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.

Systematic Screening of Penetratin’s Protein Targets by Yeast Proteome Microarrays

Cell-penetrating peptides (CPPs) have distinct properties to translocate across cell envelope. The key property of CPPs to translocation with attached molecules has been utilized as vehicles for the delivery of several potential drug candidates that illustrate the significant effect in in-vitro experiment but fail in in-vivo experiment due to selectively permeable nature of cell envelop. Penetratin, a well-known CPP identified from the third α-helix of Antennapedia homeodomain of Drosophila, has been widely used and studied for the delivery of bioactive molecules to treat cancers, stroke, and infections caused by pathogenic organisms. Few studies have demonstrated that penetratin directly possesses antimicrobial activities against bacterial and fungal pathogens; however, the mechanism is unknown. In this study, we have utilized the power of high-throughput Saccharomyces cerevisiae proteome microarrays to screen all the potential protein targets of penetratin. Saccharomyces cerevisiae proteome microarrays assays of penetratin followed by statistical analysis depicted 123 Saccharomyces cerevisiae proteins as the protein targets of penetratin out of ~5800 Saccharomyces cerevisiae proteins. To understand the target patterns of penetratin, enrichment analyses were conducted using 123 protein targets. In biological process: ribonucleoprotein complex biogenesis, nucleic acid metabolic process, actin filament-based process, transcription, DNA-templated, and negative regulation of gene expression are a few significantly enriched terms. Cytoplasm, nucleus, and cell-organelles are enriched terms for cellular component. Protein-protein interactions network depicted ribonucleoprotein complex biogenesis, cortical cytoskeleton, and histone binding, which represent the major enriched terms for the 123 protein targets of penetratin. We also compared the protein targets of penetratin and intracellular protein targets of antifungal AMPs (Lfcin B, Histatin-5, and Sub-5). The comparison results showed few unique proteins between penetratin and AMPs. Nucleic acid metabolic process and cellular component disassembly were the common enrichment terms for penetratin and three AMPs. Penetratin shows unique enrichment items that are related to DNA biological process. Moreover, motif enrichment analysis depicted different enriched motifs in the protein targets of penetratin, LfcinB, Histatin-5, and Sub-5.

Two Possible Strategies for Drug Modification of Gemcitabine and Future Contributions to Personalized Medicine

Drug repurposing is an emerging strategy, which uses already approved drugs for new medical indications. One such drug is gemcitabine, an anticancer drug that only works at high doses since a portion is deactivated in the serum, which causes toxicity. In this review, two methods were discussed that could improve the anticancer effect of gemcitabine. The first is a chemical modification by conjugation with cell-penetrating peptides, namely penetratin, pVEC, and different kinds of CPP6, which mostly all showed an increased anticancer effect. The other method is combining gemcitabine with repurposed drugs, namely itraconazole, which also showed great cancer cell inhibition growth. Besides these two strategies, physiologically based pharmacokinetic models (PBPK models) are also the key for predicting drug distribution based on physiological data, which is very important for personalized medicine, so that the correct drug and dosage regimen can be administered according to each patient’s physiology. Taking all of this into consideration, it is believed that gemcitabine can be repurposed to have better anticancer effects.

Cell-Penetrating Peptides: Emerging Tools for mRNA Delivery

Messenger RNAs (mRNAs) were previously shown to have great potential for preventive vaccination against infectious diseases and therapeutic applications in the treatment of cancers and genetic diseases. Delivery systems for mRNAs, including lipid- and polymer-based carriers, are being developed for improving mRNA bioavailability. Among these systems, cell-penetrating peptides (CPPs) of 4–40 amino acids have emerged as powerful tools for mRNA delivery, which were originally developed to deliver membrane-impermeable drugs, peptides, proteins, and nucleic acids to cells and tissues. Various functionalities can be integrated into CPPs by tuning the composition and sequence of natural and non-natural amino acids for mRNA delivery. With the employment of CPPs, improved endosomal escape efficiencies, selective targeting of dendritic cells (DCs), modulation of endosomal pathways for efficient antigen presentation by DCs, and effective mRNA delivery to the lungs by dry powder inhalation have been reported; additionally, they have been found to prolong protein expression by intracellular stabilization of mRNA. This review highlights the distinctive features of CPP-based mRNA delivery systems.

A Nanogel with Effective Blood-Brain Barrier Penetration Ability through Passive and Active Dual-Targeting Function

Clinically, surgery assisted by chemotherapy is the most effective treatment of cancer. But from our clinical observation, the median survival of patients with glioblastoma is still not so good with only 15-16 months. The low therapeutic index is mainly due to the blood-brain barrier (BBB) which significantly hindered the chemotherapeutic drug accumulation in tumor tissue. One main composition of the BBB is astrocyte, which contains a lipophilic cell membrane, which prevents more than 98% of small-molecule drugs from entering the brain. Previously, we found that the nanogel with passive targeting function can increase the BBB penetration ability, which indicates that it could be used to overcome the above mentioned in vivo obstacles which promoted drug accumulation in the tumor. In this study, thermosensitive targeted nanogel delivery systems (DPPC) with cell-penetrating peptides (CPP) are introduced onto the particle surface for active astrocyte breaking. The hydrodynamic radius of DPPC is around 300 nm, the potential is about 0-5 mV, and the TEM and DLS studies further confirm its well spherical morphology and uniform distribution. The DPPC is verified as the biocompatible carriers for further application by cell viability tests. The in vitro-constructed BBB model successfully proves that DPPC can efficiently penetrate the BBB, which is attributed to both the temperature-sensitive passive targeting and the active CPP penetration. Consequently, the intracellular doxorubicin (DOX) promotes such functional DPPC at the relatively high temperature inside tumor microenvironment (TME) (~42°C), which obviously improves intratumor drug accumulation and tumor cell-killing effects. The dual-targeted nanogel delivery systems designed in this study provides a more effective strategy for the treatment of glioma.

SynB3 conjugated QBP1 passes blood-brain barrier models and inhibits polyQ protein aggregation

Background: Polyglutamine diseases are degenerative diseases in the central nervous system caused by CAG trinucleotide repeat expansion which encodes polyglutamine tracts, leading to the misfolding of pathological proteins. Small peptides can be designed to prevent polyglutamine diseases by inhibiting the polyglutamine protein aggregation, for example, polyglutamine binding peptide 1(QBP1). However, the transportation capability of polyglutamine binding peptide 1 across the blood-brain barrier is less efficient. We hypothesized whether its therapeutic effect could be improved by increasing the rate of membrane penetration. Objectives: The objective of the study was to explore whether polyglutamine binding peptide 1 conjugated cell-penetrating peptides could pass through the blood-brain barrier and inhibit the aggregation of polyglutamine proteins. Methods: n order to investigate the toxic effects, we constructed a novel stable inducible PC12 cells to express Huntington protein that either has 11 glutamine repeats or 63 glutamine repeats to mimic wild type and polyglutamine expand Huntington protein, respectively. Both SynB3 and TAT conjugated polyglutamine binding peptide 1 was synthesized, respectively, and we tested their capabilities to pass through a Trans-well system and subsequently studied the counteractive effects on polyglutamine protein aggregation. Results: The conjugation of cell-penetrating peptides to SynB3 and TAT enhanced the transportation of polyglutamine binding peptide 1 across the mono-cell layer and ameliorated polyglutamine-expanded Huntington protein aggregation; moreover, SynB3 showed better delivery efficiency than TAT. Interestingly, it has been observed that polyglutamine binding peptide 1 specifically inhibited polyglutamine-expanded protein aggregation rather than affected other amyloidosis proteins, for example, β-Amyloid. Conclusion: Our study indicated that SynB3 could be an effective carrier for polyglutamine binding peptide 1 distribution through the blood-brain barrier model and ameliorate the formation of polyglutamine inclusions, thus SynB3 conjugated polyglutamine binding peptide 1 could be considered as a therapeutic candidate for polyglutamine diseases.

Recent Developments of Recombinant Protein Expression for Therapeutic Purposes

In the most recent seven to eight years, the therapeutic recombinant proteins have rapidly expanded in the biotechnology domain due to its wide variety of needs. There has been significant development in the mammalian expression system for fine purification and increased level of expressed recombinant proteins [1,2]. Many drugs like tetracycline have been demonstrated on the Chinese Hamster Ovary cell line for promising multi control strategies and effective cytotoxicity. Mammalian expression system improves the proper glycosylation of recombinant proteins which are very helpful to increase solubility of product [3-6]. Meanwhile on the prokaryotic expression system, E. coli has proven to be an easier to handle, friendly and economical strain [2]. Recently these expression systems are using to work on antibody fragment productions and their proper folding with co-expression of chaperones [7]. Moreover E. coli has been used for the production of cancer cell penetrating peptides which promises the targeted delivery of drugs to specific effector cells only. Yeast systems are also being used for the antibody fragments production and the high level production of insulin. Interestingly cell free expression systems are also participating in this game and that would be very fascinating to see in the coming years about cell extract medium for production of high level recombinant protein [8, 9]. Purification and optimization of recombinant protein has always been a challenging situation for scientists and they paid more attention to increase the overall yield of the product. Many affinity chromatography techniques has been introduced for efficient purification of protein of interest [10]. Despite these research and developments in methodologies to produce and purify the recombinant therapeutic protein, scientists still face the hurdles and challenges with all expression systems. Rationally E. coli produces inclusion bodies and many mammalian cell types do not show the same results with the same recombinant protein. [11]. So there is a requirement for adding the appropriate features to the expression systems focused to better improvising recovery, production and purification of recombinant protein. Copyright(c) The Author