Intracellular Delivery Of Bioactive Molecules Research Articles

Arginine-Rich Cell-Penetrating Peptides Induce Lipid Rearrangements for Their Active Translocation across Laterally Heterogeneous Membranes.

Arginine-rich cell-penetrating peptides (CPPs) have emerged as valuable tools for the intracellular delivery of bioactive molecules, but their membrane perturbation during cell penetration is not fully understood. Here, nona-arginine (R9)-mediated membrane reorganization that facilitates the translocation of peptides across laterally heterogeneous membranes is directly visualized. The electrostatic binding of cationic R9 to anionic phosphatidylserine (PS)-enriched domains on a freestanding lipid bilayer induces lateral lipid rearrangements; in particular, in real-time it is observed that R9 fluidizes PS-rich liquid-ordered (Lo) domains into liquid-disordered (Ld) domains, resulting in the membrane permeabilization. The experiments with giant unilamellar vesicles (GUVs) confirm the preferential translocation of R9 through Ld domains without pore formation, even when Lo domains are more negatively charged. Indeed, whenever R9 comes into contact with negatively charged Lo domains, it dissolves the Lo domains first, promoting translocation across phase-separated membranes. Collectively, the findings imply that arginine-rich CPPs modulate lateral membrane heterogeneity, including membrane fluidization, as one of the fundamental processes for their effective cell penetration across densely packed lipid bilayers.

Enhancing Membrane-Disruptive Activity via Hydrophobic Phenylalanine and Lysine Tethered to Poly(aspartic acid).

Amphiphilic polymers with pH-responsive abilities have been widely used as carriers for intracellular delivery of bioactive substances, while their membrane-disruptive activity exerted on cells is a critical characteristic that determines delivery efficiency. Herein, we present a novel method to prepare amphiphilic and pH-responsive polymers by chemically tethering l-phenylalanine methyl ester and followed by Nε-carbobenzyloxy-l-lysine benzyl ester to the side carboxylic acid groups of poly(aspartic acid). The obtained phenylalanine- and lysine-grafted polymer (PAsp- g-Phe)- g-Lys demonstrated enhanced membrane-disruptive activity at pH 7.4 in comparison with that of PAsp- g-Phe. Moreover, the pH-responsive behavior of the grafted polymers caused by the significantly intensified hydrophobicity could be modulated by the tethered amount of hydrophobic amino acids with phenyl groups. The prepared amphiphilic (PAsp- g-Phe)- g-Lys could facilitate entry of calcein into NIH/3T3 and HeLa cells at physiological pH values, possibly due to local chemical destabilization of cell membranes by the interaction between the polymer and membrane bilayers. Therefore, we have provided a feasible approach to prepare pH-responsive polymers with enhanced membrane-disruptive activity, and the phenylalanine- and lysine-grafted polymers could be a potential candidate for intracellular delivery of bioactive molecules in biomedical applications.

Acylation of the S413-PV cell-penetrating peptide as a means of enhancing its capacity to mediate nucleic acid delivery: Relevance of peptide/lipid interactions

BackgroundCell-penetrating peptides (CPPs) have been extensively exploited in gene therapy approaches as vectors for intracellular delivery of bioactive molecules. The ability of CPPs to be internalized into cells and their capacity to complex nucleic acids depend on their molecular structure, both primary and secondary, namely regarding hydrophobicity/hydrophilicity. CPP acylation has been used as a strategy to improve this structural feature. MethodsAcyl groups (from 6 to 18 carbon atoms) were attached to the S413-PV peptide and their effects on the peptide competence to complex siRNAs and to mediate gene silencing in glioblastoma (GBM) cells were studied. A systematic characterization of membrane interactions with S413-PV acyl-derivatives was also conducted, using different biophysical techniques (surface pressure-area isotherms in Langmuir monolayers, DSC and 31P NMR) to unravel a relationship between CPP biological activity and CPP effects on membrane stability and lipid organization. ResultsA remarkable concordance was noticed between acylated-S413-PV peptide competence to promote gene silencing in GBM cells and disturbance induced in membrane models, the lauroyl- and myristoyl-S413-PV peptides being the most effective. A cut-off effect was described for the first time regarding the influence of acyl-chain length on CPP bioactivity. ConclusionsC12-S413-PV showed high capacity to destabilize lipid bilayers, to escape from lysosomal degradation and to mediate gene silencing without promoting cytotoxicity. General significanceBesides unraveling a new CPP with high potential to be employed as a gene delivery vector, this work emphasizes the benefit from allying biophysical and biological studies towards a proper CPP structural refinement for successful pre-clinical/clinical application.

ChemInform Abstract: Current Understanding of Direct Translocation of Arginine‐Rich Cell‐Penetrating Peptides and Its Internalization Mechanisms

AbstractReview: 57 refs.

Syndecan-4 Is a Receptor for Clathrin-Mediated Endocytosis of Arginine-Rich Cell-Penetrating Peptides.

Arginine-rich cell-penetrating peptides (CPPs) such as Tat and oligoarginine peptides have been widely used as carriers for intracellular delivery of bioactive molecules. Despite accumulating evidence for involvement of endocytosis in the cellular uptake of arginine-rich CPPs, the primary cell-surface receptors for these peptide carriers that would initiate endocytic processes leading to intracellular delivery of bioactive cargoes have remained poorly understood. Our previous attempt to identify membrane receptors for octa-arginine (R8) peptide, one of the representative arginine-rich CPPs, using the photo-cross-linking probe bearing a photoreactive diazirine was not successful due to considerable amounts of cellular proteins nonspecifically bound to the affinity beads. To address this issue, here we developed a photo-cross-linking probe in which a cleavable linker of a diazobenzene moiety was employed to allow selective elution of cross-linked proteins by reducing agent-mediated cleavage. We demonstrated that introduction of the diazobenzene moiety into the photoaffinity probe enables efficient purification of cross-linked proteins with significant reduction of nonspecific binding proteins, leading to successful identification of 17 membrane-associated proteins that would interact with R8 peptide. RNAi-mediated knockdown experiments in combination with the pharmacological inhibitors revealed that, among the proteins identified, syndecan-4, one of the heparan sulfate proteoglycans, is an endogenous membrane-associated receptor for the cellular uptake of R8 peptide via clathrin-mediated endocytosis. This syndecan-4-dependent pathway was also involved in the intracellular delivery of bioactive proteins mediated by R8 peptide. These results reveal that syndecan-4 is a primary cell-surface target for R8 peptide that allows intracellular delivery of bioactive cargo molecules via clathrin-mediated endocytosis.

Current Understanding of Direct Translocation of Arginine-Rich Cell-Penetrating Peptides and Its Internalization Mechanisms

Arginine-rich cell-penetrating peptides (CPPs) including Tat, Penetratin and oligoarginine peptides are a series of short peptides that can be efficiently internalized into cells and have been widely used as carriers for intracellular delivery of bioactive molecules. In the early phase of the study, CPPs, as well as their conjugates, were thought to rapidly enter cells by direct penetration through membranes, which was later found to be an experimental artifact that was concluded from observations in fixed cells. Although re-evaluation using living unfixed cells revealed that endocytosis has a major role in internalization of these peptides, there are a number of studies reporting that, even if fixation is avoided, direct translocation across plasma membranes and cytosolic distribution of arginine-rich CPPs are still observed in cells without membrane perturbation. In addition, amphiphilic counteranions such as pyrenebutyrate dramatically accelerate direct translocation of these peptides into cells. These results suggest that there are at least two pathways, i.e., endocytosis and direct translocation, both of which would contribute to cellular internalization of arginine-rich CPPs. In this review, we first introduce the story of fixation artifact, which indeed led to the critical progress in CPP study, and then summarize the current understanding for direct translocation of arginine-rich CPPs. Comprehensive understanding of direct translocation of these peptides and its mechanistic elucidation would provide useful knowledge for developing methodologies that would enable efficient intracellular delivery.

Impact of Polyethylenimine Conjugation Mode on the Cell Transfection Efficiency of Silica Nanovectors.

The conjugation of polyethylenimine (PEI) to silica nanoparticles has emerged as a useful strategy in gene delivery. Here we investigate the influence of the PEI conjugation mode on the transfection ability of plain silica nanoparticles. Surface functionalization with sulfonate- and chloride-bearing silanes modulates the amount and conformation of PEI and therefore the particles' affinity for the plasmid, without impacting on cytotoxicity. However, transfection efficiency in both immortalized and primary cells is more directly correlated to the nature and strength of the particle-PEI interactions. It suggests that PEI detachment from the particle surface at the stage of endosomal escape is a key event in the plasmid delivery process. These data should provide fruitful guidelines for the fine tuning of colloidal surfaces intended for intracellular delivery of bioactive molecules.

Liposomes: Targeted and Controlled Delivery System

Novel drug delivery systems (NDDS) delivered the active molecules to the target site in a defined manner for produce the desired effects without disturbing the delicate bio-environment. Among many available colloidal drug delivery systems, Liposomes (a class based on phospholipids) have attracted more attention than other systems due to many meritorious features such as excellent chemical & biological stability, good solubilisation power, promote intracellular delivery of bio-active molecules, reduce the uptake of macrophages and encapsulate both hydrophilic as well as lipophillic drug molecule. In this review article, I try to give some basic knowledge about these carrier systems in tabular form, which is more understandable concept as compared to simple text form.

Fibroblast Growth Factor-12 (FGF12) Translocation into Intestinal Epithelial Cells Is Dependent on a Novel Cell-penetrating Peptide Domain: INVOLVEMENT OF INTERNALIZATION IN THE IN VIVO ROLE OF EXOGENOUS FGF12

The extracellular effect of fibroblast growth factor-12 (FGF12) remains unknown because FGF12 cannot activate any fibroblast growth factor receptors (FGFRs), and FGF12 is not currently thought to be released from cells. We reported previously that FGF12 plays an intracellular role in the inhibition of radiation-induced apoptosis. In this study, we demonstrated that recombinant FGF12 was able to be internalized into the cytoplasm of a rat intestinal epithelial cell line, IEC6, and this process was dependent on two novel cell-penetrating peptide (CPP) domains (CPP-M and CPP-C). In particular, CPP-C, composed of ∼10 amino acids, was identified as a specific domain of FGF12 and its subfamily in the C-terminal region (residues 140-149), although CPP-M was a common domain in the internal region of the FGF family. The absence of CPP-C from FGF12 or a mutation (E142L) in the CPP-C domain drastically reduced the internalization of FGF12 into cells. Therefore, CPP-C played an essential role in the internalization of FGF12. In addition, CPP-C was able to deliver other polypeptides into cells as a CPP because an FGF1/CPP-C chimeric protein was internalized into IEC6 cells more efficiently than wild-type FGF1. Finally, intraperitoneally added FGF12 inhibited radiation-induced apoptosis in the intestinal epithelial cells of BALB/c mice, and deletion of the CPP-C domain decreased the inhibition of the apoptosis. These findings suggest that exogenous FGF12 can play a role in tissues by translocating into cells through the plasma membrane, and the availability of this novel CPP provides a new tool for the intracellular delivery of bioactive molecules.

De Novo Designed Protein Transduction Domain Mimics from Simple Synthetic Polymers

Protein transduction domains (PTDs) that readily transverse cellular membranes are of great interest and are attractive tools for the intracellular delivery of bioactive molecules. Learning to program synthetic polymers and oligomers with the appropriate chemical information to capture adequately the biological activity of proteins is critical to our improved understanding of how these natural molecules work. In addition, the versatility of these synthetic mimics provides the opportunity to discover analogs with superior properties compared with their native sequences. Here we report the first detailed structure-activity relationship of a new PTD family of polymers based on a completely abiotic backbone. The synthetic approach easily allows doubling the density of guanidine functional groups, which increases the transduction efficiency of the sequences. Cellular uptake studies on three different cell lines (HEK 293T, CHO, and Jurkat T cells) confirm that these synthetic analogs are highly efficient novel protein transduction domain mimics (PTDMs), which are more effective than TAT(49-57) and nonaarginine (R9) and also highlight the usefulness of polymer chemistry at the chemistry-biology interface.

Abstract 4441: Intracellular delivery of therapeutic siRNA via an antennapedia-RNA-binding domain fusion peptide

Abstract A key characteristic in tumorigenesis is the aberrant expression of various oncogenes, whose cellular products drive various anti-apoptotic/pro-survival pathways. The RNAi pathway is an endogenous cellular pathway that transiently inhibits the translation of mRNA into protein. It therefore has the potential to selectively arrest the production of mutated proteins. As the Intracellular delivery of bioactive molecules is still a major challenge, we are exploring the possible ability of the Antenapedia homeodomain (Antp) to act as an effective transporter of conjugated cargo across biological membranes. We have previously shown that Antp delivers both p21 into p21-null tumors in order to restore cell-cycle regulation, as well as a truncated form of MAML to inhibit Notch activation and induce apoptosis. To test whether in silico-designed siRNA has the ability to silence mutated forms of genes, we have created a bifunctional fusion protein, TR7, which incorporates Antp and an RNA-binding protein. TR7 attempts to both neutralize the anionic nature of siRNA as well as introduce it to the cytoplasm, thus initiating the RNAi process in a non-cytotoxic manner. In summary, this project, utilizing Antp, aims to solve the issue of systemic RNAi delivery, as well as harness the endogenous RNAi pathway to effectively halt the production of abnormal proteins in cancer and other life threatening illnesses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4441. doi:10.1158/1538-7445.AM2011-4441

高分子の表面修飾による機能性リポソームの設計

For establishment of safe and effective therapy, carriers that achieve high-precision drug delivery are required. Liposomes are one of the most promising drug delivery systems. To increase usefulness of liposomes as drug delivery systems, we have attempted to provide various functions to liposomes via surface surface modification of liposomes with functional polymers. Functions of polymer-modified liposomes were based on their interactions. Therefore, surface-modification of liposomes with temperature-sensitive polymers can give temperature-responsive liposomes whose drug release is triggered by mild heating. Also, surface modification of liposomes with pH-sensitive polymers can generate pH-responsive liposomes whose destabilization is induced in weakly acidic environments. In addition, modification with several kinds of polymers with different functions can generate multifunctional liposomes. Here, we describe design, preparation and performance of functional liposomes based on the surface modification with functional polymers, such as temperature-sensitive, pH-sensitive, magnetic resonance-detectable polymers for the production of liposomes for high-precision site-specific delivery and/or intracellular delivery of bioactive molecules.

Direct and Rapid Cytosolic Delivery Using Cell-Penetrating Peptides Mediated by Pyrenebutyrate

Intracellular delivery of bioactive molecules using arginine-rich peptides, including oligoarginine and HIV-1 Tat peptides, is a recently developed technology. Here, we report a dramatic change in the methods of internalization for these peptides brought about by the presence of pyrenebutyrate, a counteranion bearing an aromatic hydrophobic moiety. In the absence of pyrenebutyrate, endocytosis plays a major role in cellular uptake. However, the addition of pyrenebutyrate results in direct membrane translocation of the peptides yielding diffuse cytosolic peptide distribution within a few minutes. Using this method, rapid and efficient cytosolic delivery of the enhanced green fluorescent protein (EGFP) was achieved in cells including rat hippocampal primary cultured neurons. Enhancement of bioactivity on the administration of anapoptosis-inducing peptide is also demonstrated. Thus, coupling arginine-rich peptides with this hydrophobic anion dramatically improved their ability to translocate cellular membranes, suggesting the great impact of this approach on exploring and controlling cell function.

Studies on the Internalization Mechanism of Cationic Cell-penetrating Peptides

A great deal of data has been amassed suggesting that cationic peptides are able to translocate into eucaryotic cells in a temperature-independent manner. Although such peptides are widely used to promote the intracellular delivery of bioactive molecules, the mechanism by which this cell-penetrating activity occurs still remains unclear. Here, we present an in vitro study of the cellular uptake of peptides, originally deriving from protegrin (the SynB peptide vectors), that have also been shown to enhance the transport of drugs across the blood-brain barrier. In parallel, we have examined the internalization process of two lipid-interacting peptides, SynB5 and pAntp-(43-58), the latter corresponding to the translocating segment of the Antennapedia homeodomain. We report a quantitative study of the time- and dose-dependence of internalization and demonstrate that these peptides accumulate inside vesicular structures. Furthermore, we have examined the role of endocytotic pathways in this process using a variety of metabolic and endocytosis inhibitors. We show that the internalization of these peptides is a temperature- and energy-dependent process and that endosomal transport is a key component of the mechanism. Altogether, our results suggest that SynB and pAntp-(43-58) peptides penetrate into cells by an adsorptive-mediated endocytosis process rather than temperature-independent translocation.

Receptor-targeted delivery of an intracellular toxin to outer hair cells by fibroblast growth factor

The presence and distribution of functional, high-affinity receptors for fibroblast growth factors (FGFs) in the neonatal organ of Corti were probed using the intracellular toxin saporin conjugated to basic FGF (FGF-2). FGFs that bind to high-affinity FGF receptors are internalized as part of the normal process of receptor inactivation. The receptor can thus be used for the targeted delivery of molecules conjugated to FGF into the cytoplasm. Incubation of postnatal day 5 (P5) rat organ of Corti cultures with FGF-saporin caused a dose dependent destruction of outer hair cells, Deiters cells and outer pillar cells. Inner hair cells and other cells were unaffected. Organ of Corti cultures at P0 and P10 showed much less damage than at P5. The results suggest that outer hair cells and adjacent supporting cells in the organ of Corti transiently express high-affinity FGF receptors, and that these receptors can mediate the intracellular delivery of bioactive molecules.