Bio-Membrane Internalization Mechanisms of Arginine-Rich Cell-Penetrating Peptides in Various Species

Recently, membrane-active peptides or proteins that include antimicrobial peptides (AMPs), cytolytic proteins, and cell-penetrating peptides (CPPs) have attracted attention due to their potential applications in the biomedical field. Among them, CPPs have been regarded as a potent drug/molecules delivery system. Various cargoes, such as DNAs, RNAs, bioactive proteins/peptides, nanoparticles and drugs, can be carried by CPPs and delivered into cells in either covalent or noncovalent manners. Here, we focused on four arginine-rich CPPs and reviewed the mechanisms that these CPPs used for intracellular uptake across cellular plasma membranes. The varying transduction efficiencies of them alone or with cargoes were discussed, and the membrane permeability was also expounded for CPP/cargoes delivery in various species. Direct membrane translocation (penetration) and endocytosis are two principal mechanisms for arginine-rich CPPs mediated cargo delivery. Furthermore, the amino acid sequence is the primary key factor that determines the cellular internalization mechanism. Importantly, the non-cytotoxic nature and the wide applicability make CPPs a trending tool for cellular delivery.

Protein charge parameters that influence stability and cellular internalization of polyelectrolyte complex micelles

Proteins are an important class of biologics, but there are several recurring challenges to address when designing protein-based therapeutics. These challenges include: the propensity of proteins to aggregate during formulation, relatively low loading in traditional hydrophobic delivery vehicles, and inefficient cellular uptake. This last criterion is particularly challenging for anionic proteins as they cannot cross the anionic plasma membrane. Here we investigated the complex coacervation of anionic proteins with a block copolymer of opposite charge to form polyelectrolyte complex (PEC) micelles for use as a protein delivery vehicle. Using genetically modified variants of the model protein green fluorescent protein (GFP), we evaluated the role of protein charge and charge localization in the formation and stability of PEC micelles. A neutral-cationic block copolymer, POEGMA79-b-qP4VP175, was prepared via RAFT polymerization for complexation and microphase separation with the panel of engineered anionic GFPs. We found that isotropically supercharged proteins formed micelles at higher ionic strength relative to protein variants with charge localized to a polypeptide tag. We then studied GFP delivery by PEC micelles and found that they effectively delivered the protein cargo to mammalian cells. However, cellular delivery varied as a function of protein charge and charge distribution and we found an inverse relationship between the PEC micelle critical salt concentration and delivery efficiency. This model system has highlighted the potential of polyelectrolyte-complexes to deliver anionic proteins intracellularly as well as the importance of correlating solution structure and desired functional activity.

Intracellularly Released Cholesterol from Polymer-Based Delivery Systems Alters Cellular Responses to Pneumolysin and Promotes Cell Survival

Cholesterol is highly abundant within all human body cells and modulates critical cellular functions related to cellular plasticity, metabolism, and survival. The cholesterol-binding toxin pneumolysin represents an essential virulence factor of Streptococcus pneumoniae in establishing pneumonia and other pneumococcal infections. Thus, cholesterol scavenging of pneumolysin is a promising strategy to reduce S. pneumoniae induced lung damage. There may also be a second cholesterol-dependent mechanism whereby pneumococcal infection and the presence of pneumolysin increase hepatic sterol biosynthesis. Here we investigated a library of polymer particles varying in size and composition that allow for the cellular delivery of cholesterol and their effects on cell survival mechanisms following pneumolysin exposure. Intracellular delivery of cholesterol by nanocarriers composed of Eudragit E100–PLGA rescued pneumolysin-induced alterations of lipid homeostasis and enhanced cell survival irrespective of neutralization of pneumolysin.

Synergistic Interaction of CPP2 Coupled with Thiazole Derivates Combined with Clotrimazole and Antineoplastic Drugs in Prostate and Colon Cancer Cell Lines

Cell-penetrating peptides (CPPs) are small peptide sequences used mainly as cellular delivery agents that are able to efficiently deliver cargo into cells. Some CPPs also demonstrate intrinsic anticancer properties. Previously, our group developed a new family of CPP2-thiazole conjugates that have been shown to effectively reduce the proliferation of different cancer cells. This work aimed to combine these CPP2-thiazole conjugates with paclitaxel (PTX) and 5-fluorouracil (5-FU) in PC-3 prostate and HT-29 colon cancer cells, respectively, to evaluate the cytotoxic effects of these combinations. We also combined these CPP2-thiazole conjugates with clotrimazole (CLZ), an antifungal agent that has been shown to decrease cancer cell proliferation. Cell viability was evaluated using MTT and SRB assays. Drug interaction was quantified using the Chou–Talalay method. We determined that CPP2 did not have significant activity in these cells and demonstrate that N-terminal modification of this peptide enhanced its anticancer activity in both cell lines. Our results also showed an uneven response between cell lines to the proposed combinations. PC-3 cells were more responsive to the combination of CPP2-thiazole conjugates with CLZ than PTX and were more sensitive to these combinations than HT-29 cells. In addition, the interaction of drugs resulted in more synergism in PC-3 cells. These results suggest that N-terminal modification of CPP2 results in the enhanced anticancer activity of the peptide and demonstrates the potential of CPPs as adjuvants in cancer therapy. These results also validate that CLZ has significant anticancer activity both alone and in combination and support the strategy of drug repurposing coupled to drug combination for prostate cancer therapy.

The Bcl-2 silencing with an Antisense Oligonucleotide: Increase early Apoptosis

Background Breast cancer is a heterogenic disease and hormone dependence. Estrogen receptor is positive in more than seventy percent of breast cancer patients. Tamoxifen is an estrogen receptor (ER) antagonist and used as the first line of treatment. Drug resistance is a main reason in failure of cancer treatment and progression of the disease. Combination drug therapy is a method to treatment but is not sufficient. New approaches like molecular therapy reveal new insight to cancer therapy. Studies shown, Bcl-2 gene family inhibitors and ER blockers enhance recovery. Interfering molecules such as antisense can inhibit the expression of Bcl-2 and push the cancer cells to apoptosis. Nevertheless, their effectiveness is low, mostly due to their direct use. Methods Our team designed an Antisense Oligonucleotides (ASO). The MCF-7 and the MDA-MB-231breast cancer cell lines used to evaluate cellular proliferation. Liposome and cationic nano-complex (Niosome) used to increase cellular delivery of ASO and Tamoxifen. We also investigated the cytotoxicity and apoptotic effects of Tamoxifen, naked ASO and Nano-packed ASO. Results The ASO functional potency to assess apoptosis and expression of Bcl-2 mRNA compared in different groups. The results indicated, significant down regulation of Bcl-2 gene and inhibition of MCF-7 and MDA-MB-231 cellular proliferation. Flow-cytometry showed early apoptosis in all groups. Conclusions The ASO reduced the expression of Bcl-2 gene. It also had the synergistic effect with the Tamoxifen. In all studied groups, it was able to push cancer cells to apoptosis. The cationic nano-complex (Niosome) was more efficient than the liposome in delivering designed oligo antisense Bcl-2 into the cancer cells.

Vasoactive Intestinal Peptide Amphiphile Micelle Chemical Structure and Hydrophobic Domain Influence Immunomodulatory Potentiation

Vasoactive intestinal peptide (VIP) is a neuropeptide capable of downregulating innate immune responses in antigen presenting cells (APCs) by suppressing their pro-inflammatory cytokine secretion and cell surface marker expression. Though VIP's bioactivity could possibly be leveraged as a treatment for autoimmune disorders and transplant tolerance, drug delivery innovation is required to overcome its intrinsically limited cellular delivery capacity due to its short in vivo lifetime. One option is to employ peptide amphiphiles (PAs) which are lipidated peptides capable of self-assembling into micelles in water that can enhance cellular association. With this approach in mind, a series of triblock VIP amphiphiles (VIPAs) has been synthesized to explore the influence of block arrangement and hydrophobicity on micelle biocompatibility and bioactivity. VIPA formulation has been found to influence the shape, size, and surface charge of VIPA micelles (VIPAMs) as well as their cytotoxicity and immunomodulatory effects. Specifically, the enclosed work provides strong evidence that cylindrical VIPAMs with aspect ratios of 1.5 - 150 and moderate positive surface charge are able to potentiate the bioactivity of VIP limiting TNF-a; secretion and MHC II and CD86 surface expression on APCs. With this criteria, we have identified PalmK-(EK)4-VIP as our lead formulation, which showed comparable or enhanced anti-inflammatory effects relative to the unmodified VIP at all dosages evaluated. Additionally, the relationships between peptide block location and lipid block size provide further information on the chemistry-structure-function relationships of peptide amphiphile micelles for the delivery of VIP as well as potentially for other peptides more broadly.