore not be inhibited completely by competition with free folate. Taken together, these results indicated FR-mediated endocytosis which was significantly enhanced for folate-conjugated micelleplexes and reduced in presence of free folate.

Figure 4. Cellular uptake of micelleplexes/polyplexes made of Alexa488-labeled siRNA into SKOV-3 cells after four hours of incubation as measured by flow cytometry.


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Furthermore, the subcellular localizations of micelleplexes inside cells were evaluated by confocal laser scanning microscopy. As shown in Fig. 5, SKOV-3 cells treated with the folateconjugated micelleplexes (PEI-PCL-PEG-Fol/siRNA) exhibited more and brighter green fluorescent dots than those with folate-negative micelleplexes (PEI-PCL-PEG/siRNA) or cells treated in presence of competing free folic acid. This observation may be attributed to the targeted uptake of folate-conjugated micelleplexes via specific FR-mediated endocytosis. Importantly, the confocal images also showed that fluorescently labeled siRNA was distributed within the cytoplasm mostly, which is a prerequisite to achieve functional RNA interference. The targeted cell uptake and optimized intercellular siRNA delivery into the cytosol mediated by PEI-PCL-PEG-Fol should benefit efficient siRNA delivery for functional gene silencing.

Figure 5. Confocal images showing the subcellular distribution of micelleplexes/polyplexes


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made of Alexa488-labeled siRNA (green) incubation with SKOV-3 cells for 4 h after transfection. DAPI-stained nuclei are shown in blue and TRITC-WGA stained membrane in red.

In vitro Gene Silencing To further investigate the targeting effect of siRNA delivery with PEI-PCL-PEG-Fol, in vitro gene silencing of GAPDH-siRNA containing polyplexes at N/P ratio of 5 was tested in SKOV-3 cells by the RT-PCR measurements. In comparison to previously reported FR-targeted delivery systems that were used at N/P 2433 or weight/weight ratio 50,32 we were able to use a comparably low N/P ratio while avoiding the a large excess of polymer. The commercial transfection reagent lipofectamine™2000 and PEI25k were chosen as the positive controls. Both GAPDH and betaactin are housekeeping genes expressed in all cells of an organism. As shown in Fig. 6, significant inhibition of GAPDH expression was obtained by PEI-PCL-PEG-Fol, and targeted micelleplexes enhanced the gene silencing efficiency by about 60% compared to non-targeted micelleplexes at the same N/P ratio. Moreover, their GAPDH silence efficacies were strongly decreased when cells were incubated in the folate-enriched medium, which indicates that free folic acid would inhibit the knockdown activity of folate-conjugated micelleplexes. Comparing our results to the recent literature, we obtained stronger inhibition of gene knockdown upon competition with free folic acid than others. Benoit et al. also used the housekeeping gene GAPDH for proof-of-concept experiments and reported 80% gene knockdown with their targeted delivery system at charge ratio 4:1 in comparison to 60% gene knockdown after competition with free folic acid.35 It is possible that the strong inhibition of gene silencing in our case, compared to the weak inhibition of uptake, reflects that targeted polyplexes that are taken


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up by mechanisms other than receptor-mediated endocytosis are trapped in intracellular vesicles and do not mediate gene knockdown. Our results indicate that PEI-PCL-PEG-Fol is a targeted siRNA vector that, in absence of high concentrations of competing folic acid, is most probably taken up via specific cellular folate-FR interaction that leads to better intracellular availability of siRNA than upon delivery with PEI-PCL-PEG. Interestingly, the siRNA transfection efficacy of PEI-PCL-PEG was lower than that of PEI 25k. This finding is in contrast with an earlier report where different PEI-PCL-PEG copolymers were used25 and with the uptake efficiency shown here. However, the possible reason may lay in the different endosomal escape efficacies between micelleplexes and polyplexes. Unmodified PEI is known to exhibit a stronger buffering capacity than modified PEI which helps the ‘proton-sponge-effect’.36 With the introducing of folic acid as targeting ligand, PEI-PCL-PEG-Fol showed significantly (***p < 0.001) higher gene silencing efficiency than PEI25kDa which could be a result of differences in the endocytosis mechanism. Additionally, PEI-PCL-PEG-Fol delivered siRNA showed an increased silencing efficiency over lipofectamine™2000 in SKOV-3 cells. The high knockdown activity of PEI-PCL-PEG-Fol most likely results from the targeted recognition of folate, the suitable size as well as the good stability, and the low cytotoxicity.


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Figure 6. siRNA-mediated GAPDH gene knock down in SKOV-3 cells. (***p

Efficient and Tumor Targeted siRNA Delivery by Polyethylenimine

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