Noncovalently Associated Cell Penetrating Peptides for Nonviral Gene Delivery

Gene therapy has become a promising strategy for treatment of numerous diseases such as cancer, hemophilia, and neurodegenerative diseases. Glybera® (alipogene tiparvovec) became the first gene therapy approved in the European Union, for the treatment of lipoprotein lipase deficiency (LPLD). Promising late-stage clinical trials of this drug may herald the first gene therapy to be approved in the United States. The advancement of vectors (viral and nonviral) for efficient and safe gene delivery has garnered significant attention recently. Although viral vectors (e.g., retroviruses and adenoviruses) are the most effective vectors, applied in 70% of gene therapy clinical trials, they present several notable challenges including safety concerns (e.g., immunogenicity and pathogenicity), production difficulties, and rapid clearance from circulation. In contrast, nonviral vectors could be promising gene carriers due to their low cost, ease of synthesis, and decreased immunogenicity relative to viral vectors. Key progress has been made in the development of several nonviral gene delivery vectors. The use of cell penetrating peptides (CPPs) to deliver genetic materials for gene therapy has been a topic of interest for more than 20 years. One strategy is through covalent conjugation of CPPs with genetic materials, which requires complex synthesis procedures. In contrast, electrostatic complexation of CPPs with genetic materials is relatively simple and has been demonstrated to improve gene delivery both in vitro and in vivo. Reported herein, a simple method to generate small CPP complexes (100-200 nm) capable of high transfection efficiency was explored. Positively charged CPPs (e.g., polyarginine 9 [R9] and polylysine 9 [K9]) were complexed with plasmid DNA (pDNA), which resulted in unstable large particles (~1 micron). These were then condensed into small nanoparticles using Ca2+. CPPs also displayed negligible cytotoxicity. These CPP-pDNA-Ca2+ complexes showed high transfection efficiency and low cytotoxicity in vitro (human and mouse cell lines) and in vivo (syngeneic mice). Thus, Ca2+-condensed CPP complexes emerged as simple, attractive candidates for future studies on nonviral gene delivery. Futhermore, the relationships between transfection efficiency and polyarginine molecular weight, polyarginine-pDNA charge ratios, and calcium concentrations were studied. Polyarginine 7 was significantly more effective than other polyarginines under most formulation conditions, suggesting a link between molecular weight and transfection efficiency. Furthermore, Polylysine 9 was complexed with angiotensin II type 2 receptor (AT2R) plasmid DNA (pAT2R). The polylysine 9 complexe (K9-pAT2R-Ca2+) showed high transfection efficiency and negligible in vitro cytotoxicity towards human and mouse cell lines. This complex demonstrated cancer-targeted gene delivery in vivo when administered via intravenous injection or intratracheal spray. A single administration of this complex markedly attenuated lung cancer growth. Mechanistic understanding of CPP-mediated membrane insertion and intracellular translocation of nonviral gene complexes would allow rational design of next-generation CPPs for gene delivery. To this aim, we employed zwitterionic and anionic phospholipid monolayers as models to mimic the membrane composition of the outer leaflet of cell plasma and intracellular vesicular membranes at relevant intracellular pH. Subsequently, we investigated the membrane insertion potential of CPPs and gene complexes (CPP-pDNA-Ca2+ complexes) into model membranes. The insertion potential of CPPs and complexes were recorded using a Langmuir monolayer approach that records peptides and complexes adsorption to model membranes. Results showed that small changes to amino acids and peptide sequences resulted in dramatically different insertion potentials and membrane reorganization. Lastly, the effect of CPP charge type, charge spacing, and hydrophobicity on transfection efficiency was investigated by replacing three residues of the polyarginine 9 with a hydrophilic residue (histidine) or hydrophobic residues (alanine, leucine, and tryptophan) at positions 3, 4, and 7. R9 and RW9 complexes appeared especially effective compared to other CPP complexes, whereas RH9, RA9, and RL9 complexes seemed to have moderate- to low-gene expression. Initially, this suggested CPPs with better membrane penetration yielded higher gene expression. After further exploration, we discovered the charge spacing of CPPs affected the ability of CPPs to complex with nucleic acids and this property correlated to gene expression levels. In conclusion, our complexes emeraged as simple, attractive candidates for further in vivo studies on nonviral gene delivery

Polyarginine Molecular Weight Determines Transfecion Efficiency of Calcium Condensed Complexes

Cell penetrating peptides (CPPs) have been extensively studied in polyelectrolyte complexes as a means to enhance the transfection efficiency of plasmid DNA (pDNA). Increasing the molecular weight of CPPs often enhances gene expression, but poses a risk of increased cytotoxicity and immunogenicity compared to low molecular weight CCPs. Conversely, low molecular weight CPPs typically have low transfection efficiency due to large complex size. Complexes made using low molecular weight CPPs were found to be condensed to a small size by adding calcium. In this study, complexes of low molecular weight polyarginine and pDNA were condensed with calcium. These complexes showed high transfection efficiency and low cytotoxicity in A549 carcinomic human alveolar basal epithelial cells. The relationship between transfection efficiency and polyarginine size (5, 7, 9 or 11 amino acids), polyarginine/pDNA charge ratios, and calcium concentrations were studied. Polyarginine 7 was significantly more effective than other polyarginines under most formulation conditions suggesting a link between cell penetration ability and transfection efficiency

Dynamic Measurements of Membrane Insertion Potential of Synthetic Cell Penetrating Peptide/pDNA/Ca2+ Complexes

This is the published version. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.Noncovalent complexation of plasmid DNA (pDNA) using cell penetrating peptides (CPPs) has been less explored due to the relatively large complex size formed and the low-level gene expression. Here, condensing synthetic CPP polyplexes using CaCl2 produced small and stable complexes, which show higher level of in vitro gene expression. Anionic (i.e., POPS and POPG) or zwitterion (i.e., POPC) phospholipid monolayers at the air-water interface are used as model cell membranes to monitor the membrane insertion potential of synthetic CPPs. The insertion potential of complexes having different cationic (dTAT, H9, K9, R9, and RH9) and amphiphilic (RA9, RL9, and RW9) peptides were recorded using a Langmuir monolayer approach that records complexes adsorption to model membranes. Further, to mimic the pH of early endosome and late endosome and lysosome, phospholipid complex interactions were recorded at normal (pH 7.4) and low (pH 4.4) pH. All the complexes studied induced disruptions in phospholipid packing, which were most pronounced for the complexes having amphiphilic CPPs (i.e., RW9 and RL9). Particularly, the surface pressure of the complexes was significantly lower at normal pH when compared to acidic pH in the presence of POPC and POPS monolayers, except for RL9 and RW9 complexes. In contrast, the surface pressure of the complexes was significantly higher at normal pH when compared to acidic pH in the presence of POPG monolayer. Since the late endosomes contain an abundance of PC lipids and low pH, these results may be highly relevant to understand the efficiency of endosomal escape of these complexes

Effect of Lipid Headgroup Charge and pH on the Stability and Membrane Insertion Potential of Calcium Condensed Gene Complexes

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Langmuir, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/la504970n.Noncovalently condensed complexes of genetic material, cell penetrating peptides (CPPs), and calcium chloride present a nonviral route to improve transfection efficiency of nucleic acids (e.g., pDNA and siRNA). However, the exact mechanisms of membrane insertion and delivery of macromolecule complexes to intracellular locations as well as their stability in the intracellular environment are not understood. We show that calcium condensed gene complexes containing different hydrophilic (i.e., dTAT, K9, R9, and RH9) and amphiphilic (i.e., RA9, RL9, and RW9) CPPs formed stable cationic complexes of hydrodynamic radii 100 nm at neutral pH. However, increasing the acidity caused the complexes to become neutral or anionic and increase in size. Using zwitterionic and anionic phospholipid monolayers as models that mimic the membrane composition of the outer leaflet of cell membranes and intracellular vesicles and pHs that mimic the intracellular environment, we study the membrane insertion potential of these seven gene complexes (CPP/pDNA/Ca2+ complexes) into model membranes. At neutral pH, all gene complexes demonstrated the highest insertion potential into anionic phospholipid membranes, with complexes containing amphiphilic peptides showing the maximum insertion. However, at acidic pH, the gene complexes demonstrated maximum monolayer insertion into zwitterionic lipids, irrespective of the chemical composition of the CPP in the complexes. Our results suggest

Scaffold hopping of α-rubromycin enables direct access to FDA-approved cromoglicic acid as a SARS-CoV-2 M^Pro inhibitor

The COVID-19 pandemic is still active around the globe despite the newly introduced vaccines. Hence, finding effective medications or repurposing available ones could offer great help during this serious situation. During our anti-COVID-19 investigation of microbial natural products (MNPs), we came across α-rubromycin, an antibiotic derived from Streptomyces collinus ATCC19743, which was able to suppress the catalytic activity (IC50 = 5.4 µM and Ki = 3.22 µM) of one of the viral key enzymes (i.e., MPro). However, it showed high cytotoxicity toward normal human fibroblasts (CC50 = 16.7 µM). To reduce the cytotoxicity of this microbial metabolite, we utilized a number of in silico tools (ensemble docking, molecular dynamics simulation, binding free energy calculation) to propose a novel scaffold having the main pharmacophoric features to inhibit MPro with better drug-like properties and reduced/minimal toxicity. Nevertheless, reaching this novel scaffold synthetically is a time-consuming process, particularly at this critical time. Instead, this scaffold was used as a template to explore similar molecules among the FDA-approved medications that share its main pharmacophoric features with the aid of pharmacophore-based virtual screening software. As a result, cromoglicic acid (aka cromolyn) was found to be the best hit, which, upon in vitro MPro testing, was 4.5 times more potent (IC50 = 1.1 µM and Ki = 0.68 µM) than α-rubromycin, with minimal cytotoxicity toward normal human fibroblasts (CC50 > 100 µM). This report highlights the potential of MNPs in providing unprecedented scaffolds with a wide range of therapeutic efficacy. It also revealed the importance of cheminformatics tools in speeding up the drug discovery process, which is extremely important in such a critical situation

Natural product-inspired synthesis of coumarin–chalcone hybrids as potential anti-breast cancer agents

Twelve novel neo-tanshinlactone–chalcone hybrid molecules were constructed through a versatile methodology involving the Horner–Wadsworth–Emmons (HWE) olefination of 4-formyl-2H-benzo [h]chromen-2-ones and phosphonic acid diethyl esters, as the key step, and evaluated for anticancer activity against a series of four breast cancers and their related cell lines, viz. MCF-7 (ER + ve), MDA-MB-231 (ER-ve), HeLa (cervical cancer), and Ishikawa (endometrial cancer). The title compounds showed excellent to moderate in vitro anti-cancer activity in a range of 6.8–19.2 µM (IC50). Compounds 30 (IC50 = 6.8 µM and MCF-7; IC50 = 8.5 µM and MDA-MB-231) and 31 (IC50 = 14.4 µM and MCF-7; IC50 = 15.7 µM and MDA-MB-231) exhibited the best activity with compound 30 showing more potent activity than the standard drug tamoxifen. Compound 30 demonstrated a strong binding affinity with tumor necrosis factor α (TNF-α) in molecular docking studies. This is significant because TNFα is linked to MCF-7 cancer cell lines, and it enhances luminal breast cancer cell proliferation by upregulating aromatase. Additionally, virtual ADMET studies confirmed that hybrid compounds 30 and 31 met Lipinski’s rule; displayed high bioavailability, excellent oral absorption, favorable albumin interactions, and strong penetration capabilities; and improved blood–brain barrier crossing. Based on the aforementioned results, compound 30 has been identified as a potential anti-breast cancer lead molecule

Targeted gene silencing of CCL2 inhibits triple negative breast cancer progression by blocking cancer stem cell renewal and M2 macrophage recruitment

Triple negative breast cancers are an aggressive subtype of breast cancer, characterized by the lack of estrogen receptor, progesterone receptor and Her2 expression. Triple negative breast cancers are non-responsive to conventional anti-hormonal and Her2 targeted therapies, making it necessary to identify new molecular targets for therapy. The chemokine CCL2 is overexpressed in invasive breast cancers, and regulates breast cancer progression through multiple mechanisms. With few approaches to target CCL2 activity, its value as a therapeutic target is unclear. In these studies, we developed a novel gene silencing approach that involves complexing siRNAs to TAT cell penetrating peptides (Ca-TAT) through non-covalent calcium cross-linking. Ca-TAT/siRNA complexes penetrated 3D collagen cultures of breast cancer cells and inhibited CCL2 expression more effectively than conventional antibody neutralization. Ca-TAT/siRNA complexes targeting CCL2 were delivered to mice bearing MDA-MB-231 breast tumor xenografts. In vivo CCL2 gene silencing inhibited primary tumor growth and metastasis, associated with a reduction in cancer stem cell renewal and recruitment of M2 macrophages. These studies are the first to demonstrate that targeting CCL2 expression in vivo may be a viable therapeutic approach to treating triple negative breast cancer

Capsaicin ameliorate pulmonary fibrosis via antioxidant Nrf-2/ PPAR- γ pathway activation and inflammatory TGF-β1/ NF-κB/COX II pathway inhibition

Bleomycin is an effective antibiotic with a significant anticancer properties, but its use is limited due to its potential to induce dose-dependent pulmonary fibrosis. Therefore, this study aimed to assess the therapeutic potential of Capsaicin as an additional treatment to enhance patient tolerance to Bleomycin compared to the antifibrotic drug Pirfenidone. Pulmonary fibrosis was induced in rats through by a single intratracheal Bleomycin administration in day zero, followed by either Capsaicin or Pirfenidone treatment for 7 days. After the animals were sacrificed, their lungs were dissected and examined using various stains for macroscopic and histopathological evaluation. Additionally, the study assessed various antioxidant, anti-inflammatory, and antifibrotic parameters were assessed. Rats exposed to Bleomycin exhibited visible signs of fibrosis, histopathological alterations, increased collagen deposition, and elevated mucin content. Bleomycin also led to heightened increased inflammatory cells infiltration in the bronchoalveolar lavage, elevated fibrosis biomarkers such as hydroxyproline, alpha-smooth muscle actin (α-SMA) and transforming growth factor-beta (TGF-β1), increased inflammatory markers including tumor necrosis factor-alpha (TNF-α), interlukine-6 (Il-6), interlukine-1β (Il-1β) nuclear factor-kappa B (NF-κB), and Cyclooxygenase-2 (COX-2), and transforming growth factor-beta (TGF-β1),. Furthermore, it reduced the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ), increased oxidative stress biomarkers like nitric oxide (NO), malondialdehyde (MDA), myeloperoxidase (MPO) and protein carbonyl. Bleomycin also decreased the expression of nuclear factor erythroid 2–related factor 2 (Nrf-2), reduced glutathione (GSH), total antioxidant capacity, and the activities of catalase and superoxide dismutase (SOD). Treating the animals with Capsaicin and Pirfenidone following Bleomycin exposure resulted in improved lung macroscopic and microscopic characteristics, reduced collagen deposition (collagen I and collagen III) and mucin content, decreased inflammatory cell infiltration, lowered levels of hydroxyproline, α-SMA, and TGF-β1, decreased TNF-α, Il-6, Il-1β, NF-κB, and COX-2, increased PPAR-γ and Nrf-2 expression, and improvement improved in all oxidative stress biomarkers. In summary, Capsaicin demonstrates significant antifibrotic activity against Bleomycin-induced lung injury that may be attributed, at least in part, to the antioxidant and anti-inflammatory activities of Capsaicin mediated by upregulation of PPAR-γ and Nrf-2 expression and decreasing. TGF-β1, NF-κB and COX II proteins concentrations

The CCL2 chemokine is a negative regulator of autophagy and necrosis in liminal B breast cancer cells

Luminal A and B breast cancers are the most prevalent forms of breast cancer diagnosed in women. Compared to luminal A breast cancer patients, patients with luminal B breast cancers experience increased disease recurrence and lower overall survival. The mechanisms that regulate the luminal B subtype remain poorly understood. The chemokine CCL2 is overexpressed in breast cancer, correlating with poor patient prognosis. The purpose of this study was to determine the role of CCL2 expression in luminal B breast cancer cells. Breast tissues, MMTV-PyVmT and MMTV-Neu transgenic mammary tumors forming luminal B-like lesions, were immunostained for CCL2 expression. To determine the role of CCL2 in breast cancer cells, CCL2 gene expression was silenced in mammary tumor tissues and cells using TAT cell-penetrating peptides non-covalently cross linked to siRNAs (Ca-TAT/siRNA). CCL2 expression was examined by ELISA and flow cytometry. Cell growth and survival were analyzed by flow cytometry, immunocytochemistry, and fluorescence microscopy. CCL2 expression was significantly increased in luminal B breast tumors, MMTV-PyVmT and MMTV-Neu mammary tumors, compared or normal breast tissue or luminal A breast tumors. Ca-TAT delivery of CCL2 siRNAs significantly reduced CCL2 expression in PyVmT mammary tumors, and decreased cell proliferation and survival. CCL2 gene silencing in PyVmT carcinoma cells or BT474 luminal B breast cancer cells decreased cell growth and viability associated with increased necrosis and autophagy. CCL2 expression is overexpressed in luminal B breast cancer cells and is important for regulating cell growth and survival by inhibiting necrosis and autophagy

Optimized D-α-tocopherol polyethylene glycol succinate/phospholipid self-assembled mixed micelles: A promising lipid-based nanoplatform for augmenting the antifungal activity of fluconazole

Fluconazole (FLZ) is the most widely used antifungal agent for treating cutaneous candidiasis. Although oral FLZ has been proved to be effective, the incidence of side effects necessitates the development of an effective formulation that could surpass the pitfalls associated with systemic availability. Accordingly, this research aimed at developing a self-assembled mixed micelles topical delivery system to enhance the topical delivery of the drug. Self-assembled mixed micelles were developed using D-α-tocopheryl polyethylene glycol 1000 succinate and phospholipids and optimized using Box-Behnken design. The optimized formulation with minimized size was then tested in vivo for the antifungal activity against C. albicans in immunocompromised mice. Treatment with the optimized formulation led to decreased peripheral erythema as well as lesions due to fungal infection in comparison to raw FLZ loaded gel. Therefore, the developed formulation was found to be a promising vehicle for the treatment of cutaneous candidiasis