Prostate cancer cell-specific BikDDA delivery by targeted polymersomes

In this paper, we report the polymersome-mediated intracellular delivery of pro-apoptotic BikDDA gene using two different peptide–copolymer covalent conjugation strategies specific for prostate cancer targeting. The BikDDA gene was used as a therapeutic agent on prostate cancer cells. The transfection efficiency of BikDDA-loaded poly[oligo(ethyleneglycol) methacrylate]-co-poly[2-(diisopropylamino) ethyl methacrylate] (P(OEG10MA)20-PDPA100) polymersomes revealed that they could serve as a suitable non-viral gene transfection tool. The targeted delivery of BikDDA into prostate cancer cells (LNCaP) using polymersomes was successfully carried out by conjugating the PSMA-targeting moiety (peptide 563) to P(OEG10MA)20-PDPA100 copolymer using either succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) as a bifunctional linker between the thiol-bearing targeting peptide and amino-bearing P(OEG10MA)20-PDPA100 copolymer or attaching a maleimide-modified targeting peptide onto a thiol-terminated P(OEG10MA)20-PDPA100 copolymer. The pH-responsive and biocompatible polymersomes, conjugated with peptide 563, exhibited an enhanced cellular uptake by LNCaP cells in comparison to the healthy prostate epithelial cell line PNT1A, thus indicating the cell-specific delivery. The increased Bik mRNA expression and cell death in these LNCaP cells indicates high effectiveness of the targeting polymersomes. According to these results, we believe more efficient gene delivery systems via specifically targeted pH-sensitive polymersomes can be a promising approach and promote the development of novel therapies against prostate cancer.


Introduction
Prostate cancer is the second most common type of cancer amongst men, with 1.1 million cases recorded worldwide (International Agency for Research on Cancer 2012). Depending on the patient's condition, the treatment of prostate cancer usually includes surgery, radiation therapy, hormone therapy, and/or chemotherapy. Although the conventional combination chemotherap ies are usually preferred by physicians, the heterogeneity of prostate cancer cells and toxicity/s ide effects of drugs on healthy cells are the limiting factors for the treatment (Beer and Raghavan 2000). Therefore, conventional treatment protocols require novel tools to overcome these problems. In order to eliminate the shortcomings of conventional therapies and to replace the standard treatment protocols, employment of the novel nanomaterials ranging from polymer / lipid based particles (e.g. polymersomes, dendrimers, micelles, mesoporous silica particles, and liposomes) to metallic particles (e.g. gold / silver particles), resulting in major improveme nts.
These nanoscale tools can be utilized to offer controlled drug release, enhance drugs' pharmacokinetic profiles, reduce dosage amounts and frequency, augment cell permeability, enable targeted molecule delivery and improve therapeutic efficacy/safety (Banik et al. 2016).
From this point of view, the targeted delivery of pro-apoptotic genes and/or drugs to specific cancer cells using nanomaterials represents a promising approach.
The Bik gene is a member of the Bcl-2 pro-apoptotic gene family and functions either by inducing the activity of other pro-apoptotic Bcl-2 genes, or by inhibiting the anti-apoptotic Bcl-2 proteins (Chinnadurai et al. 2009). For this reason, it has been proposed as an apoptosis-potentia ting therapeutic gene in human cancers (Zou et al. 2002). BikDD protein, a mutant form of Bik protein, behaves as the constitutively phosphorylated and active form of Bik protein, with an enhanced binding affinity to anti-apoptotic members. When overexpressed in human cancer cells, BikDD 4 exhibits a greater pro-apoptotic activity in comparison to its wild type counterpart, leading to a two-fold decrease in tumor growth in in vivo assays (Li et al. 2003;Lang et al. 2011). BikDD not only strongly induces apoptosis, but also inhibits cell proliferation when expressed in breast (Lang et al. 2011), pancreatic (Xie et al. 2007), liver (Li et al. 2010), and prostate cancers (Xie et al. 2014). In a recent work, the inoculation of male nude mice with prostate cancer cells expressing BikDD led to a reduction in tumor growth and extended survival with low toxicity (Xie et al. 2014). To increase the half-life of BikDD protein, the BikDDA gene was generated by mutating serine 124 of BikDD, which was shown to be a more efficient therapeutic gene than BikDD in the treatment of triple-negative breast cancer (Jiao et al. 2014).Thus BikDDA expresses a protein which exhibits an extended half-life and higher therapeutic efficacy than the product codified by the wild-type Bik or mutant BikDD (Jiao et al. 2014). Therefore, in our study, the BikDDA gene was loaded into pH-sensitive polymersomes functionalized with peptides that exhibit high affinity to prostate specific membrane antigen (PSMA) (Shen et al. 2013).
Over the past 20 years, the deepening of the understanding of polymer chemistry allowed scientists to engineer biologically-inspired vesicles formed by the self-assembly of copolymers, also called polymersomes (Discher et al. 1999). These are used for various applications, such as cargo delivery tools [for drugs (Ahmed et al. 2006), genes (Lomas et al. 2010) and proteins (Rameez et al. 2008)], nanoreactors (Gaitzsch et al. 2012), and for diagnostic purposes (Huang et al. 2015). In our previous reports we demonstrated the encapsulation and delivery of pDNA (Lomas et al. 2007(Lomas et al. , 2008Oz et al. 2019), mechanisms of cellular entry (Colley et al. 2014), rapid and effic ie nt endosomal release of the payload from PDPA-based polymersomes (Lomas et al. 2010). In addition, this study is the first application of pDNA-loaded targeted polymersomes as non-viral vectors for gene delivery.

Poly[oligo(ethyleneglycol) methacrylate]-co-poly[2-(diisopropylamino) ethyl methacryla te]
(P(OEG10MA)20-PDPA100, also denoted as POEGMA-PDPA) copolymer, was employed to produce polymersomes. The unique properties of P(OEG10MA)20-PDPA100 in terms of stealth ability in the blood stream and endosomal escape when internalized by cells, allowed developing smart polymersomes for intracellular cargo delivery. Upon cell-specific recognition of polymersomes, internalization by endocytosis occurs where polymersomes are confined in an endosomal membrane, forming early endosomes to start their cytosolic journey. Here, the endosome acid lumen (pH ~6) induces the fast disassembly of the polymersomes (PDPA pKa =6.2). The consequent dramatic increase in the number of species from one vesicle to its building blocks and cargo generates an osmotic shock, which leads to the temporary rupture of the endosomal membrane and the subsequent release of any encapsulated gene into the cytosol (Tian et al. 2015).
Active targeting of the cancer cells mainly exploits the overexpression of a receptor element on the plasma membrane so that the targeted particles are solely internalized by the related cancer cells. Here, we focused our attention on PSMA, a membrane-expressed and prostate-cancer specific target (Elsässer-Beile et al. 2009). To accomplish this objective, a small peptide, "peptide563" (GRFLTGGTGRLLRIS), which exhibits high binding affinity to PSMA (Shen et al. 2013), was conjugated to the P(OEG10MA)20-PDPA100 copolymer and used to decorate polymersomes for targeting prostate cancer cells.
We recently explored different metal-free conjugation methods for amphiphilic copolymers and concluded that click methods, such as ring-strain promoted azide-alkyne reaction (SPAAC) or Diels-Alder reaction of a 1,2,4-triazoline-3,5-dione (TAD) are extremely selective and effic ie nt (Gaitzsch et al. 2016). However, these methods are sometimes incompatible with pre-6 polymerization approaches in which their insaturation can interfere/or become degraded during radical polymerizations (Gaitzsch et al. 2016). Furthermore, click methods involve the dual modification of the copolymers and the targeted peptide with consequent complications. We observed that in aprotic solvents, the most efficient is the amine/NHS ester reaction, while in protic solvents, the thiol/Maleimide is the most effective (Gaitzsch et al. 2016). The latter is also better suited for most proteins and complex peptides where amine bearing lysine residuals are often involved in binding and protein folding while cysteines tend to be only structural and not functio na l (Ruoslahti 2012). We synthesized maleimide-bearing initiators for both the polymerization of PMPC and POEGMA copolymers (Tian et al. 2015;Gaitzsch et al. 2016) and while this strategy allows the functionalization of peptides, its limited yield permits only few ligands per polymersomes.
Our study demonstrates the targeted delivery potential of P(OEG10MA)20-PDPA100 polymersomes functionalized with peptide563, on prostate cancer cells. The copolymer-peptide conjugatio ns were successfully carried out utilizing different thiol/Maleimide strategies. After BikDDA encapsulation, the targeting and transfection efficacy of the polymersomes were investigated on human prostatic adenocarcinoma cells (LNCaP) and on the PNT1A cell line, which serves as the model for normal prostate epithelium. The transfection efficiency and cell specific delivery of the peptide563 modified P(OEG10MA)20-PDPA100 polymersomes was compared with pristine polymersomes to reveal the ability of cell specific gene delivery.  The resulting dispersion was freeze-dried giving a white powder. The resulting copolymer composition was analyzed by 1 H-NMR in CDCl3:CD3OD (3:1, v/v) and the polydispersity was determined by gel permeation chromatography in H2O + 0.25% v/v TFA. In a typical procedure ( Figure S4), maleimide terminated peptide563 (purchased from Proteogenix) (2.0 mg, 0.00087 mmol, 2 eq.) and PDPA100-P(OEG10MA)20-S-S-P(OEG10MA)20-PDPA100 (27.0 mg, 0.00044 mmol, 1 eq.) were placed in a flask. A complete dissolution was obtained by adding EtOH (4 mL) then purged with N2 for 1 h. Afterwards, triphenylphosp hine (0.2 mg, 0.00076 mmol, 1.5 eq.) was added and the solution was continually stirred for 48 h at room temperature. Then, the solution was passed through silica and dialyzed (MWCO 10 kDa)

Reaction of PDPA
against EtOH (two times) and then against H2O (three times).
The final dispersion of the conjugate was freeze-dried and conjugation was confirmed by fluorescence detection via HPLC analysis.

Reaction of NH 2 -P(OEG 10 MA) 20 -PDPA 100 with cysteine terminated Peptide563 via SMCC
First the synthesis and purification of cysteine terminated peptide563 were performed according to previously published procedures (Mustapa et al. 2009) and confirmed by Waters Micromass MALDI-TOF ( Figure S6) and analytical HPLC ( Figure S7).
Peptide conjugation to H2N-P(OEG10MA)20-PDPA100 was carried out via using succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) (Figure 1). SMCC exhibits thiol and amino sensitive groups on opposite sides of its structure; therefore, as a first step H2N-P(OEG10MA)20-PDPA100 (0.0075 mmol, 1eq) was dissolved in 2 mL of DMF and purged with nitrogen for 1 hour. Freshly prepared SMCC (0.015 mmol, 2eq) solution in 0.5 mL of DMF was added to the polymer solution and purged with nitrogen 30 min more. A few drops of N,Ndiisopropylethylamine (DIPEA) were added to the solution and left to react for 2 h at 25 °C. Then, purification was carried out by dialysis (MWCO 1kDa) against DCM for two days, during which time the dialysis medium was changed four times to remove the non-reacted SMCC. The product was dissolved in DMF again after DCM was evaporated from purified product by rotary evaporation.
In the second step of conjugation, targeting peptide sequence (powder form, 2 eq) was added in SMCC-H2N-P(OEG10MA)20-PDPA100 (1eq) solution in DMF and purged with nitrogen for 1 h then PPh3 (1.5 eq) was added in solution and left to react over 48 h under nitrogen. The peptide conjugated polymer was purified by dialysis (MWCO 10kDa) against pH 7.4 water for two days (dialysis medium was changed four times) and final product was obtained after freeze drying.

Preparation, purification and characterization of polymersomes
The polymersome formulations were prepared using the pH-switch method. To prepare the peptide563 targeted polymersome (tPS) formulations, a mixture of peptide563-P(OEG10MA)20-PDPA100 / ME-P(OEG10MA)20-PDPA100 at 1:9 ratio was used. Following a typical procedure, first, the polymer mixture was dissolved (10 mg/mL) in a pH 2.0 phosphate buffered saline (PBS). The pH was increased by adding 0.5 M NaOH at a 10 μL/min rate via a syringe pump. When the pH reached 6.0, the flow rate of pH 2.0 PBS was decreased to 5 μL/min until the pH reached 7.4. After this process, the dispersion was stirred for 1 day to stabilize the vesicles.
The polymersome formulation was then purified by sonicating the cloudy dispersion for 45 min to ensure that all the tubular formations were broken down into spherical vesicles, and then all the remaining aggregates and large particles were removed by centrifugation (1000 g, 10 min). The vesicle dispersion was purified by SEC via a Sepharose 4B column to remove micellar structures.
The polymer concentration was evaluated after the purification step by dissolving the polymer in PBS pH 2.0 and performing a UV-Vis spectroscopy measurement at 220 nm. The average diameter size and polydispersity of polymersomes (0.5 mg/mL) were analyzed using the Dynamic Light Scattering (DLS) method and the zeta potential was analyzed with electrophoretic light scattering method to assess the surface charge in 5 replicates.
The size, surface morphology and topology of polymersomes were visualized via Transmiss io n Electron Microscopy (TEM, Tecnai 10, Philips, Netherlands) at 80 kV. The visualization was carried out on the phosphotungustic acid (PTA) stained polymersomes (Lopresti et al. 2011), in which 5 µL of polymersome dispersion (0.5 mg/mL) was deposited onto glow-discharged carbon coated copper grids for 1 min. A 0.75% (w/v) PTA solution at pH 7.4 was used for staining. The excess amounts of solutions were blotted with filter paper and samples were dried under vacuum after each step. After the reaction was completed, unreacted labeling reagent was removed by EtOH precipitatio n. After purification, the encapsulated amount of BikDDA was analyzed by a fluorometer and presented as number of BikDDA per polymersome ).

Cell culture and measurement of cell viability
PNT1A and LNCaP cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum.
In addition, the cell viability of LNCaP cells transfected with the BikDDA gene loaded tPS (tPS-BikDDA) was evaluated after 72 h. For this purpose, cells were seeded in 96-well plates at 5 × 10 3 cells/well and subjected to an MTS assay at each 24 h interval following the standard protocol provided by the manufacturer. The absorbance was read with a microplate reader at 490 nm.

Fluorescence microscopy
LNCaP and PNT1A (1 × 10 4 cells/well) cell lines were treated with 25, 50, 75 and 100 µg/mL concentrations of PS-BikDDA or tPS-BikDDA. The cell images (from at least four random fields covering a minimum of 100 cells/field) were taken under the Zeiss Axio Vert.A1 fluoresce nce microscope at 20× objective at 24, 48 and 72 h following the treatment. The transfection efficie nc y was presented as the percentage ratio of EGFP expressing cells to the total number of cells in the field.
After 4 h incubation, for confocal microscopy cell monolayers were washed in serum free medium, fixed with 2% formaldehyde, stained with 5μg/mL of DAPI and visualized with a Zeiss LSM 700 confocal microscope. For flow cytometry, after analysis the incubation cells were dislodged from their substratum via trypsinization and analyzed immediately in a BD FACS Calibur flow cytometer.

Quantitative reverse transcription PCR (qRT-PCR)
The relative Bik/18sRNA mRNA expression using real time PCR in LNCaP cell line transfected with tPS-BikDDA, empty PS and non-treated LNCaP cells (control) were analyzed as described previously (Erdem et al. 2014). Primer sequences used for Bik were sense 5'-GAGACATCTTGATGGAGACC-3' and antisense 5'-TCTAAGAACATCCCTGATGT-3'. As a housekeeping gene to ensure equal loading 18sRNA reference gene was used and the data was analyzed using 18sRNA as normalization control.

Statistical analysis
Statistical analyses were performed using GraphPad Prism Software (version 6.01). Error bars were obtained represent the standard error of the mean, and the data sets were compared using ttest. The significance level was set at p < 0.05.

Synthesis of P(OEG 10 MA) 20 -PDPA 100 copolymers and peptide conjugation strategies
The synthesis of P(OEG10MA)20-PDPA100 copolymers (plain and bearing an amino or thiol end groups) was carried out according to previously published procedures (Lomas et al. 2007;Simón-Gracia et al. 2016). 1 H-NMR analyses were performed to confirm the molecular structures of the polymers ( Figure S1b, S2b and S3b). The molecular weight and distribution were determined by gel permeation chromatography (GPC) analysis, and a size distribution was observed with a polydispersity index of 1.2 ( Figure S5).
After synthesis, the copolymers were conjugated with peptides via two different strategies. For the first reaction, thiol terminated polymers were conjugated with a PMSA-targeting peptide sequence (Tamra-GRFLTGGTGRLLRISK-Maleimide), exploiting the one-pot synthesis reaction between the thiol group of the copolymer and the maleimide group of the peptide sequence (Gaitzsch et al. 2016;Simón-Gracia et al. 2016). As shown in Figure 2a, the P(OEG10MA)20-PDPA100 copolymer does not exhibit any florescence intensity, while the fluorescence peak of the Tamra-bearing peptide sequence was observed during the 13th min of analysis. The peptide conjugation to copolymer was confirmed (Figure 2) by detecting the fluorescence peak of Tamra by HPLC analysis using the fluorescence detector set at 541 nm excitation and 568 nm emissio n 15 wavelengths. The disappearance of the peptide's fluorescence peak at 13th min and observation of the strong fluorescence peak in the 18th min of the HPLC chromatogram (Figure 2a) of the peptidecopolymer conjugation reaction product, although the POEGMA-PDPA copolymer itself has no fluorescence signal, suggests that the Tamra-bearing peptide sequence was successfully conjugated to the copolymer. As the second conjugation strategy, the NH2-P(OEG10MA)20-PDPA100 copolymer was primarily reacted with the succinimide group of SMCC by the using NHS ester amidification reaction in DMF to functionalize the copolymer with the maleimide group, then purified by dialysis to remove unreacted SMCC. After this process, the purified copolymer product bearing maleimide group, was conjugated with synthesized cysteine terminated peptide563 via thiol-maleimide click reaction, in DMF again (Figure 1). HPLC analyses were performed to reveal the conjugation by setting the fluorescence detector to detect phenylalanine's excitation (257 nm) and emission (282 nm) wavelengths. The disappearance of the peptide's fluorescence peak at the 13th min and observation of the strong fluorescence peak in the 17th min of the HPLC chromatogram (Figure 2b) of the peptide-copolymer conjugation reaction product confirms the peptide-copolymer conjugation and indicates the successful utilization of SMCC as a linker for peptide-copolymer conjugation. The reason for the shifting of fluorescence intensity peaks from ~13min, to ~17 and 18 min (Figure 2a-b) is the increment of the total molecular weight after fluorescent peptide sequences were conjugated with copolymers.
The aim of proposing two counter-approaches for the same reaction is to bring a deeper insight to the commonly utilized thiol-maleimide conjugation strategy through presence of the same functional groups on different moieties and to reveal the application potential of SMCC as a crosslinker for conjugation compared to direct conjugation. Among the peptide-copolymer conjugation approaches, the advantage of using SMCC is to eliminate the challenges of maleimide terminated polymer synthesis, such as the necessity of protecting the maleimide group. However, the application of direct thiol-maleimide reaction for peptide-copolymer coupling was found to be advantageous compared to SMCC as a crosslinker, considering that the whole conjugation is completed in one-pot synthesis with higher yields and precludes the possibility of undesired reaction pathways leading to byproducts. Although we successfully achieved the synthesis of peptide-copolymer conjugates with both strategies, we preferred the utilization of the one pot synthesis thiol-maleimide coupling for further peptide-copolymer conjugations. Apart from the conjugation strategy, prostate cancer targeted polymersomes were formulated with Tamra-Peptide563-P(OEG10MA)20-PDPA100 conjugates since the presence of Tamra creates strong fluorescence intensity that is necessary in carrying out further microscopy and flow cytometry analyses.

Self-Assembly, BikDDA loading and characterization of polymersomes
The preparations for the self-assembly P(OEG10MA)20-PDPA100 polymersomes (PS) and peptide functionalized polymersomes (tPS) were all carried out using the pH switch method (Robertson et al. 2016) as presented in Figure 3. The mass ratio of targeting peptide to the P(OEG10MA)20-PDPA100copolymer used during the self-assembly was 10% (w/w). After the vesicle-forma tio n process, the polymersomes were sonicated and purified by centrifugation and size exclusio n chromatography (SEC) was used to eliminate unwanted structures. The spherical vesicular structure of polymersomes was confirmed by transmission emission microscope (TEM) imaging ( Figure 4a, 4b). The average polymersome diameter, measured by dynamic light scattering (DLS), was 181.6±3.4 nm for PS-BikDDA and 205.8±2.5 nm for tPS-BikDDA (Figure 4c, 4d), suggesting non-significant differences, which is in agreement with previous reports (Simón-Gracia et al. 17 2016). The polymersome surface was assessed by zeta potential analysis at 25±0.5 °C, was -1.54±0.31 mV for PS-BikDDA and -2.49±0.18 for tPS-BikDDA to be neutral for all formulatio ns Hence, we can infer that polymersome cellular uptake is controlled by the binding of the given peptide rather than unspecific interactions. The encapsulated amount of Cy5-labelled pEGFP-BikDDA plasmid into polymersomes was determined by HPLC using fluorescence detection, which showed the number of encapsulated BikDDA per polymersome as 1.2 for PS and tPS. The final BikDDA and polymer concentrations are presented in Table S2. The cytotoxicity assay results showed that P(OEG10MA)20-PDPA100 polymersomes did not exhibit any significant cytotoxicity on the PNT1A and LNCaP cell lines, confirming the biocompatibility of the vesicles as reported in earlier studies (Lomas et al. 2007). Recent studies demonstrated that polymersomes could serve as excellent gene delivery carrier (Lomas et al. 2010). The comparison of the formulated P(OEG10MA)20-PDPA100

Prostate cancer targeting specificity of tPS
polymersomes with its targeted form demonstrated that targeted P(OEG10MA)20-PDPA100 polymersomes could be used for in vitro cell specific gene delivery.

BikDDA loaded tPS (tPS-BikDDA) increase the levels of Bik target genes and cell death activity in the LNCaP cell line
The relative Bik/18sRNA mRNA expression of LNCaP cells treated and non-treated with tPS-BikDDA were analyzed using qRT-PCR. The results showed that LNCaP cells treated with tPS-BikDDA expressed the Bik gene twice as much as the untreated cells (Figure 8a). Cell viability analysis indicates that almost 50% of the LNCaP cell lines transfected with tPS-BikDDA were not vital after 72 h (Figure 8b). These results confirm that tPS-BikDDA is effective in increasing the Bik gene expression into LNCaP cells to induce cell death.

Conclusion
The purpose of this study was to develop novel peptide-copolymer conjugation strategies and demonstrate the application of pro-apoptotic BikDDA-loaded polymersomes as targeted gene delivery tools for prostate cancer cells. Peptide-copolymer conjugation was achieved by the reaction between maleimide-thiol groups and using SMCC as a linker between the peptide cysteine residual and a primary amine expressed in the copolymer. SMCC has recently been employed for conjugation in aqueous medium (El-Sayed et al. 2016) but we developed a novel reaction strategy which takes place in organic solvent (DMF) and demonstrated the peptide-copolymer conjugate by fluorometric HPLC detection.
The TEM images of electroporated and purified polymersomes proved that the integrity of the polymersome membrane structure was preserved after electroporation, whilst the transfectio n experiments indicated that BikDDA remained stable after electroporation without any degradation.
Both these results are in agreement with our previously published work .
Given that peptide563 has high binding affinity to PSMA protein, which is expressed on the surface of prostate cancer cells (such as LNCaP), it was used to direct P(OEG10MA)20-PDPA100 polymersomes specifically to LNCaP cells. Polymersomes decorated on their surface with peptide563, revealed specificity and high binding affinity to PSMA-expressing LNCaP cells in comparison to PNT1A cell line. PSMA-targeting peptide-decorated polymersomes were prepared and their cellular uptake by LNCaP and PNT1A cells were demonstrated. The targeted polymersomes not only exhibited a higher cellular uptake by LNCaP cells, but also were not internalized by PNT1A cells. This data might suggest that targeting peptide "peptide563" acts as a steric barrier against the polymersome internalization by PNT1A cells.

The overexpression of BikDD in various human cancer cells lines results in strong apoptosis
induction (Beer and Raghavan 2000;Li et al. 2003), and BikDD expression was recently shown to kill androgen dependent prostate cancer LNCaP cell lines (Lang et al. 2011 This study proposed different peptide-copolymer conjugation strategies and demonstrates the effectiveness of targeted P(OEG10MA)20-PDPA100 polymersomes loaded with BikDDA to enable cell specific delivery with enhanced gene transfection ability. This represents a novel, nontoxic and highly effective gene therapy strategy for the prostate cancer LNCaP cell line.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.