Vaccine delivery systems and administration routes: Advanced biotechnological techniques to improve the immunization efficacy.

Since the first use of vaccine tell the last COVID-19 pandemic caused by spread of SARS-CoV-2 worldwide, the use of advanced biotechnological techniques has accelerated the development of different types and methods for immunization. The last pandemic showed that the nucleic acid-based vaccine, especially mRNA, has an advantage in terms of development time; however, it showed a very critical drawback namely, the higher costs when compared to other strategies, and its inability to protect against new variants. This showed the need of more improvement to reach a better delivery and efficacy. In this review we will describe different vaccine delivery systems including, the most used viral vector, and also variable strategies for delivering of nucleic acid-based vaccines especially lipid-based nanoparticles formulation, polymersomes, electroporation and also the new powerful tools for the delivery of mRNA, which is based on the use of cell-penetrating peptides (CPPs). Additionally, we will also discuss the main challenges associated with each system. Finlay, the efficacy and safety of the vaccines depends not only on the formulations and delivery systems, but also the dosage and route of administration are also important players, therefore we will see the different routes for the vaccine administration including traditionally routes (intramuscular, Transdermal, subcutaneous), oral inhalation or via nasal mucosa, and will describe the advantages and disadvantage of each administration route.

Comparative Study of the Potential Cell-Penetrating Peptide ∆M4 on Apoptosis Cell Signaling in A375 and A431 Cancer Cell Lines.

In recent yearsjajajj, peptide-based therapeutics have attracted increasing interest as a potential approach to cancer treatment. Peptides are characterized by high specificity and low cytotoxicity, but they cannot be considered universal drugs for all types of cancer. Of the numerous anticancer-reported peptides, both natural and synthetic, only a few have reached clinical applications. However, in most cases, the mechanism behind the anticancer activity of the peptide is not fully understood. For this reason, in this work, we investigated the effect of the novel peptide ∆M4, which has documented anticancer activity, on two human skin cancer cell lines. A novel approach to studying the potential induction of apoptosis by anticancer peptides is the use of protein microarrays. The results of the apoptosis protein study demonstrated that both cell types, skin malignant melanoma (A375) and epidermoid carcinoma (A431), exhibited markers associated with apoptosis and cellular response to oxidative stress. Additionally, ∆M4 induced concentration- and time-dependent moderate ROS production, triggering a defensive response from the cells, which showed decreased activation of cytoplasmic superoxide dismutase. However, the studied cells exhibited a differential response in catalase activity, with A375 cells showing greater resistance to the peptide action, possibly mediated by the Nrf2 pathway. Nevertheless, both cell types showed moderate activity of caspases 3/7, suggesting that they may undergo partial apoptosis, although another pathway of programmed death cannot be excluded. Extended analysis of the mechanisms of action of anticancer peptides may help determine their effectiveness in overcoming chemoresistance in cancerous cells.

Evidence That a Peptide-Drug/p53 Gene Complex Promotes Cognate Gene Expression and Inhibits the Viability of Glioblastoma Cells.

Glioblastoma multiform (GBM) is considered the deadliest brain cancer. Conventional therapies are followed by poor patient survival outcomes, so novel and more efficacious therapeutic strategies are imperative to tackle this scourge. Gene therapy has emerged as an exciting and innovative tool in cancer therapy. Its combination with chemotherapy has significantly improved therapeutic outcomes. In line with this, our team has developed temozolomide-transferrin (Tf) peptide (WRAP5)/p53 gene nanometric complexes that were revealed to be biocompatible with non-cancerous cells and in a zebrafish model and were able to efficiently target and internalize into SNB19 and U373 glioma cell lines. The transfection of these cells, mediated by the formulated peptide-drug/gene complexes, resulted in p53 expression. The combined action of the anticancer drug with p53 supplementation in cancer cells enhances cytotoxicity, which was correlated to apoptosis activation through quantification of caspase-3 activity. In addition, increased caspase-9 levels revealed that the intrinsic or mitochondrial pathway of apoptosis was implicated. This assumption was further evidenced by the presence, in glioma cells, of Bax protein overexpression-a core regulator of this apoptotic pathway. Our findings demonstrated the great potential of peptide TMZ/p53 co-delivery complexes for cellular transfection, p53 expression, and apoptosis induction, holding promising therapeutic value toward glioblastoma.

Neuroprotective Activity of a Non-Covalent Imatinib+TP10 Conjugate in HT-22 Neuronal Cells In Vitro.

This study evaluated the probable relevance of a non-covalent conjugate of imatinib with TP10 in the context of a neuroprotective effect in Parkinson's disease. Through the inhibition of c-Abl, which is a non-receptor tyrosine kinase and an indicator of oxidative stress, imatinib has shown promise in preclinical animal models of this disease. The poor distribution of imatinib within the brain tissue triggered experiments in which a conjugate was obtained by mixing the drug with TP10, which is known for exhibiting high translocation activity across the cell membrane. The conjugate was tested on the HT-22 cell line with respect to its impact on MPP+-induced oxidative stress, apoptosis, necrosis, cytotoxicity, and mortality. Additionally, it was checked whether the conjugate activated the ABCB1 protein. The experiments indicated that imatinib+PEG4+TP10 reduced the post-MPP+ oxidative stress, apoptosis, and mortality, and these effects were more prominent than those obtained after the exposition of the HT-22 cells to imatinib alone. Its cytotoxicity was similar to that of imatinib itself. In contrast to imatinib, the conjugate did not activate the ABCB1 protein. These favorable qualities of imatinib+PEG4+TP10 make it a potential candidate for further in vivo research, which would confirm its neuroprotective action in PD-affected brains.

Potential of Cell-Penetrating Peptide-Conjugated Antisense Oligonucleotides for the Treatment of SMA.

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder that is caused by mutations in the survival motor neuron 1 (SMN1) gene, hindering the production of functional survival motor neuron (SMN) proteins. Antisense oligonucleotides (ASOs), a versatile DNA-like drug, are adept at binding to target RNA to prevent translation or promote alternative splicing. Nusinersen is an FDA-approved ASO for the treatment of SMA. It effectively promotes alternative splicing in pre-mRNA transcribed from the SMN2 gene, an analog of the SMN1 gene, to produce a greater amount of full-length SMN protein, to compensate for the loss of functional protein translated from SMN1. Despite its efficacy in ameliorating SMA symptoms, the cellular uptake of these ASOs is suboptimal, and their inability to penetrate the CNS necessitates invasive lumbar punctures. Cell-penetrating peptides (CPPs), which can be conjugated to ASOs, represent a promising approach to improve the efficiency of these treatments for SMA and have the potential to transverse the blood-brain barrier to circumvent the need for intrusive intrathecal injections and their associated adverse effects. This review provides a comprehensive analysis of ASO therapies, their application for the treatment of SMA, and the encouraging potential of CPPs as delivery systems to improve ASO uptake and overall efficiency.

A proteomic approach supports the clinical relevance of TAT-Cx43266-283 in glioblastoma.

Glioblastoma (GBM) is the most frequent and aggressive primary brain cancer. The Src inhibitor, TAT-Cx43266-283, exerts antitumor effects in in vitro and in vivo models of GBM. Because addressing the mechanism of action is essential to translate these results to a clinical setting, in this study we carried out an unbiased proteomic approach. Data-independent acquisition mass spectrometry proteomics allowed the identification of 190 proteins whose abundance was modified by TAT-Cx43266-283. Our results were consistent with the inhibition of Src as the mechanism of action of TAT-Cx43266-283 and unveiled antitumor effectors, such as p120 catenin. Changes in the abundance of several proteins suggested that TAT-Cx43266-283 may also impact the brain microenvironment. Importantly, the proteins whose abundance was reduced by TAT-Cx43266-283 correlated with an improved GBM patient survival in clinical datasets and none of the proteins whose abundance was increased by TAT-Cx43266-283 correlated with shorter survival, supporting its use in clinical trials.

Cell-penetrating peptides for sustainable agriculture.

Cell-penetrating peptides (CPPs) are short (typically 5-30 amino acids), cationic, amphipathic, or hydrophobic peptides that facilitate the cellular uptake of diverse cargo molecules by eukaryotic cells via direct translocation or endocytosis across the plasma membrane. CPPs can deliver a variety of bioactive cargos, including proteins, peptides, nucleic acids, and small molecules into the cell. Once inside, the delivered cargo may function in the cytosol, nucleus, or other subcellular compartments. Numerous CPPs have been used for studies and drug delivery in mammalian systems. Although CPPs have many potential uses in plant research and agriculture, the application of CPPs in plants remains limited. Here we review the structures and mechanisms of CPPs and highlight their potential applications for sustainable agriculture.

Improving corneal permeability of dexamethasone using penetration enhancing agents: First step towards achieving topical drug delivery to the retina.

With an ever-increasing burden of vision loss caused by diseases of the posterior ocular segment, there is an unmet clinical need for non-invasive treatment strategies. Topical drug application using eye drops suffers from low to negligible bioavailability to the posterior segment as a result of static and dynamic defensive ocular barriers to penetration, while invasive delivery systems are expensive to administer and suffer potentially severe complications. As the cornea is the main anatomical barrier to uptake of topically applied drugs from the ocular surface, we present an approach to increase corneal permeability of a corticosteroid, dexamethasone sodium-phosphate (DSP), using a novel penetration enhancing agent (PEA). We synthesised a novel polyacetylene (pAc) polymer and compared its activity to two previously described cell penetrating peptide (CPP) based PEAs, TAT and penetratin, with respect to increasing transcorneal permeability of DSP in a rapid ex-vivo porcine corneal assay over 60 min. The transcorneal apparent permeability coefficients (Papp) for diffusion of pAc, and fluorescein isothiocyanate (FITC) conjugated TAT and penetratin were up to 5 times higher (p < 0.001), when compared to controls. When pAc was used in formulation with DSP, an almost 5-fold significant increase was observed in Papp of DSP across the cornea (p = 0.0130), a significant 6-fold increase with TAT (p = 0.0377), and almost 7-fold mean increase with penetratin (p = 0.9540). Furthermore, we investigated whether the PEAs caused any irreversible damage to the barrier integrity of the corneal epithelium by measuring transepithelial electrical resistance (TEER) and immunostaining of tight junction proteins using zonula occludens-1 (ZO-1) and occludin antibodies. There was no damage or structural toxicity, and the barrier integrity was preserved after PEA application. Finally, an in-vitro cytotoxicity assessment of all PEAs in human retinal pigment epithelium cells (ARPE-19) demonstrated that all PEAs were very well-tolerated, with IC50 values of 64.79 mM for pAc and 1335.45 µM and 87.26 µM for TAT and penetratin, respectively. Our results suggest that this drug delivery technology could potentially be used to achieve a significantly higher intraocular therapeutic bioavailability after topical eye drop administration, than currently afforded.

Functionalized nanoparticles to deliver nucleic acids to the brain for the treatment of Alzheimer's disease.

Brain-targeted gene delivery across the blood-brain barrier (BBB) is a significant challenge in the 21st century for the healthcare sector, particularly in developing an effective treatment strategy against Alzheimer's disease (AD). The Internal architecture of the brain capillary endothelium restricts bio-actives entry into the brain. Additionally, therapy with nucleic acids faces challenges like vulnerability to degradation by nucleases and potential immune responses. Functionalized nanocarrier-based gene delivery approaches have resulted in safe and effective platforms. These nanoparticles (NPs) have demonstrated efficacy in protecting nucleic acids from degradation, enhancing transport across the BBB, increasing bioavailability, prolonging circulation time, and regulating gene expression of key proteins involved in AD pathology. We provided a detailed review of several nanocarriers and targeting ligands such as cell-penetrating peptides (CPPs), endogenous proteins, and antibodies. The utilization of functionalized NPs extends beyond a singular system, serving as a versatile platform for customization in related neurodegenerative diseases. Only a few numbers of bioactive regimens can go through the BBB. Thus, exploring functionalized NPs for brain-targeted gene delivery is of utmost necessity. Currently, genes are considered high therapeutic potential molecules for altering any disease-causing gene. Through surface modification, nanoparticulate systems can be tailored to address various diseases by replacing the target-specific molecule on their surface. This review article presents several nanoparticulate delivery systems, such as lipid NPs, polymeric micelles, exosomes, and polymeric NPs, for nucleic acids delivery to the brain and the functionalization strategies explored in AD research.

Charge-switchable cell-penetrating peptides for rerouting nanoparticles to glioblastoma treatment.

Glioblastoma (GB) is one of the most lethal types of neoplasms with unique anatomic, physiologic, and pathologic features that usually persist after exposure to standard therapeutic modalities. It is biologically aggressive, and the existence of the blood-brain barrier (BBB) limits the efficacy of standard therapies. In this work, we hypothesize the potential of surface-functionalized ultra-small nanostructured lipid carriers (usNLCs) with charge-switchable cell-penetrating peptides (CPPs) to overcome this biological barrier and improve targeted delivery to brain tumor tissues. The big question is: what is the potential of CPPs in directing nanoparticles toward brain tumor tissue? To answer this question, the usNLCs were functionalized with distinct biomolecules [five CPPs, c(RGDfK) and transferrin, Tf] through electrostatic interaction and its ability as a targeting approach to BBB (HBMEC) and glioma cells (U87 cells) evaluated in terms of physicochemical properties, cellular uptake, permeability in a 2D-BBB model, and tumor growth inhibition. Monte Carlo simulations elucidated CPP adsorption patterns. The permeability studies revealed that targeted usNLCs, especially usNLCsTf and usNLCsCPP4, exhibited an increased permeability coefficient compared to the non-targeted usNLCs. Functionalized usNLCs evidenced enhanced uptake in BBB cells, with smaller CPPs showing higher internalization (CPP1 and CPP2). Similarly, functionalized usNLCs exhibited more significant cytotoxicity in glioma cells, with specific CPPs promoting favorable internalization. Analysis of the endocytic pathway indicated that usNLCsCPPs were mainly internalized by direct translocation and caveolae-mediated endocytosis. Optimal usNLCs with dual targeting capabilities to both BBB and GB cells provide a promising therapeutic strategy for GB.

Development of TRIB3-Based Therapy as a Gene-Independent Approach to Treat Retinal Degenerative Disorders.

Inherited retinal degeneration (RD) constitutes a heterogeneous group of genetic retinal degenerative disorders. The molecular mechanisms underlying RD encompass a diverse spectrum of cellular signaling, with the unfolded protein response (UPR) identified as a common signaling pathway chronically activated in degenerating retinas. TRIB3 has been recognized as a key mediator of the PERK UPR arm, influencing various metabolic pathways, such as insulin signaling, lipid metabolism, and glucose homeostasis, by acting as an AKT pseudokinase that prevents the activation of the AKT → mTOR axis. This study aimed to develop a gene-independent approach targeting the UPR TRIB3 mediator previously tested by our group using a genetic approach in mice with RD. The goal was to validate a therapeutic approach targeting TRIB3 interactomes through the pharmacological targeting of EGFR-TRIB3 and delivering cell-penetrating peptides targeting TRIB3 → AKT. The study employed rd10 and P23H RHO mice, with afatinib treatment conducted in p15 rd10 mice through daily intraperitoneal injections. P15 P23H RHO mice received intraocular injections of cell-penetrating peptides twice at a 2-week interval. Our study revealed that both strategies successfully targeted TRIB3 interactomes, leading to an improvement in scotopic A- and B-wave ERG recordings. Additionally, the afatinib-treated mice manifested enhanced photopic ERG amplitudes accompanied by a delay in photoreceptor cell loss. The treated rd10 retinas also showed increased PDE6ß and RHO staining, along with an elevation in total PDE activity in the retinas. Consequently, our study demonstrated the feasibility of a gene-independent strategy to target common signaling in degenerating retinas by employing a TRIB3-based therapeutic approach that delays retinal function and photoreceptor cell loss in two RD models.

Efficient systemic CNS delivery of a therapeutic antisense oligonucleotide with a blood-brain barrier-penetrating ApoE-derived peptide.

Antisense oligonucleotide (ASO) has emerged as a promising therapeutic approach for treating central nervous system (CNS) disorders by modulating gene expression with high selectivity and specificity. However, the poor permeability of ASO across the blood-brain barrier (BBB) diminishes its therapeutic success. Here, we designed and synthesized a series of BBB-penetrating peptides (BPP) derived from either the receptor-binding domain of apolipoprotein E (ApoE) or a transferrin receptor-binding peptide (THR). The BPPs were conjugated to phosphorodiamidate morpholino oligomers (PMO) that are chemically analogous to the 2'-O-(2-methoxyethyl) (MOE)-modified ASO approved by the FDA for treating spinal muscular atrophy (SMA). The BPP-PMO conjugates significantly increased the level of full-length SMN2 in the patient-derived SMA fibroblasts in a concentration-dependent manner with minimal to no toxicity. Furthermore, the systemic administration of the most potent BPP-PMO conjugates significantly increased the expression of full-length SMN2 in the brain and spinal cord of SMN2 transgenic adult mice. Notably, BPP8-PMO conjugate showed a 1.25-fold increase in the expression of full-length functional SMN2 in the brain. Fluorescence imaging studies confirmed that 78% of the fluorescently (Cy7)-labelled BPP8-PMO reached brain parenchyma, with 11% uptake in neuronal cells. Additionally, the BPP-PMO conjugates containing retro-inverso (RI) D-BPPs were found to possess extended half-lives compared to their L-counterparts, indicating increased stability against protease degradation while preserving the bioactivity. This delivery platform based on BPP enhances the CNS bioavailability of PMO targeting the SMN2 gene, paving the way for the development of systemically administered neurotherapeutics for CNS disorders.

Enhancing Cellular Uptake of Native Proteins through Bio-Orthogonal Conjugation with Chemically Synthesized Cell-Penetrating Peptides.

The potential for native proteins to serve as a platform for biocompatible, targeted, and personalized therapeutics in the context of genetic and metabolic disorders is vast. Nevertheless, their clinical application encounters challenges, particularly in overcoming biological barriers and addressing the complexities involved in engineering transmembrane permeability. This study is dedicated to the development of a multifunctional nanoentity in which a model therapeutic protein is covalently linked to a cell-penetrating peptide, NickFect 55, with the objective of enhancing its intracellular delivery. Successful binding of the nanoentity fragments was achieved through the utilization of an intein-mediated protein-trans splicing reaction. Our research demonstrates that the fully assembled nanoentity-containing protein was effectively internalized by the cells, underscoring the potential of this approach in overcoming barriers associated with protein-based therapeutics for the treatment of genetic disorders.

Copper-Chelated Chitosan Microgels for the Selective Enrichment of Small Cationic Peptides.

Copper-chelated chitosan microgels were investigated as an immobilized metal affinity chromatography (IMAC) phase for peptide separation. The copper-crosslinked chitosan beads were shown to strongly interact with a range of amino acids, in a wide range of pH and saline conditions. The beads exhibited an affinity that seemed to depend on the isoelectric point of the amino acid, with the extent of uptake increasing with decreasing isoelectric point. This selective interaction with anionic amino acids resulted in a significant relative enrichment of the supernatant solution in cationic amino acids. The beads were then studied as a novel fractionation system for complex milk hydrolysates. The copper chitosan beads selectively removed larger peptides from the hydrolysate aqueous solution, yielding a solution relatively enriched in medium and smaller peptides, which was characterized both quantitatively and qualitatively by size exclusion chromatography (SEC). Liquid chromatography-mass spectrometry (LCMS) work provided comprehensive data on a peptide sequence level and showed that a depletion of the anionic peptides by the beads resulted in a relative enrichment of the cationic peptides in the supernatant solution. It could be concluded that after fractionation a dramatic relative enrichment in respect to small- and medium-sized cationic peptides in the solution, characteristics that have been linked to bioactivities, such as anti-microbial and cell-penetrating properties. The results demonstrate the use of the chitosan copper gel bead system in lab scale fractionation of complex hydrolysate mixtures, with the potential to enhance milk hydrolysate bioactivity.

Combating bone marrow failure with polymer materials.

Bone marrow failure (BMF) has become one of the most studied autoimmune disorders, particularly due to its prevalence both as an inherited disease, but also as a result of chemotherapies. BMF is associated with severe symptoms such as bleeding episodes and susceptibility to infections, and often has underlying characteristics, such as anemia, thrombocytopenia, and neutropenia. The current treatment landscape for BMF requires stem cell transplantation or chemotherapies to induce immune suppression. However, there is limited donor cell availability or dose related toxicity associated with these treatments. Optimizing these treatments has become a necessity. Polymer-based materials have become increasingly popular, as current research efforts are focused on synthesizing novel cell matrices for stem cell expansion to solve limited donor cell availability, as well as applying polymer delivery vehicles to intracellularly deliver cargo that can aid in immunosuppression. Here, we discuss the importance and impact of polymer materials to enhance therapeutics in the context of BMF.

Cell-penetrating peptides with nanoparticles hybrid delivery vectors and their uptake pathways.

Cell-penetrating peptides (CPPs) are molecules that improve the cellular uptake of various molecular payloads that do not easily traverse the cellular membrane. CPPs can be found in pharmaceutical and medical products. The vast majority of cell-penetrating chemicals that are discussed in published research are peptide based. The paper also delves into the various applications of hybrid vectors. Because CPPs are able to carry cargo across the cellular membrane, they are a viable candidate for use as a suitable carrier for a wide variety of cargoes, such as siRNA, nanoparticles, and others. In which we discuss the CPPs, their classification, uptake mechanisms, hybrid vector systems, nanoparticles and their uptake mechanisms, etc. Further in this paper, we discuss CPPs conjugated to Nanoparticles, Combining CPPs with lipids and polymeric Nanoparticles in A Conjugated System, CPPs conjugated to nanoparticles for therapeutic purposes, and potential therapeutic uses of CPPs as delivery molecules. Also discussed the preclinical and clinical use of CPPS, intracellular trafficking of nanoparticles, and activatable and bioconjugated CPPs.

Molecular determinants for brain targeting by peptides: a meta-analysis approach with experimental validation.

Blood-brain barrier (BBB) peptide-shuttles (BBBpS) are able to translocate the BBB and reach the brain. Despite the importance of brain targeting in pharmacology, BBBpS are poorly characterized. Currently, their development relies on the empiric assumption that cell-penetrating peptides (CPPs), with proven ability to traverse lipid membranes, will likewise behave as a BBBpS. The relationship between CPPs/BBBpS remains elusive and, to the best of our knowledge, has not hitherto been subject to thorough experimental scrutiny. In this work, we have identified/quantified the main physicochemical properties of BBBpS and then searched for CPPs with these properties, hence potential BBBpS. The specific features found for BBBpS are: (i) small size, (ii) none or few aromatic residues, (iii) hydrophobic, and (iv) slight cationic nature. Then, we selected the 10 scoring best in an ordinary least squares analysis, and tested them in vitro and in vivo. Overall, we identified the molecular determinants for brain targeting by peptides, devised a methodology that can be used to assist in the design of peptides with potential brain penetration from amino acid residue sequences, and found four new BBBpS within the CPP library.

Influence of Aza-Glycine Substitution on the Internalization of Penetratin.

The cell-penetrating peptide (CPP) penetratin has gained much attention over many years due to its potential role as a transporter for a broad range of cargo into cells. The modification of penetratin has been extensively investigated too. Aza-peptides are peptide analogs in which one or more of the amino residues are replaced by a semicarbazide. This substitution results in conformational restrictions and modifications in hydrogen bonding properties, which affect the structure and may lead to enhanced activity and selectivity of the modified peptide. In this work, the Trp residues of penetratin were substituted by aza-glycine or glycine residues to examine the effect of these modifications on the cellular uptake and the internalization mechanism. The substitution of Trp48 or Trp48,56 dramatically reduced the internalization, showing the importance of Trp48 in cellular uptake. Interestingly, while aza-glycine in the position of Trp56 increased the cellular uptake, Gly reduced it. The two Trp-modified derivatives showed altered internalization pathways, too. Based on our knowledge, this is the first study about the effect of aza-amino acid substitution on the cell entry of CPPs. Our results suggest that aza-amino acid insertion is a useful modification to change the internalization of a CPP.

Investigating the impact of thiol reactivity and disulfide formation on cellular uptake of cell-permeable peptides.

Cell-penetrating peptides (CPPs) have been explored as versatile tools to transport various molecules into cells. The uptake mechanism of CPPs is still not clearly understood and most probably depends on several factors like the nature of the CPP itself, the attached cargo, the investigated cell system, and other experimental conditions, such as temperature and concentration. One of the first steps of internalization involves the interaction of CPPs with negatively charged molecules present at the outer layer of the cell membrane. Recently, thiol-mediated uptake has been found to support the effective translocation of sulfhydryl-bearing substances that would actually not be cell-permeable. Within this work, we aimed to understand the relevance of thiol reactivity for the uptake mechanism of cysteine-containing CPPs that we have developed previously in our group. Therefore, we compared the two peptides, sC18-Cys and CaaX-1, in their single reduced and dimeric disulfide versions. Cytotoxicity, intracellular accumulation, and impact on the internalization process of the disulfides were investigated in HeLa cells. Both disulfide CPPs demonstrated significantly stronger cytotoxic effects and membrane activity compared with their reduced counterparts. Notably, thiol-mediated uptake could be excluded as a main driver for translocation, showing that peptides like CaaX-1 are most likely taken up by other mechanisms.

Extracellular Microvesicles Modified with Arginine-Rich Peptides for Active Macropinocytosis Induction and Delivery of Therapeutic Molecules.

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), transfer bioactive molecules from donor to recipient cells in various pathophysiological settings, thereby mediating intercellular communication. Despite their significant roles in extracellular signaling, the cellular uptake mechanisms of different EV subpopulations remain unknown. In particular, plasma membrane-derived MVs are larger vesicles (100 nm to 1 µm in diameter) and may serve as efficient molecular delivery systems due to their large capacity; however, because of size limitations, receptor-mediated endocytosis is considered an inefficient means for cellular MV uptake. This study demonstrated that macropinocytosis (lamellipodia formation and plasma membrane ruffling, causing the engulfment of large fluid volumes outside cells) can enhance cellular MV uptake. We developed experimental techniques to induce macropinocytosis-mediated MV uptake by modifying MV membranes with arginine-rich cell-penetrating peptides for the intracellular delivery of therapeutic molecules.