Folate receptor‐targeted aminoglycoside‐derived polymers for transgene expression in cancer cells

Abstract Targeted delivery of anticancer therapeutics can potentially overcome the limitations associated with current chemotherapeutic regimens. Folate receptors are overexpressed in several cancers, including ovarian, triple‐negative breast and bladder cancers, making them attractive for targeted delivery of nucleic acid therapeutics to these tumors. This work describes the synthesis, characterization and evaluation of folic acid‐conjugated, aminoglycoside‐derived polymers for targeted delivery of transgenes to breast and bladder cancer cell lines. Transgene expression was significantly higher with FA‐conjugated aminoglycoside‐derived polymers than with Lipofectamine, and these polymers demonstrated minimal cytotoxicty. Competitive inhibition using free folic acid significantly reduced transgene expression efficacy of folate‐targeted polymers, suggesting a role for folate receptor‐mediated uptake. High efficacy FA‐targeted polymers were employed to deliver a plasmid expressing the TRAIL protein, which induced death in cancer cells. These results indicate that FA‐conjugated aminoglycoside‐derived polymers are promising for targeted delivery of nucleic acids to cancer cells that overexpress folate receptors.

Aminoglycosides are a group of antibiotics which are used in the treatment of various bacterial infections, 26,27 although their use is somewhat limited because of concerns regarding nephrotoxicity. These molecules typically contain three to five sugar moieties, each of which contain multiple amines and/or hydroxyls. Aminoglycosides demonstrate antibacterial activity through selective binding to the bacterial ribosomal RNA regions. 28,29 In addition, aminoglycosides have also been shown to have the capability to bind eukaryotic RNA and plasmid DNA (pDNA). [30][31][32][33] Given their chemical diversity, we employed aminoglycosides as starting materials for the generation of bioseparation ligands, 30 DNA-binding ligands, 30,33 and microbeads. 34 Recently, we employed parallel synthesis and chemical informatics modeling for the generation and rapid identification of aminoglycoside-derived polymers and lipopolymers for delivering transgenes to cancer cells. [35][36][37][38] These studies have led to the identification of polymers and lipopolymers that demonstrate higher efficacies for transgene expression than commonly used reagents (e.g., branched pEI).
In the present work, we have developed a targeted delivery approach with an eye toward facilitating the selective delivery of pDNA to cancer cells. In particular, we hypothesized that modification of efficacious aminoglycoside-derived polymers with FA would be a powerful approach for delivery to cancer cells that overexpress the FR (Scheme 1). Four aminoglycoside-derived polymers were derivatized with FA and their efficacy for transgene expression was evaluated in breast and bladder cancer cells. Effective polymers were then evaluated for delivering a plasmid that expresses the tumor necrosis factoralpha related apoptosis-inducing ligand (TRAIL) protein, and the extent of cancer cell death was determined.

| Synthesis of aminoglycoside-derived polymers
The synthesis and characterization of aminoglycoside-derived polymers (neomycin-GDE, paromomycin-GDE, neomycin-RDE, and paromomycin-RDE) were based on modifications made to methods described in our previous reports. 37 Briefly, sulphate-containing aminoglycosides neomycin and paromomycin were dissolved in nanopure water and passed through a Clion exchange resin for 3 hr. The resulting sulphatefree aminoglycosides were reacted with GDE or RDE in a 1:2.2 molar ratio in a solvent mixture of water and N,N-dimethylformamide (DMF), (1.5:1 v/v) for 5 hr at 608C. The reaction mixture was precipitated by using acetone, and was further purified by dialysis using a 3.5 kDa molecular weight cutoff (MWCO) dialysis membrane for 48 hr to remove unreacted aminoglycosides and diglycidylethers. The dialyzed material was lyophilized to obtain the purified polymers. Neomycin-GDE, paromomycin-GDE, neomycin-RDE, and paromomycin-RDE polymers are abbrevated as NG, PG, NR, and PR, respectively. These untargeted polymers and are also called parental polymers collectively in subsequent usage. were added to this flask and the contents were stirred for 30 min at room temperature. A unique polymer solution (i.e., NG, PG, NR, or PR) in DMSO was added to this reaction mixture, and the contents were stirred for 48 h at room temperature. The FA-conjugated polymer product was obtained following precipitation with diethyl ether. The resulting polymer product was dissolved in water and dialyzed against Nanopure water for 48 hr using a 3.5 kDa MWCO tubing. The dialyzed material was lyophilized to obtain the final FA-conjugated polymer products, which were abbreviated as NGFA, PGFA, PRFA, or NRFA (FA 5 folic acid).
Structural information on FA-conjugated polymers dissolved in DMSO-d 6 was obtained using 1 H NMR spectroscopy (Varian 500 instrument operating at 500 MHz in the Fourier transform mode); tetramethyl silane was employed as an internal reference. FT-IR spectra were recorded with a Bruker IFS 66V/S 32 mm instrument using a round cell window (KBr), to further confirm FA conjugation onto parental aminoglycoside-derived polymers.

| GPC for molecular weight measurement
GPC was used to measure the molecular weights of parental and FAconjugated polymers. The Waters 1515 GPC system is equipped with an ultrahydrogel 250 column (Waters Corporation, MA) and a refractive index detector (Waters 2410). An aqueous solvent containing 0.1% trifloroacetic acid and 40% acetonitrile was used as the mobile phase. The mobile phase in the column was operated at a flow rate of 0.5 mL/min, and the column temperature was set at 358C. Poly(2-vinylpyridine) standards (MWs: 3,300, 7,600, 12,800, 35,000, and 70,000 Da) were employed for molecular weight (MW) calibration. All chromatograms were analyzed using the Waters Millennium 32 GPC software.
To further investigate the MW range, the parental polymers were first dialyzed through a 3.5 kDa MWCO membrane to remove any unreacted monomers. Following this first step for 48 hr, the retentate was dialyzed through a 10 kDa MWCO membrane. Samples of retentates from the 3.5 and 10 kDa MWCO dialysis membranes were subjected to ninhydrin analysis which reports for reactive amine content.
A calibration using glycine as a standard was used to determine the amine content of polymers, and the relative amounts of amines left behind in the retentates were compared to determine the MW range of the synthesized polymers.

| Preparation of polymer-pDNA complexes (polyplexes)
Stock solutions of parental (untargeted) and FA-conjugated (targeted) polymers were prepared at a concentration of 2 mg/mL in 13 PBS (pH 7.4). These solutions were filtered through 0.2 mm filters before use. pDNA stock solutions were prepared in EB buffer (Qiagen, Germany) at a concentration of 50 ng/mL. For transgene expression studies, a total of 75 ng pDNA were complexed with varying amounts of polymers in 1X PBS buffer for 20 min, resulting in polyplexes at different polymer:pDNA weight ratios (w/w) ranging from 1:1 to 100:1.

| Competitive inhibition of transgene expression with soluble FA in media
Cells were treated with free FA prior to the delivery of polyplexes as a competitive inhibition assay, to investigate the role of FR in transgene expression efficacy. MDA-MB-231 and UMUC3 cells were pre-treated with 5 mM free FA [39][40][41] in media for 6 hr, removed media and washed with 1 X PBS, followed by treatment with FA-conjugated polymers and unconjugated parental polymers at an optimal polymer: pGL4.5 weight ratio of 25:1. After 48 hr, cells were investigated for their transgene (luciferase) expression efficacies in the presence of serum. As before, Lipofectamine-3000 was used as a standard for comparison. The concentration of FA added to the media was significantly higher than that originally present in the media formulation from the vendor (FA in media 5 9 mM).

| Immunostaining for folate receptor alpha
Expression of FR-a in UMUC3 cells was visualized using immunostaining, and T24 human bladder cancer cells were used as the positive control since expression of the FR is known in these cells 42,43 ; in addition, the expression of FR-a in MDA-MB-231 cells is well established. 44,45 300,000 UMUC3 or T24 cells/well were seeded onto cover slips and incubated for 24 hr and stained using 1:100 dilution of primary antibody against FR-a (Mouse API3005AA, Biocare Medical) at room temperature followed by incubation with a 1:200 dilution of MACH 4 anti-mouse probe (Rabbit UP534G, Biocare Medical) at room temperature for 1h. The cells were then incubated with an FITC-tagged antirabbit antibody using 1:400 dilution (Goat Alexa Fluor-488, Thermo-Fisher Scientific) for 1 hr. The cells were twice washed with 13 PBS containing 2% FBS for 15 min before and after each antibody incubation step and visualized using a Leica TCS SP5 AOBS Spectral Confocal Microscopy System using 103, 203, and 403 (oil immersion) objectives; cells were excited at 488 nm and emission wavelengths of 530 nm were recorded. The same procedure was followed without the addition of the primary antibody as a negative control in these studies.

| Cytotoxicity of polyplexes
The MTT ((3-(4,5-Dimethylthiazol-2-yl)22,5-diphenyltetrazolium bromide)) cell viability assay is a metabolic assay used to determine cell proliferation, and can be employed as an indirect reporter for cell viability. The MTT assay was employed for determining the cytotoxicity of polymer:pDNA complexes (weight ratios 5:1 to 25:1) in MDA-MB-231 and UMUC3 cells. Cell seeding and polyplex formation procedures were similar to those described in section 2E. Untreated control wells were treated only with media (i.e., no polymer). After 48 hr, 10 mL of the MTT reagent were added to the cells and they were incubated for 3 hr at 378C following which, 50 mL methanol:dimethylsulfoxide (1:1) were added and incubated at room temperature for 30 min. After incubation, contents in the wells were thoroughly mixed, and absorbance at 570 nm was determined using a plate reader (Bio-Tek Synergy 2).

| Construction of the pEF-TRAIL plasmid
To construct the pEF-TRAIL plasmid, full-length human TRAIL was first PCR-amplified from the pEGFP-TRAIL plasmid, kindly provided by Prof. Christina Voelkel Johnson and described here. 46 Figure S1).

| Delivery of pEF-TRAIL to cancer cells using FAconjugated aminoglycoside-derived polymers
We determined the loss of viability in MDA-MB-231 and UMUC3 cells following delivery of the pEF-TRAIL plasmid using NR, PR, NRFA and PRFA polymers. Two optimal polymer:pDNA weight ratios, 15:1 and 25:1, were employed in these experiments. The pEF-GFP expression vector, which expresses EGFP, was employed as a control in the study since it expresses EGFP which should not induce death in cancer cells.
Polyplex formation and other experimental procedures employed in these studies were similar to that described previously in this section.    These results serve to further confirm molecular weights determined using GPC (Table 1).    Although cationic polymers are effective in facilitating high levels of transgene expression, their cytotoxicity can limit their application.

| Transgene expression efficacy and cytotoxicity of FA-conjugated polymers
For example, polyethyleneimine (pEI) can be an effective gene delivery polymer in certain cases, but its high toxicity is a limiting concern. 47-50 FA-conjugated polymers and parental polymers demonstrated negligible toxicities even at relatively high concentrations employed (e.g., polymer:pDNA weight ratios of 25:1; Figure 3). The cytotoxicity of FAconjugated polymers was similar to that of unconjugated polymers.
Taken together, our results indicate that FA-conjugated polymers demonstrate higher transgene expression efficacies compared to their

| Delivery of TRAIL-expressing pDNA
TRAIL is a protein that has been shown to induce programed cell death in cancer cells with minimal effect on normal cells, which makes it an attractive therapeutic for cancer diseases. 46

| DISCUSSION
The FR is overexpressed on several cancer cell types including breast, ovarian, endometrial, brain, and bladder cancer. 16,25,42 FA is a small molecule (441 Da) and is internalized in cells either by binding to FRs alpha, beta, and gamma (FOLR1-3) or by a solute carrier protein SLC19A1 (RFC-1) that can transport folate via a proton pump. 23,56 Targeting the FR using FA is an attractive strategy for delivering therapeutic cargo (e.g., small molecules and nucleic acids) specifically to cancer cells. 25 Folate targeting has also been previously explored for delivering nucleic acids (antisense oligonucleotides, pDNA, or siRNA) using different macromolecular formulations, [57][58][59][60][61][62][63][64] and folate-targeted lipids have also been investigated for delivering genes in vivo. 65 In addition, folatetargeted therapeutics have been investigated in clinical trials, which indicates the translational promise of this strategy. 66 We have previously demonstrated that aminoglycoside-derived polymers and lipopolymers demonstrated effective levels of transgene expression. [36][37][38] Although aminoglycoside antibiotics can be nephrotoxic in their mono- Our results indicate that FA conjugated, aminoglycoside-derived polymers may be promising vehicles for delivering nucleic acids and imaging agents to cancer cells that overexpress this receptor.