The delivery of therapeutic oligonucleotides into cells remains a significant challenge in gene therapy. This study investigates the use of Xentry, a cell-penetrating peptide, in combination with peptides XK and XP to facilitate the delivery of Cx43AsODN, an antisense oligonucleotide targeting connexin 43 (Cx43). Connexin 43 is a key protein involved in cell communication and is implicated in various pathological conditions. Effective delivery of Cx43AsODN could offer therapeutic benefits by modulating Cx43 expression. This article, relevant to research conducted around Xentry 2018, delves into the formation, characterization, and cellular uptake of Cx43AsODN complexes with XK and XP peptides, highlighting their potential for targeted gene therapy.
Characterization of Cx43AsODN Complex Formation and Size Using Zetasizer Technology
To effectively deliver Cx43AsODN into cells, it is crucial to form stable complexes that can protect the oligonucleotide from degradation and facilitate cellular entry. In this study, Cx43AsODN complexes were formed with two different peptides, XK and XP, utilizing non-covalent electrostatic interactions. This method leverages the inherent charges of the molecules to create complexes, as previously described in studies focusing on DNA and peptide interactions. The rationale behind using XK and XP peptides stems from their cationic nature and prior success in nucleic acid binding and cell permeabilization. Furthermore, XK itself has demonstrated efficacy in transporting siRNA into mammalian cells, making it a promising candidate for Cx43AsODN delivery.
To determine the optimal conditions for complex formation, a range of charge ratios between Cx43AsODN and the peptides (XK and XP) was tested. The goal was to identify the point at which a net positive charge is achieved, indicating complete binding of Cx43AsODN with the peptides. Zetasizer measurements were employed to assess both the zeta potential and the size of the formed complexes.
Zeta Potential Analysis of Cx43AsODN:XK and Cx43AsODN:XP Complexes
Zeta potential measurements provide insights into the surface charge of the complexes, which is crucial for cellular interactions. For Cx43AsODN:XK complexes, the transition from a net negative to a net positive charge occurred at a charge ratio of 1:2 (Cx43AsODN:XK). This indicates that at a 1:2 ratio, sufficient XK peptide is present to neutralize the negative charge of Cx43AsODN and impart a positive surface charge to the complex.
In contrast, Cx43AsODN:XP complexes reached a net positive charge at a lower charge ratio of 1:1.2 (Cx43AsODN:XP). This difference is likely attributed to the higher density of positive charges in XP compared to XK. XP contains more than double the positive charges of XK, requiring less XP to achieve charge neutralization and a net positive charge for the complex.
Size Measurements of Cx43AsODN Complexes
Alongside zeta potential, size is another critical parameter for effective cellular uptake. Size measurements revealed that while there were no significant size variations across different charge ratios for either Cx43AsODN:XK or Cx43AsODN:XP complexes, notable differences existed in the overall size between the two complex types.
Cx43AsODN:XK complexes exhibited a significantly larger size (approximately 1106.36 nm) compared to Cx43AsODN:XP complexes (approximately 463.65 nm). This size disparity can be attributed to several factors. Firstly, the higher positive charge density of XP, due to its greater number of arginine residues (13 in XP vs. 6 lysine residues in XK), allows for complex neutralization with fewer XP molecules, leading to a smaller complex size. Secondly, arginine residues are known to promote more compact DNA condensation compared to lysine residues due to their inherent structural and chemical properties. Furthermore, XP is a shorter peptide than XK, contributing to the smaller overall size of the Cx43AsODN:XP complexes. Despite the larger size of Cx43AsODN:XK complexes, the Xentry peptide component is known for its ability to deliver large molecules, suggesting that size alone might not be a limiting factor for cellular uptake.
Gel Shift Mobility Assay Confirms Complete Complex Formation
Beyond size and charge, confirming complete complex formation is vital to ensure that Cx43AsODN is efficiently protected and delivered. Unbound oligonucleotides are vulnerable to degradation in biological systems, potentially reducing therapeutic efficacy. The gel shift mobility assay was employed to verify the complete complexation of Cx43AsODN with XK and XP peptides. This technique is a standard method to study DNA-protein interactions.
In a gel shift assay, unbound DNA migrates freely through the agarose gel, whereas peptide-bound DNA complexes exhibit reduced mobility or remain in the well, depending on the extent of binding. Increasing charge ratios of XK and XP were mixed with Cx43AsODN and loaded onto an agarose gel.
The results demonstrated that complete complex formation for Cx43AsODN:XK occurred at a charge ratio of 1:2.5. At this ratio, no free Cx43AsODN migration was observed, indicating complete binding to XK. For Cx43AsODN:XP, complete complex formation was observed at a slightly lower charge ratio of 1:2.2, consistent with the zeta potential findings.
Interestingly, while zeta potential measurements suggested positive surface charge at lower ratios, the gel shift assay indicated slightly higher ratios for complete complex formation. This discrepancy could be attributed to the different solvents used: ultrapure water for zeta potential and PBS for the gel shift assay. The medium’s ionic strength can influence complex formation, potentially requiring higher charge ratios in PBS for complete complexation. Previous studies using similar oligonucleotides and KALA peptides also reported comparable charge ratios for preventing DNA migration due to complex formation, supporting the findings of this study. This suggests that incorporating Xentry into KALA does not negatively impact its electrostatic binding capability, and effective complex formation is achieved.
Cellular Uptake of Cx43AsODN:XK and Cx43AsODN:XP in ARPE-19 Cells
Having characterized the complexes, the next crucial step was to assess their ability to deliver Cx43AsODN into cells. ARPE-19 cells, a human retinal pigment epithelium cell line known to express Cx43, were chosen for cellular uptake studies. Based on the gel shift assay results, charge ratios of 1:2.5 for Cx43AsODN:XK and 1:2.2 for Cx43AsODN:XP were selected for these experiments. Cx43AsODN was labeled with Cyanine-3 (Cy3) to enable visualization of cellular uptake using fluorescence microscopy.
Cells treated with media alone or free Cx43AsODN showed minimal red fluorescence, indicating negligible cellular uptake of free Cx43AsODN. This is likely due to degradation of the oligonucleotide in serum-containing media and/or limited uptake below the assay’s detection threshold.
In stark contrast, cells treated with Cx43AsODN:XK or Cx43AsODN:XP complexes exhibited detectable red fluorescence within the cells, confirming successful cellular uptake. Cx43AsODN:XK uptake showed a punctate, circular pattern, suggesting endosomal localization. This endosomal entrapment might hinder the therapeutic efficacy of Cx43AsODN as it needs to reach the cytoplasm to interact with mRNA. While KALA, a component of XK, is known to promote endosomal escape at higher charge ratios, the ratios used in this study might not have been sufficient for effective endosomal release for Cx43AsODN:XK. Despite this, Xentry’s reported ability to deliver siRNA effectively suggests that the Xentry component itself is not inherently limiting endosomal escape. The larger size of Cx43AsODN:XK complexes and the lower positive charge of lysine-rich KALA might contribute to the observed endosomal localization.
Notably, Cx43AsODN:XP uptake displayed a different intracellular distribution. While some punctate vesicles were observed, a significant portion of the Cy3 fluorescence was dispersed throughout the cytoplasm, indicating successful endosomal escape and cytoplasmic delivery of Cx43AsODN:XP. This cytoplasmic delivery is crucial for Cx43 mRNA targeting and subsequent protein knockdown. Given its superior cellular uptake and endosomal escape, Cx43AsODN:XP was chosen for further investigation.
Assessing Cell Morphology Changes with Increasing XP Peptide Concentrations
Before evaluating therapeutic efficacy, it is essential to assess potential cytotoxicity. Highly cationic peptides, like XP, can induce cell morphology changes at higher concentrations. Therefore, ARPE-19 cells were treated with increasing concentrations of XP peptide alone to examine any morphological alterations.
At lower concentrations (1-5 µM), XP did not induce noticeable changes in cell morphology compared to untreated cells. However, at 10 µM, some cells began to exhibit condensation, appearing rounded and darker. At higher concentrations (20 and 25 µM), a dose-dependent increase in condensed cells was observed, indicating peptide-induced morphological changes.
Quantification of condensed cells confirmed a significant increase at 20 and 25 µM XP compared to untreated cells. While XP alone can induce morphological changes at higher concentrations, it is important to note that when complexed with Cx43AsODN, the overall net positive charge of the complex is reduced, potentially mitigating these effects. Furthermore, protamine, a peptide with similar positive charge characteristics to XP, has been used clinically with minimal toxicity, including in formulations like neutral protamine Hagedorn (NPH) insulin. This suggests that XP, particularly when complexed with Cx43AsODN at optimized ratios, could be used safely.
Cx43 Protein Expression Knockdown Post Cx43AsODN:XP Uptake
To determine if the delivered Cx43AsODN:XP complexes were functionally active, Western blot analysis was performed to assess Cx43 protein knockdown in ARPE-19 cells. Cells were treated with Cx43AsODN:XP complexes and then analyzed for Cx43 protein levels.
Western blot results showed a significant reduction in Cx43 protein levels in cells treated with Cx43AsODN:XP compared to untreated control cells. Quantification of band intensities confirmed a statistically significant decrease in Cx43 expression in treated cells.
This demonstrates that Cx43AsODN:XP complexes effectively deliver Cx43AsODN into cells, leading to target mRNA interaction and subsequent Cx43 protein knockdown. The substantial reduction in Cx43 protein suggests a potent effect, potentially requiring careful dose optimization for therapeutic applications to avoid disrupting normal gap junction communication. However, previous in vivo studies have used higher doses of Cx43AsODN for chronic conditions, suggesting that dose adjustments are crucial depending on the specific therapeutic context (acute vs. chronic conditions).
Cx43 Protein Expression Modulation Under Hypoxic Conditions
Cx43 expression is known to be upregulated under hypoxic stress. To assess the efficacy of Cx43AsODN:XP in a disease-relevant context, Cx43 knockdown was investigated under hypoxic conditions. ARPE-19 cells were subjected to hypoxia using HAIR solution and then treated with Cx43AsODN:XP complexes.
Under hypoxic conditions, untreated cells exhibited increased Cx43 protein levels compared to normal cells, as expected. In normal cells, Cx43AsODN:XP treatment did not significantly alter baseline Cx43 levels, indicating minimal impact on normal Cx43 expression at the lower concentrations used.
However, in hypoxic cells, Cx43AsODN:XP treatment effectively reduced the hypoxia-induced upregulation of Cx43 protein, bringing expression levels closer to baseline. This demonstrates that Cx43AsODN:XP can selectively knockdown the excess Cx43 protein produced under hypoxic stress without significantly affecting normal baseline levels. This targeted knockdown in hypoxic conditions highlights the therapeutic potential of Cx43AsODN:XP for conditions involving Cx43 upregulation in response to cellular stress.
Conclusion
This study demonstrates the successful formation and characterization of Cx43AsODN complexes with Xentry-derived peptides XK and XP. Cx43AsODN:XP complexes exhibit smaller size, better cellular uptake, and effective endosomal escape compared to Cx43AsODN:XK. Critically, Cx43AsODN:XP effectively delivers Cx43AsODN into ARPE-19 cells, resulting in significant Cx43 protein knockdown, particularly under hypoxic conditions where Cx43 is upregulated. These findings suggest that xentry 2018 based peptide delivery systems, specifically utilizing XP, hold significant promise for targeted gene therapy applications requiring modulation of Cx43 expression. Further research should focus on in vivo evaluation and dose optimization of Cx43AsODN:XP for specific disease models.
