Spermidine and Chloroquine Enhance Peptide-Based pDNA Transfection Efficiency within the GRP78-Overexpressing DU145 Prostate Cancer Cells

doi:10.21203/rs.3.rs-4966373/v1

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Spermidine and Chloroquine Enhance Peptide-Based pDNA Transfection Efficiency within the GRP78-Overexpressing DU145 Prostate Cancer Cells

https://doi.org/10.21203/rs.3.rs-4966373/v1

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This study describes a peptide-based formulation for plasmid gene (pDNA) delivery within the GRP78-overexpressing prostate cancer (DU145) cells. The GRP78-targeting sequence (WIFPWIQL) extended with the nona-arginine (R9) cell-penetrating peptide (WIFPWIQL-R9) effectively enabled capture and release of pDNA, while conferring serum stability, according to agarose gel electrophoresis. Characterization studies based on transmission electron microscopy revealed the formation of stably condensed peptide:pDNA nanoparticles (<200 nm) at excess peptide nitrogen (N) to pDNA phosphate (P) stoichiometric ratios (10:1 N/P) and with CaCl₂ functioning as an ionic stabilizer, that rendered the nanoparticles applicable to cell biology. Confocal imaging of the FITC-labeled peptide:pDNA formulation indicated cell uptake and intracellular entrapment within endosomes that restricted pDNA gene expression in the DU145 cells. Optimization studies with a reporter plasmid Green Fluorescent Protein (pGFP) revealed spermidine and chloroquine as the most effective additives for enhancing peptide-based transfection efficiency. Furthermore, blocking with anti-GRP78 confirmed the GRP78-dependent mechanism for cell uptake. Thus, peptide-targeting of GRP78 allows for selective binding and entry within the GRP78-overexpressing cancer cells for gene (pDNA) delivery. Transfection of the tumor suppressor p53-expression vector using the optimized peptide-based transfection conditions revealed elevated levels of p53 within the DU145 prostate cancer cells. These findings indicate the potential anti-cancer utility of the GRP78-targeting peptide gene (pDNA) delivery system.

cancer-targeting peptides

GRP78

polyarginine

cell-penetrating peptides

chloroquine

spermidine

pDNA

Peptides form an interesting class of small- to intermediate-sized biomolecules that can be readily synthesized by conventional solid-phase synthesis procedures (Coin et al. 2007).¹ Peptide modifications (peptidomimetics), including the incorporation of un-natural amino acids, cross-linking agents, spacers and linkers, as well as branching and cyclization motifs that also template supramolecular self-assembly have produced a vast array of synthetic peptide mimicry for investigating structure-activity relationships (Wynne et al. 2024).² Furthermore, synthetic peptides have been combined with various (bio)molecular (e.g., nucleic acids, polysaccharides, lipids and proteins) probes and cargo (e.g., fluorophores, quantum dots, nanoparticles, and radionuclides) that enables diagnostic visualization and homing capabilities with receptor and cellular targets (Ilies et al. 2024).³ These include the cell-penetrating and cell-targeting peptides (CPPs and CTPs) functionalized with biomolecules, synthetic polymers and small molecule ligands that have been used for cell uptake and intracellular biological activity studies (Shah et al. 2020).⁴ Arginine-rich CPPs have shown favorable cell translocation activities due to the cationic guanidinium side chain group of arginine that confers favorable electrostatic interactions with the negatively charged phospholipid membrane bilayer of cells (at physiological pH ~ 7). Cell permeability for arginine-rich cell-penetrating peptides occurs via a variety of cell uptake mechanisms. Arginine-rich CPPs can demonstrate sequence- (polyarginine length) and concentration-dependent cell uptake by membrane recognition, destabilization, pore formation and translocation into the cytosol of the cell (Herce et al. 2009).⁵ The CPPs can also interact favorably with a variety of cell membrane-spanning glycoprotein receptors (e.g., heparan sulfate proteoglycans) that favor energy-dependent caveolae and clathrin-mediated endocytosis as well as macropinocytosis cell uptake pathways (Madani et al. 2011).⁶ The inclusion of intracellular localization domains in cell-penetrating peptides, such as mitochondrial (MLS) and nuclear localization sequences (NLS), enables peptide intracellular localization into specific organelles (Xu et al. 2016 and Jean et al. 2016).^7,8 Considering the amphiphilic, polycationic nature of the CPPs, they can also favor stable electrostatic interactions with negatively charged nucleic acids (DNA and RNA), creating condensed ionic complexes suitable for the delivery of genetic therapies in cells, tissues and in vivo (Cardoso et al. 2013 and Lehto et al. 2011).^9,10 Of particular interest are the arginine-rich peptides that have been investigated for their ability to condense plasmid DNA (pDNA) into small, easy to transfect nanoparticles for gene expression. For example, the TAT (RKKRRQRRR) CPP formed a stable complex with pDNA, that effected cell-uptake and entry into the nucleus with a nuclear localization sequence (NLS, PKKKRKV) resulting in vascular endothelial growth factor (VEGF) expression and angiogenic effects in a hindlimb ischemia in vivo model (Qu et al. 2013).¹¹ A comparative study in between various arginine-rich CPPs, indicated that the TAT and polyarginine (R9), peptides improved cell uptake and expression of the Green Fluorescent Protein (GFP) reporter in the HEK-293 (a virus-immortalized kidney cell line) and HCC827 (a human lung cancer cell line) (Milech et al. 2015).¹² The introduction of non-polar/hydrophobic residues (Trp, His, and Ala), polymer scaffolds, peptide stapling, branching and cyclization from medium-to-large-sized macrocycles produce modified arginine-rich peptides with enhanced cell uptake, endosomal escape and gene expression capacity (Szabo et al. 2002).¹³ Despite their cell-based and in vivo activities, the arginine-rich CPPs remain non-specific gene delivery agents, thereby limiting efficacy and raising toxicity concerns in therapeutic applications (Zhang et al. 2015).¹⁴

The CTPs function as an important class of cancer-targeting agents, that selectively bind to cancer cell surface receptors for specific detection and treatment of tumors (Zhang et al. 2023).¹⁵ A variety of selection methods, such as phage display biopanning, combinatorial library and parallel (micro)array peptide screening, as well as in silico molecular modeling and docking studies have led to the discovery of novel CTPs against key receptor and cellular cancer targets (Todaro et al. 2023 and Liu et al. 2017).^16,17 The CTPs have been conjugated with a variety of imaging and therapeutic agents for cancer-targeted therapy (Yang et al. 2022).¹⁸ Of specific interest to this study, the W peptide sequence, WIFPWIQL, is a GRP78-targeting peptide capable of binding to cell surface Glucose Regulated Protein 78 (csGRP78), an endoplasmic reticulum stress-inducible chaperone protein that is overexpressed and translocated to surface of a variety of cancer cell lines, but is absent or minimally presented on the surface of non-malignant cell types (Arap et al. 2004).¹⁹ The W-peptide selected by phage display biopanning to target csGRP78 on prostate and breast tumor tissues (Arap et al. 2004),¹⁹ demonstrates specific GRP78 binding and not to other related, homologous chaperones (e.g., heat shock proteins 70, Hsp70, and 90, Hsp90), moderate dose (50 µM) binding activity with rapid association/dissociation kinetics (k_on/k_off) according to surface plasmon resonance (Wang et al. 2016),²⁰ at the peptide binding domain of GRP78, based on molecular modeling and docking studies (Wang et al. 2016 and 2018).^21,22 The W-peptide also displayed GRP78-specific binding on the surface of prostate (DU145) cancer cells, with decreased binding detected in competition with soluble GRP78 and a blocking polyclonal anti-GRP78 (Arap et al. 2004).¹⁹ Accumulation of the W-peptide was also observed in the DU145 cells and at the localized tumor site within mice xenograft models, with notable regression in tumor growth upon administration of the chimeric W-peptide and the pro-apoptotic (D-(KLAKLAK)₂) sequence (Arap et al. 2004).¹⁹ Despite its in vitro and in vivo anti-cancer activities, the GRP78-targeting W-peptide retains poor cell uptake efficiency and is unable to deliver gene therapeutics directly into cancer cells.

To address this limitation, this work reveals that the addition of the CPP sequence, R9, to the GRP78-targeting W-peptide, produces a novel polyarginine-derived GRP78-targeting peptide sequence (FITC-Ahx-WIFPWIQL-GG-R9-GG, Fig. 1A). Peptide synthesis and characterization have been reported in our previous study (Daniel et al. 2024).²³ This peptide is also conjugated to the fluorochrome fluorescein isothiocyanate (FITC), linked to an aminohexanoic acid (Ahx) linker at the N-terminus for imaging cell binding and permeability of the peptide in combination with pDNA expression vectors. The incorporation of Gly (-GG-) spacers in between the CTP (W-peptide) and CPP (R9) domains and at the peptide C-terminus provides spatial distance in between each peptide functional domain that enables gene delivery directly within csGRP78-presenting DU145 prostate cancer cells (Fig. 1B). Formulation studies of the peptide:pDNA (nano)particles examined the influence of stoichiometry (peptide nitrogen to pDNA phosphorus (N:P) ratios), annealing buffer and serum stability in media, as well as the role of additives (e.g., CaCl₂, heparin, spermidine and chloroquine, among others) on the formation of stably condensed, and reversible peptide:pDNA ionic complexes, that function as dynamic capture and release gene delivery systems. This characterization study using mock (pUC19 and pBR32) and reporter (pGFP) plasmid (pDNA) expression vectors led to the discovery of an optimized peptide-based transfection protocol. Peptide-based transfections of the wild-type p53 tumor suppressor expression vector within the GRP78-presenting DU145 prostate cancer cells indicated noticeable p53 induction, underscoring their promising anti-cancer utility.

Formulation of Peptide:pDNA Nanoparticles

The optimization of the peptide:pDNA formulation was initially based on screening the favorable stoichiometry of the polycationic peptide nitrogen to the polyanionic pDNA phosphorous (1:1-20:1, N:P) mole ratios that promotes stable peptide:pDNA ionic complex formation in annealing (50 mM Tris-HCl and 150 mM NaCl, pH 7.5) buffer. An agarose gel-shift electrophoretic mobility assay in between peptide (polyarginine CPP, R9, vs. CTP-CPP, W-R9) and the pUC19 (2,686 base-pair) pDNA vector, revealed complete disappearance of the pDNA band alone and formation of a more retained complex with excess (10-20:1) N:P peptide:pDNA ratios that effectively form stable ionic complexes (Fig. 2A). This provides a binary approach for confirming the ideal N:P stoichiometric ratio in buffer, by demonstrating formation of the more retained, higher-order peptide:pDNA ionic complex that also excludes external dye (ethidium bromide) binding and staining of the pDNA vector. A similar outcome was observed with the FITC-labeled R9 peptide and the larger (4,361 base-pairs) pBR322 pDNA, that also suggests samples in unbound (FITC-R9) vs. bound (FITC-R9:pDNA) states, highlighting the versatility of the peptides to form stable complexes with various-sized vectors (Fig. 2B). Interestingly, these results also show that the hydrophilic, polycationic R9 CPP can effectively form stable ionic complexes with pUC19 at a 1:1 N:P ratio, presumably due to the unimpeded electrostatic interactions, while the amphiphilic W-R9 CTP-CPP requires a 10:1 N:P ratio to form the stable peptide:pDNA complex (Fig. 2A). The hydrophobic GRP78-targeting W peptide sequence may hinder the formation of stable peptide:pDNA ionic complexes at stoichiometric equivalence. However, this limitation is overcome with excess peptide:pDNA N:P ratios, while the amphiphilic nature of the W-R9 peptide may also enable more favorable csGRP78 receptor and membrane interactions for gene delivery (Lozanda et al. 2023 and Alkhamy et al. 2016).^24,25

An optimal N:P stoichiometric ratio is thus essential for achieving efficient peptide condensation of pDNA, while maintaining peptide:pDNA ionic complex stability that may also ensure protection against enzymatic degradation that prolongs transfection efficiency. The stability of the peptide:pDNA complex was next investigated against enzymes such as nucleases or proteases present within serum containing media used in cell culture transfection conditions (Adami et al. 1998).²⁶ The peptide:pDNA samples were incubated in Eagle’s Minimal Essential Medium (EMEM) containing 10% fetal bovine serum (FBS), and aliquots at various time points (1-24 h.) were collected and analyzed on agarose gel electrophoresis (Fig. 2C). The stability of pUC19 in FBS:EMEM at short (1 h.) and extended (24 h.) incubation times indicated some changes in the integrity of the pDNA band, by the appearance of more-retained, smear of bands with slower electrophoretic mobility on gel vs. pUC19 alone (Fig. 2C). This may be an indication of nick(s) in the pDNA resulting in the formation of linear, knotted or catenated DNA structures that transitions circular into open-chained DNA with slower electrophoretic mobility compared to the unnicked condensed circular pDNA vector (Cebrian et al. 2015).²⁷ This outcome confirms that the pDNA is unstable in the FBS-containing media conditions. In contrast, the peptide:pDNA complex (W-R9:pUC19) shows good stability in FBS:EMEM throughout the entire 24 hour incubation period (Fig. 2C). This sample displayed only very faint, slower moving pDNA bands, with no visible changes in intensities over the 24-hour incubation period in FBS:EMEM. The faint bands may be due to minimal quantities of unbound pDNA, that were absent of stable ionic complex with peptide and enzyme exposed for degradation. However, the W-R9 peptide sufficiently forms stable ionic complex with pDNA and provides substantial protection from enzymatic digestion in serum-containing media during the 24-hour incubation period, thereby bolstering its potential transfection utility.

Heparin is a polyanionic sulfated glycosaminoglycan polysaccharide that is commonly used in displacement assays to confirm the reversible capture-release nature of non-covalent, ionic complexes (Ryu et al. 2011).²⁸ In this assay (Fig. 2D), heparin sodium salt was added to outcompete the binding capacity of the cationic peptide (W-R9 and R9) for the anionic pDNA (pUC19), thereby releasing the pDNA vector in its free form. The heparin displacement assay validates the reversibility of the peptide:pDNA ionic complex, that is applicable to the capture and release of pDNA for gene expression. Heparin competitive binding displacement of the pUC19 pDNA vector from the peptides, W-R9 (Fig. 2D) and R9 (Fig. S1, ESI), in a dose-dependent (1-10 mg) manner, was confirmed by the reappearance of the pDNA band upon heparin addition, when compared to the peptide:pDNA ionic complex (10:1 N:P ratio) without heparin. The fainter pUC19 band intensities with lower-dose (1 µg) compared to higher-doses (2.5-10 µg) of heparin indicates that some peptide remains bound to the pUC19 at lower heparin treatment conditions, and with complete displacement of pDNA with increased addition of heparin. This result aligns with literature precedence, showing the ability of heparin to displace peptides from pDNA vectors (Ryu et al. 2011).²⁸ Overall, this result indicates that W-R9 forms a reversible ionic complex with a pUC19 pDNA, that effectively functions as a capture and release system for gene delivery.

Transmission electron microscopy (TEM) imaging (Fig. 3) of the peptide:pDNA ionic complexes (FITC-W-R9 combined with pUC19 at a 10:1 N:P ratio in milliQ H₂O) was next conducted to gain a visual representation of the relative sizes and shapes of the (nano)particles with (Fig. 3B) and without (Fig. 3A) CaCl₂, functioning as ionic stabilizer (Alkhamy et al. 2016 and Baoum et al. 2012).^25,29 The peptide:pDNA complex aloneshows a disordered aggregated assembly of large particles, whereas CaCl₂condensed the peptide:pDNA ionic complex into smaller (< 200 nm), discrete nanoparticles that may be applicable to gene delivery. In the absence of CaCl₂, the peptide:pDNA complex appears to form larger aggregated structures, that may impede cell uptake and reduce gene delivery efficacy (Cultrara et al. 2019).³⁰ Therefore, the ability to condense stable and reversible peptide:pDNA ionic complexes into small, discrete and uniform nanoparticles is another important requirement for effective gene delivery (Su et al. 2022).³¹ Various conditions exist for condensing pDNA-based ionic complexes, including CaCl₂, that also improves cell uptake efficiency when used in combination with cell penetrating peptides (Su et al. 2022, Alkhamy et al. 2016 and Baoum et al. 2012).^25,29,31 Furthermore, CaCl₂ has also been reported to enhance the endosomal escape capacity of nucleic acid cargo resulting in enhanced transfection efficacy (Khondee et al. 2011).³² Mechanistic studies revealed the combined Ca²⁺ effects on the peptide:pDNA ionic complex that promotes membrane destabilization and pore formation for enhancing cell uptake, while also promoting endosomal rupture and release of pDNA for gene expression (Alkhamy et al. 2016).²⁵ Thus, CaCl₂ has been effectively shown to condense the peptide:pDNA ionic complexes into stable nanoparticles that improves transfection efficiency.

Cell Biology of Peptide:pDNA Nanoparticles

A preliminary cell uptake study was conducted to test the transfection capabilities of the fluorescein-labeled polyarginine-derived GRP78-targeting peptide (FITC-W-R9) in combination with a mock pBR322 vector within the DU145 prostate cancer cells (Fig. 4). Confocal fluorescence microscopy was used to confirm cell uptake and determine the subcellular localization of the polyarginine-derived GRP78-targeting peptide:pDNA (FITC-W-R9:pBR322). Punctate areas of green fluorescence (green arrows, Fig. 4) were detected in the cytosol of transfected DU145 cells. This observation suggests the entrapment of the peptide-based pDNA formulation in vesicle-type endosomes may restrict pDNA release for gene expression. This result also aligns with the reported clathrin-receptor mediated endocytosis cell uptake mechanism of GRP78-targeting peptides (Kim et al. 2006 and Liu et al. 2007).^33,34 This pathway has been associated with the active peptide-binding and ATPase domains of csGRP78, that favor formation of clathrin-coated pits that detach from the plasma membrane to form intracellular vesicles via receptor-mediated endocytosis of peptide ligands (Liu et al. 2007).³⁴Although polyarginine-derived GRP78-targeting peptides can improve cell uptake (Joseph et al. 2014),³⁵ intracellular entrapment may restrict its drug (gene) delivery efficacy.

Transfection efficiency was initially tested with a reporter Green Fluorescent Protein (GFP)-expressing pDNA (pGFP) in combination with the polyarginine-derived GRP78-targeting peptide (W-R9) that formed the peptide:pDNA (W-R9:pGFP) nanoparticles. A direct comparison of the peptide-based (W-R9) pGFP transfection efficiency with the benchmark Lipofectamine™ 3000 cationic lipid transfection agent indicated poor (<5%) GFP expression signaling with the peptide:pDNA conditions (Fig. 5B), whereas Lipofectamine™ 3000 effected noticeable (25-35%) GFP detection (Fig. 5A) according to fluorescence microscopy (Fig. S2, ESI). The insignificant peptide-based pDNA transfection efficiency may be due to the large, aggregated particles apparent by TEM imaging (Fig. 3A), that inhibits cell uptake (Cutrara et al. 2019),³⁰ or incomplete endolysosomal escape, that is consistent with the punctate fluorescent (FITC) nanoparticles suggestive of peptide;pDNA entrapment within vesicle-type endosomes in the DU145 cells (Fig. 4).

A variety of additives, commonly used to improve peptide:pDNA transfection efficiency (Baoum et al. 2012, Ibanez et al. 1996, Yang et al. 2009, Griesenbach et al. 2012 and Alhakamy et al. 2013 and 2016),^25,29,36-39were screened and selected to optimize the peptide-based (W-R9) transfection conditions with the reporter pGFP vector within the DU145 cells. For example, CaCl₂ was added to condense the peptide:pDNA formulation into stable nanoparticles via favourable electrostatic interactions (Fig. 3B), that was also anticipated to enhance cell uptake (Alhakamy et al. 2016 and Baoum et al. 2012).^25,29Moreover, CaCl₂ has been used to increase endosomal escape of cationic cell-penetrating peptides and pDNA nanoparticles (Alhakamy et al. 2013).³⁹ The addition of CaCl₂ (50, 150, and 300 mM) exhibited modest effects on the peptide-based pGFP transfection, and resulted in apparent toxicity to the DU145 cells, especially at higher concentrations (300 mM) and extended (48-72 h.) incubations (Fig. S3, ESI). Trans-cyclohexane-1,2-diol (TCHD) was also tested for its ability to increase peptide-based transfection efficiency with the pGFP reporter. TCHD has been shown to enlarge the nuclear pores, thereby collapsing the permeability barrier across the nuclear membrane, for nuclear uptake of larger biomacromolecules, including pDNA for gene expression (Griesenbach et al. 2012).³⁸ The addition of TCHD (0.5, 1.0, 2.0 %) also moderately improved the peptide-based pGFP transfection efficiency according to fluorescence microscopy, while producing significant toxicity to the DU145 cells, even at low doses (Fig. S4, ESI). Due to cell toxicity, CaCl₂ and TCHD were discontinued in the optimization of peptide-based pDNA transfection studies.

Spermidine and chloroquine were next evaluated to optimize the peptide-based transfection conditions with the reporter pGFP in the GRP78-presenting DU145 cells (Wong-Baeza et al. 2010).⁴⁰ Spermidine is a polycationic polyamine that aides in the condensation of nucleic acids and favors the intracellular release of pDNA for gene expression (Ibanez et al. 1996).³⁶ Chloroquine, functions as a lysosomotropic agent that increases the lysosomal pH, thereby directly disrupting endosomal membranes contributing to endolysosomal escape of cargo for intracellular activity (Yang et al. 2009).³⁷ Transfection studies have used chloroquine and spermidine in combination with liposomal-based gene delivery agents, that served to improve transfection efficiency resulting in enhanced gene expression (Moradpour et al. 1996 and Zhang et al. 2023).^41,42 In this study, the peptide-based (W-R9) transfection efficiency of pGFP was examined in the presence and absence of chloroquine and spermidine, and compared to the benchmark Lipofectamine 3000 transfection reagent (Fig. 5). The combination of chloroquine and spermidine (70 and 17.5 µM, respectively) improved the transfection efficiency of the peptide:pDNA complex, resulting in enhanced pGFP expression (Fig. 5C) compared to the peptide:pDNA transfection conditions that were absent of the additives and displayed very little, faint fluorescence signaling (Fig. 5B). Chloroquine and spermidine also maintained good cell viability compared to the previously tested peptide-based pGFP transfection conditions (Figs. S3 and S4, ESI). In comparison, the pGFP transfection conditions with chloroquine and spermidine, but absent of peptide, displayed background GFP fluorescence, as negative control (Fig. 5D). Alternatively, the Lipofectamine:pGFP transfection condition promoted GFP expression, as positive control (Fig 5A). This outcome demonstrates the ability for the select additives, chloroquine and spermidine, to improve the peptide:pDNA transfection efficiency, while maintaining cell viability at low effective concentrations underscoring their utility as safe and effective additives for promoting peptide-based gene delivery.

The optimized peptide-based (W-R9) pGFP transfection conditions with chloroquine and spermidine were also used to confirm the GRP78-dependent mechanism of cell uptake resulting in GFP expression (Fig. 6). In competition with the primary GRP78/Bip monoclonal antibody (1D6F7), selected to block the peptide-binding domain (351-654) of csGRP78, the pGFP expression was diminished in the peptide-based transfection with chloroquine and spermidine (Fig. 6B). In comparison, the peptide-based transfection conditions with chloroquine and spermidine, in the absence of anti-GRP78 displayed an increase in GFP signaling (Fig. 6A). Alternatively, the negative control pGFP transfection conditions with chloroquine and spermidine alone, produced negligible GFP signaling (Fig. 6C). Significantly, this result provides direct evidence of the GRP78-dependent peptide:pDNA cell uptake mechanism that results in pGFP expression within the DU145 cells (Fig. 6A vs. 6B). Therefore, the GRP78-targeting polyarginine peptide (W-R9) functions as a gene delivery system within csGRP78-presenting tumors for applications in the cancer-targeted delivery of gene (pDNA) therapeutics.

The translational, anti-cancer utility of the optimized peptide-based transfection conditions was subsequently examined with a plasmid vector applicable to cancer-targeted gene therapy. The p53 pDNA expression vector was used to produce wild-type tumor suppressor protein p53 within the representative DU145 prostate cancer (PCa) cell line expressing the csGRP78 biomarker (Arap et al. 2004)¹⁹ and mutant tumor tp53 oncogene (Chappell et al. 2012).⁴³ The tumor suppressor protein, p53, is long-regarded as the guardian of the human genome, acting as a critical transcription factor of multiple (~500) gene targets, including those involved in DNA repair, cell cycle arrest, senescence and apoptosis in transformed cancer cells (Lane 1992).⁴⁴ However, the anti-tumor activity of p53 is abolished in about half of all cancers due to excessive mutations and oncogenic tumor p53 signaling driving cancer cell proliferation, spread and therapy resistance (Muller et al. 2014).⁴⁵ Therefore, an effective gene therapy strategy will activate wild-type (wtp53) gene expression to restore tumor suppression, resulting in potent anti-cancer responses (Chappell et al. 2012 and Yu et al. 2004).^43,46 The optimized peptide-based (W-R9) pDNA transfection conditions with chloroquine and spermidine were used in a preliminary study, to express the wild-type p53 tumor suppressor within the DU145 PCa cells, known to express mutant tp53 variants (Chappell et al. 2012).⁴³ Western blot indicated the protein expression levels of the wild-type p53 isoforms, detected significantly with the control Lipofectamine™ 3000 transfection agent (Fig. 7, lane 2), and to a lesser, but noticeable extent with the optimized peptide-based transfection conditions (Fig. 7, lane 4). Interestingly, the peptide-based transfection conditions in the absence of the additives, chloroquine and spermidine, did not produce detectable levels of p53 (Fig. 7, lane 3), that was consistent with the poor pGFP transfection efficiency observed for the W-R9 peptide alone (Fig. 5B). However, in combination with chloroquine and spermidine additives, an enhancement in peptide-based transfection efficiency is observed (Figs., 5C, 6A and 7, lane 4). Alternatively, that additives chloroquine and spermidine in the absence of peptide (W-R9) do not enable protein expression (Figs., 5D, 6C and 7, lane 5). Therefore, the polyarginine-derived GRP78-targeting peptide (W-R9), with chloroquine and spermidine additives, enhances gene (pDNA) delivery directly within the GRP78-overexpressing DU145 prostate cancer cells. These exploratory results also suggest the ability to restore the functional, anti-tumor responses of the wild-type p53 tumor suppressor directly within the DU145 cells, and to overcome the oncogenic effects of mutant (tp53) biomarkers, leading towards the innovation of a promising cancer-targeted gene therapy approach.

This study describes the gene delivery applications of an optimized formulation of peptide:pDNA nanoparticles for cell-based detection and treatment in the GRP78-overexpressing DU145 prostate cancer cells. A nona-arginine (R9) cell penetrating peptide was added to the GRP78-targeting peptide sequence (WIFPWIQL) to produce the (WIFPWIQL-R9) chimeric peptide that effectively formed stable pDNA ionic complexes in annealing buffer conditions. Characterization data confirmed formation of stably condensed peptide:pDNA nanoparticles (< 200 nm), at excess peptide nitrogen (N) to pDNA phosphate (P) stoichiometric ratios (10:1 N/P) and with CaCl₂ functioning as ionic stabilizer, thereby rendering the nanoparticles applicable to cancer cell biology. Confocal imaging of the FITC-labeled peptide:pDNA formulation indicated cell uptake and intracellular localization within endosomes that restricted gene expression in the DU145 cells. Optimization studies with the peptide:pGFP reporter system investigated the effects of additives (CaCl₂, spermidine and chloroquine, among others) on transfection efficiency as a function of gene (GFP) expression. This screen revealed the combined, synergistic effects of additives (chloroquine and spermidine) resulting in an enhancement in the peptide:pDNA transfection efficiency. The application of anti-GRP78 also validated the GRP78-dependent cell uptake mechanism of the peptide:pGFP formulation. Preliminary investigations into the peptide-based transfection of the wild-type p53 vector in the GRP78-presenting DU145 prostate cancer cells produced noticeable expression of the p53 tumor suppressor. Significantly, the optimization of a peptide-based transfection protocol may be broadly applicable to a variety of gene therapeutics, for gene delivery selectively within csGRP78-presenting tumors, leading to the innovation of an impactful cancer-targeted gene therapy approach.

Peptide synthesis and characterization

All peptides were designed synthesized and characterized following the recently published procedures (Daniel et. al. 2024).²³

Stoichiometric peptide nitrogen to nucleic acid phosphorus (N:P) ratios

In order to determine the optimal peptide nitrogen (N) to pDNA phosphate (P) stoichiometric ratios, the samples were prepared at various N:P ratios; 1:1, 10:1, and 20:1 following a literature protocol (Aydin et al. 2022).⁴⁷ Peptide (FITC-R9, R9 and W-R9, 3.06 x 10^-10 - 6.15 x 10^-9 mol) and pDNA (pUC19 and pBr322, 3.06 x 10^-10 mol) combinations were incubated in Tris buffer (50 mM Tris-HCl and 150 mM NaCl, pH 7.5) and diluted in dH₂O to a set volume (24 µL). All samples were incubated for 30 min. at room temperature. A sample of only pDNA was also prepared as control.

Dye exclusion agarose gel electrophoresis

A 1% non-denaturing agarose gel was prepared for 30 minutes. A tracking dye (6X TrackIt Cyan/Orange loading buffer, ThermoFisher cat. no. 10488085) was added to each peptide:pDNA sample. The peptide:pDNA test samples, along with pDNA alone and a base pair ladder (TrackIt 1Kb Plus DNA Ladder, ThermoFisher cat. no. 10488085) were added to the agarose gel. The gel was run in 1x TEA gel running buffer for 1 h. at 71 V. The gel was stained using ethidium bromide (EtBr) on a shaker for 1 hour and destained using dH₂O on a shaker at room temperature for 20 min. Gels were imaged using a Bio-Rad molecular imager and analyzed using ImageJ software.

Heparin release assay

Peptide:pDNA samples were prepared at a 10:1 N:P ratio using W-R9 peptide and pUC19 pDNA. Prior to adding the heparin, peptide:pDNA samples were incubated Tris buffer (50 mM Tris-HCl and 150 mM NaCl, pH 7.5) for 30 min. at room temperature. Heparin (1-10 µg) was prepared in Tris buffer (10 µL) then added to each peptide:pDNA sample in increasing quantities (1 µg, 2.5 µg, 5 µg, 7.5 µg, and 10 µg). Samples were incubated for 30 min. at room temperature and then diluted to volume (25 µL) using Tris buffer. Agarose gel (1%, non-denaturing) electrophoresis was run and analyzed following the procedure previously described.

Fetal bovine serum (FBS) stability

Peptide:pDNA samples were prepared at a 10:1 N:P ratio using W-R9 peptide and pUC19 pDNA. Prior to addition of FBS, peptide:pDNA samples were incubated in Tris buffer (50 mM Tris-HCl and 150 mM NaCl, pH 7.5) for 30 min. at room temperature. An aliquot (20 µL) of 20 % FBS in Eagle’s Minimum Essential Medium (EMEM) was added so each sample has a final concentration of 10% FBS in EMEM. A control peptide:pDNA sample was prepared in Tris buffer, absent of FBS in EMEM. Samples with FBS were left to incubate for 1, 2, 4, 6, and 24 hrs. at 37 ^oC. Samples of only pUC19 and FBS were prepared to a final concentration of 10% FBS and collected at 1 h and 24 hrs. The samples were centrifuged, separated, quenched with gel loading buffer (6X TrackIt Cyan/Orange loading buffer), and frozen until being resolved on an agarose gel (1%, non-denaturing) electrophoresis following the procedure previously described.

TEM imaging

Peptide:pDNA samples were prepared at a 10:1 N:P ratio in milliQ H₂O. Samples were also incubated with 50 mM CaCl₂ (5 µL) for 30 min at room temperature. An additional aliquot (7.5 µL) of 1% uranyl acetate in milliQ H₂O was added to the samples to ensure equivalent volumes. Aliquots (10 µL) of individual samples were then deposited onto a 300-mesh formvar copper grid coated with carbon (Electron Microscopy Sciences Inc., Hatfield, PA). It was allowed to evaporate for 1 h. and TEM images were obtained using the FEI Tecnai G2 F20 Transmission Electron Microscope at an accelerating voltage of 120 kV and the Gatan ORIUS TEM CCD Camera was used to capture images across the grids. The Oxford X-Max 80 mm² Energy Dispersive X-ray (EDX) detector was used alongside Aztec software for EDX analysis. Particle size measurements of width, length, and diameter were taken using ImageJ software (LOCI, University of Wisconsin).

Cell culture

The prostate cancer mammalian cell line (DU145 cells) was gratefully obtained as a gift from Dr. Bruce McKay (Department of Biology, Carleton University). The culture conditions used MultiCell’s Essential Modified Eagle’s Medium (EMEM, 320-005-CL) supplemented with 10% heat inactivated fetal bovine serum (FBS) and incubated at 37˚C, in a ThermoForma triple gas (N₂-O₂-CO₂) incubator at 21% O₂ and 5% CO₂, with 95% relative humidity. All cell work was conducted in a biosafety cabinet (ThermoScientific – 1300 Series A2 Hood).

Fluorescence microscopy

For cell uptake studies, the DU145 cells, counted and seeded at a density of 3.5 x 10⁵ cells per 35 cm plate for 24 hours, were incubated with the FITC-labeled peptide (W-R9) and pBR322 (10:1 N/P ratio in Tris buffer) for 24 hours. The cells were washed twice with 1X PBS then fixed with 4% paraformaldehyde for 15 minutes, then washed again with 1X PBS twice. DAPI and Mito-Tracker were used to stain the cells. The coverslip was then inverted onto a 1.5 mm microscope slide and imaged on a Zeiss LSM 980 confocal microscope using the 63X magnification oil lens.

Cells were seeded to in a 24-well plate to 50,000 cells/well 24 hours prior to transfection optimization studies. Varying concentrations of TCHD (0.5, 1.0, 2.0 %), spermidine (7.5, 12.5, 17.5 µM), and CaCl₂ (50, 150, 300 mM) were added to separate 10:1 N:P aliquots of FITC-W-R9 and pcDNA3.1-GFP and incubated for one hour at room temperature in annealing Tris buffer. The media was changed to Opti-MEM (31985-070, Gibco), and the peptide:pGFP formulation was added dropwise to the wells. Lipofectamine transfection of pGFP was carried out according to the manufacturer’s instructions (Thermo Fisher) and the media was replaced 4 hours post-transfection. Fluorescent images were taken 24 and 48 hours after transfection on the EVOS Fluorescence microscope (Thermo Fisher).

Transfection conditions

Cells were seeded in a 6-well plate to 250,000-350,000 cells/well 24 hours prior to transfection. The FITC-W-R9 peptide was combined with the pcDNA3.1-GFP or the pRc-cmv-p53 pDNA vector in a 10:1 M:P ratio, followed by the addition of 70 µM chloroquine (C6628, Sigma), 17.5 µM of spermidine (204-689-0, Sigma), in Tris buffer (50 µL). The solution was incubated at room temperature for 1 hour in the dark. The media was changed to Opti-MEM (31985-070, Gibco), and the peptide:pDNA formulation was added dropwise to the wells. Lipofectamine-based transfections were carried out according to the manufacturer’s instructions (Thermo Fisher) and replaced to MultiCell’s Essential Modified Eagle’s Medium (EMEM, 320-005-CL) supplemented with 10% heat inactivated fetal bovine serum (FBS) 4 hours post-transfection. Fluorescent images for detection of GFP expression were taken 24 and 48 hours after transfection on the EVOS Fluorescence microscope (Thermo Fisher).

Western blot

Following transfection (48 h.), the DU145 cells were collected and pelleted in phosphate buffered saline (PBS) at 1,200 RPM for 5 mins and then lysed with RIPA buffer (89901, Thermofisher) for 20 mins on ice while gently shaking. The cell lysates were centrifuged at 12,000 RPM for 20 mins. The protein concentration from the supernatant was determined by Bradford assay (Bio-Rad). A 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was prepared, with samples loaded with protein aliquots (20 µg). The gel was run at 120V for roughly 1.5 hours. Gel transfer on a PVDF membrane was completed with transfer buffer at 4˚C in the fridge and run overnight at 0.18 amps. The following day, the membrane was removed from the assembly, placed in a container, and blocked with 5% dry milk in 1X Tris-buffered saline-Tween 20 (TBST) for 1 hour in the fridge while shaking. After blocking, incubation with primary antibodies for probing p53 (sc-6243), PUMA (PC686, Calbiochem), p21 (OP64, EMD), and beta-actin (A5316, Sigma), were used at 1:1000 concentration 1X TBST at 4˚C overnight. The following day, the primary antibodies were collected for future use, and the membranes were washed for 25 minutes, five times with 1X TBST over a shaker at 4˚C. A secondary antibody conjugated with HRP was then added to the corresponding membrane, at 1:5000 concentration in 1X TBST at 4˚C for 1 hour. The secondary antibodies were collected, and the membranes were washed for 25 minutes, five times with 1X TBST. To visualize the membranes, a Clarity Western ECL Substrate (#1705061, Bio-Rad) was added to the membranes and allowed to sit at room temperature in the dark for 5 minutes. After which, the chemiluminescence solution was removed and the membrane was placed in a plastic sheet and imaged on the Bio-Rad ChemiDoc station to visualise the proteins. ImageLab software was used for further analysis of all Western Blots including protein band densitometry to determine equal protein loading using beta-actin relative to treatment conditions.

Acknowledgements

This work was gratefully supported by the Natural Sciences and Engineering Council Discovery Grant to D.S. (RGPIN-2024-05349) and W.G.W. (RGPIN-2017-06414) and the Canada Foundation for Innovation John R. Evans Leaders Fund and the Ontario Research Small Infrastructure Fund to D.S. (43604). The co-authors are also grateful to Dr. Jianqun Wang from the Carleton Nano Imaging Facility for assistance with TEM and EDX analysis of peptide:pDNA samples.

Author contributions

G.D., F.C., G.H. and E.R. conducted experimental investigations. W.G.W., B.C.M. and D.S., supervised and secured project funding, compiled and revised manuscript. All authors have read and approve this manuscript.

Statements and Declarations

The authors declare no conflicts of interest.

Supplementary Information^†

The online version contains supplemental material available at:

Data Availability

No datasets were generated or analyzed in this study.

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Spermidine and Chloroquine Enhance Peptide-Based pDNA Transfection Efficiency within the GRP78-Overexpressing DU145 Prostate Cancer Cells

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