Endosomolytic Agents Research Articles

Escape sites from the endo-lysosomal trafficking route manipulate exocytosis of nanoparticles in polar epithelium.

The endo-lysosomal pathway is a major barrier for the trans-epithelial transport of nanoparticles (NPs), but escape strategies could facilitate trans-epithelial delivery. Based on the polarization properties of the epithelium, different escape compartments may result in different exocytosis fates of NPs and further affect the delivery efficiency. Therefore, optimizing the escape sites is critical for trans-epithelial delivery. Here, commonly used PEG-coated-poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles were fabricated as model nanoparticles (MNPs) and the intestinal epithelium was chosen as the polarized epithelium. The MNPs were incubated with different endosomolytic agents for early endosomal escape, late endosomal escape and lysosomal escape, respectively. According to in vitro and in vivo studies, MNPs escaping from early endosomes and late endosomes exhibited stronger capacity for trans-epithelial transport than those escaping from lysosomes. By further probing into the mechanism, we surprisingly found that although MNPs escaping from early endosomes quickly egressed from the apical side of epithelia, they were subsequently followed by "reuptake" via caveolae and trafficked through the endoplasmic reticulum-Golgi apparatus (ER/GA) secretory pathway, achieving efficient trans-epithelial transport; MNPs escaping from late endosomes, which were located near the nucleus, were prone to enter the ER/GA for efficient basolateral exocytosis. However, MNPs escaping from lysosomes were detained within cells by autophagosomes. Collectively, our research suggested that early endosomes and late endosomes were ideal escape sites for trans-epithelial delivery.

Poly(2-Propylacrylic Acid) Increases In Vitro Bioactivity of Chitosan/mRNA Nanoparticles.

Chitosan-based nanoparticles have been extensively studied for the delivery of nucleic acids. Previous results suggest that these nanoparticles have limited ability to escape the endosome, one of the main cellular barriers hindering nucleic acid delivery. Escape can be improved by the addition of endosomolytic agents during the formulation process or by developing delivery systems with intrinsic properties to disrupt endosomal membranes. In this study, Poly(2-Propylacrylic Acid) (PPAA), an anionic synthetic polymer with known membrane lytic activity was added to the binary chitosan/mRNA nanoparticles to improve bioactivity. The ionization behavior of PPAA was characterized to identify conditions in which PPAA is sufficiently charged to interact electrostatically with chitosan and thus form nanoparticles. The physicochemical characteristics (hydrodynamic diameter, polydispersity index, ζ-potential) and the in vitro transfection efficiency (bioactivity) of this new family of CS/mRNA/PPAA ternary nanoparticles were evaluated. The addition of PPAA to CS/mRNA nanoparticles was shown to be an efficient strategy to augment in vitro bioactivity. The optimal formulation reached an expression level ~86% of the commercial lipid control at pH 6.5 without any signs of metabolic toxicity. In this paper, we report the effect of salt and pH on the ionization behavior of PPAA and demonstrate 1) successful incorporation of PPAA into/onto nanoparticles, 2) improved bioactivity with PPAA, and 3) that the kosmotropic effects of trehalose play a minimal role in the apparent increase in bioactivity in presence of trehalose.

Analysis of Triterpenoid Saponins Reveals Insights into Structural Features Associated with Potent Protein Drug Enhancement Effects

Plant-based saponins are amphipathic glycosides composed of a hydrophobic aglycone backbone covalently bound to one or more hydrophilic sugar moieties. Recently, the endosomal escape activity of triterpenoid saponins has been investigated as a potentially powerful tool for improved cytosolic penetration of protein drugs internalized by endocytic uptake, thereby greatly enhancing their pharmacological effects. However, only a few saponins have been studied, and the paucity in understanding the structure-activity relationship of saponins imposes significant limitations on their applications. To address this knowledge gap, 12 triterpenoid saponins with diverse structural side chains were screened for their utility as endosomolytic agents. These compounds were used in combination with a toxin (MAP30-HBP) comprising a type I ribosome-inactivating protein fused to a cell-penetrating peptide. Suitability of saponins as endosomolytic agents was assessed on the basis of cytotoxicity, endosomal escape promotion, and synergistic effects on toxins. Five saponins showed strong endosomal escape activity, enhancing MAP30-HBP cytotoxicity by more than 106 to 109 folds. These saponins also enhanced the apoptotic effect of MAP30-HBP in a pH-dependent manner. Additionally, growth inhibition of MAP30-HBP-treated SMMC-7721 cells was greater than that of similarly treated HeLa cells, suggesting that saponin-mediated endosomolytic effect is likely to be cell-specific. Furthermore, the structural features and hydrophobicity of the sugar side chains were analyzed to draw correlations with endosomal escape activity and derive predictive rules, thus providing new insights into structure-activity relationships of saponins. This study revealed new saponins that can potentially be exploited as efficient cytosolic delivery reagents for improved therapeutic drug effects.

Synthetic Polymer Tag for Intracellular Delivery of siRNA

AbstractShort interference RNA (siRNA) is capable of precisely controlling the expression levels of virtually any functional genes. Before tapping into the full potential of siRNA, however, the longstanding difficulty of cell type‐specific cytosolic delivery of siRNA must be addressed. Conventional cationic materials are unsuitable because of their toxicity, colloidal instability, large and heterogeneous complex sizes, and interference with targeting. Building on the new concept of tagging individual siRNA‐targeting ligand chimera with charge–neutral endosomolytic agents, a new class of tags based on synthetic polymers is developed. Compared to multifunctional proteins and peptides previously made for the same functions, the synthetic polymer tags are simple and inexpensive to make, compact in size, have a low immunogenic potential, and are stable in circulation. Efficient in vitro and in vivo gene silencing is achieved, opening exciting opportunities for development of targeted biologics.

Noncovalent tagging of siRNA with steroids for transmembrane delivery

Short interfering RNA (siRNA) has broad applications in biology and medicine, and holds tremendous potential to become a new class of therapeutics for many diseases. As a highly anionic macrobiomolecule, its cytosolic delivery, however, has been a major roadblock in translation. Here, we report the development of small, bifunctional chemical tags capable of transporting siRNA directly into the cytosol. The bifunctional tags consist of a siRNA-binding moiety that interacts with siRNA non-covalently, and a steroid domain that readily fuses with the mammalian cell membrane. In contrast to the conventional covalently conjugated siRNA-steroid that enters cells largely via endocytosis which substantially limits siRNA bioavailability, the non-covalently tagged siRNA is cell membrane-permeant, avoiding the endocytic pathway. This new methodology enables effective RNA interference (RNAi) without the need of cationic transfection or endosomolytic agents, opening a new avenue for intracellular delivery of native biologics.

Unlocking Endosomal Entrapment with Supercharged Arginine-Rich Peptides.

Endosomal entrapment is a common bottleneck in various macromolecular delivery approaches. Recently, the polycationic peptide dfTAT was identified as a reagent that induces the efficient leakage of late endosomes and, thereby, enhances the penetration of macromolecules into the cytosol of live human cells. To gain further insights into the features that lead to this activity, the role of peptide sequence was investigated. We establish that the leakage activity of dfTAT can be recapitulated by polyarginine analogs but not by polylysine counterparts. Efficiencies of peptide endocytic uptake increase linearly with the number of arginine residues present. In contrast, peptide cytosolic penetration displays a threshold behavior, indicating that a minimum number of arginines is required to induce endosomal escape. Increasing arginine content above this threshold further augments delivery efficiencies. Yet, it also leads to increasing the toxicity of the delivery agents. Together, these data reveal a relatively narrow arginine-content window for the design of optimally active endosomolytic agents.

Synthetic polynucleotides as endosomolytic agents and bioenergy sources

Nucleotides (NTs), such as adenosine triphosphate (ATP) and guanosine triphosphate (GTP), are signaling and bioenergy molecules to mediate a range of cellular pathways. We recently reported their significant endosomolytic activity. To evaluate whether polymeric NTs keep endosomolytic and bioenergetic functions of NTs in drug delivery and cell survival, NTs were polymerized by a coupling reaction to form polynucleotides (pNTs: pATP and pGTP) with their molecular weights around 500kDa. The cellular toxicity, indicated by IC50, of pNT was as low as that of corresponding monomeric NT. pNTs were degraded by an intracellular enzyme, alkaline phosphatase. Introduction of pNTs in a polycation-gene complex (polyplex) enhanced the extent of gene expression in cancerous, non-cancerous, and stem cells, up to 1500-fold higher than that of pNT-free polyplex. In addition, cells stored in a pATP solution resulted in a significantly higher survival rate (e.g., up to 20% increase) when exposed to low temperatures than pATP-free solution. The presence of pNT in polyplexes prevented the reduction of transfection efficiency induced by a low temperature. The findings in this study suggest that endosomolytic and bioenergetic pNTs serve as a non-toxic gene carrier component and protect cells from a cold shock or energy depletion.

Nucleotides as Nontoxic Endogenous Endosomolytic Agents in Drug Delivery

Nontoxic endogenous nucleotides such as adenosine triphosphate and guanosine triphosphate have secondary phosphate groups, causing proton-buffering capacity and/or hemolytic activity in endolysosomal pH ranges. Nucleotides co-delivered in single polymeric pDNA nanocarrier induce highly enhanced transfection efficiency with negligible cytotoxicity due to their endosomolytic functions.

Formation of Solid Lipid Nanoparticle (SLN)–Gene Vector Complexes for Transfection of Mammalian Cells In Vitro: Figure 1.

Solid lipid nanoparticles (SLNs) offer several technological advantages over standard DNA carriers such as cationic lipids or cationic polymers. However, in the absence of endosomolytic agents such as chloroquine, gene-transfer efficiency mediated by SLN-derived gene vectors consisting of optimized lipid composition remains lower compared to those achieved with standard transfection agents. This protocol describes the incorporation of a dimeric human immunodeficiency virus type-1 (HIV-1) TAT peptide into SLN gene vectors to increase gene-transfer efficiency. This results in higher transfection rates than for standard transfection agents in vitro; the ternary SLN-gene vector complexes usually result in transfection levels equal to or higher than those observed with gene vector complexes formulated with branched polyethylenimine (PEI) 25 kDa. One significant advantage of using this method is the low cytotoxicity of the SLN gene vectors. The application of the gene-transfer technique is limited to relatively low plasmid DNA (pDNA) concentrations of the resulting complexes (10 µg/mL). At higher concentrations, the particles tend to aggregate and precipitate. Therefore, their use for in vivo application, which generally requires high pDNA concentrations, is limited.

Biological activities of histidine-rich peptides; merging biotechnology and nanomedicine

Histidine-rich peptides are commonly used in recombinant protein production as purification tags, allowing the one-step affinity separation of the His-tagged proteins from the extracellular media or cell extracts. Genetic engineering makes feasible the post-purification His-tag removal by inserting, between the tag and the main protein body, a target site for trans-acting proteases or a self-proteolytic peptide with regulatable activities. However, for technical ease, His tags are often not removed and the fusion proteins eventually used in this form. In this commentary, we revise the powerful biological properties of histidine-rich peptides as endosomolytic agents and as architectonic tags in nanoparticle formation, for which they are exploited in drug delivery and other nanomedical applications. These activities, generally unknown to biotechnologists, can unwillingly modulate the functionality and biotechnological performance of recombinant proteins in which they remain trivially attached.

Diol glycidyl ether-bridged cyclens: preparation and their applications in gene delivery

Polymeric 1,4,7,10-tetraazacyclododecanes (cyclens) using diol glycidyl ether with different chain length as bridges (5a-e) were designed and synthesized from various diols, 1,7-diprotected cyclen and epichlorohydrin. The molecular weights of the title polymers were measured by GPC with good polydispersity. Agarose gel retardation and fluorescent titration using ethidium bromide showed good DNA-binding ability of 5. They could retard plasmid DNA (pDNA) at an N/P ratio of 4-6 and form polyplexes with sizes around 100-250 nm from an N/P ratio of 10 to 60 and relatively low zeta-potential values (5-22 mV). The cytotoxicity of 5 assayed by MTT is much lower than that of 20 kDa PEI. In vitro transfection against A549 and 293 cells showed that the transfection efficiency (TE) of 5c/DNA polyplexes is close to that of 20 kDa PEI at an N/P ratio of 5. Structure-activity relationship (SAR) of 5 was discussed in their DNA-binding, cytotoxicity, and transfection studies. The TE of 5c/DNA polyplexes could be improved by the introduction of 50 μM of chloroquine, the endosomolytic agents, to pretreated cells. These studies may extend the application areas of macrocyclic polyamines, especially for cyclen.

Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure-activity studies

Redirecting the splicing machinery through the hybridization of high affinity, RNase H- incompetent oligonucleotide analogs such as phosphoramidate morpholino oligonucleotides (PMO) might lead to important clinical applications. Chemical conjugation of PMO to arginine-rich cell penetrating peptides (CPP) such as (R-Ahx-R)4 (with Ahx standing for 6-aminohexanoic acid) leads to sequence-specific splicing correction in the absence of endosomolytic agents in cell culture at variance with most conventional CPPs. Importantly, (R-Ahx-R)4–PMO conjugates are effective in mouse models of various viral infections and Duchenne muscular dystrophy. Unfortunately, active doses in some applications might be close to cytotoxic ones thus presenting challenge for systemic administration of the conjugates in those clinical settings. Structure–activity relationship studies have thus been undertaken to unravel CPP structural features important for the efficient nuclear delivery of the conjugated PMO and limiting steps in their internalization pathway. Affinity for heparin (taken as a model heparan sulfate), hydrophobicity, cellular uptake, intracellular distribution and splicing correction have been monitored. Spacing between the charges, hydrophobicity of the linker between the Arg-groups and Arg-stereochemistry influence splicing correction efficiency. A significant correlation between splicing correction efficiency, affinity for heparin and ability to destabilize model synthetic vesicles has been observed but no correlation with cellular uptake has been found. Efforts will have to focus on endosomal escape since it appears to remain the limiting factor for the delivery of these splice-redirecting ON analogs.

Arginine‐rich cell penetrating peptides: Design, structure–activity, and applications to alter pre‐mRNA splicing by steric‐block oligonucleotides

Rerouting the splicing machinery with steric-block oligonucleotides (ON) might lead to new therapeutic strategies in the treatment of diseases such as beta-thalassemia, Duchenne muscular dystrophy, or cancers. Interfering with splicing requires the sequence-specific and stable hybridization of RNase H-incompetent ON as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligomers (PMO). Unfortunately, these uncharged DNA mimics are poorly taken up by most cell types and conventional delivery strategies that rely on electrostatic interaction do not apply. Likewise, conjugation to cell penetrating peptides (CPPs) as Tat, Arg9, Lys8, or Pen leads to poor splicing correction efficiency at low concentration essentially because PNA- and PMO-CPP conjugates remain entrapped within endocytotic vesicles. Recently, we have designed an arginine-rich peptide (R-Ahx-R)4 (with Ahx for aminohexanoic acid) and an arginine-tailed Penetratin derivative which allow sequence-specific and efficient splicing correction at low concentration in the absence of endosomolytic agents. Both CPPs are undergoing structure-activity relationship studies for further optimization as steric-block ON delivery vectors.

Methoxy poly(ethylene glycol) – Low molecular weight linear polyethylenimine-derived copolymers enable polyplex shielding

Targeted gene delivery relies on the development of materials that allow for the formation of small neutrally charged particles of sufficient colloidal stability preventing non-specific interactions with cells. In order to identify a copolymer composition that combines adequate plasmid DNA (pDNA) compaction with an efficient charge-shielding effect, we synthesized a series of copolymers by covalent linkage of activated 5 or 20 kDa linear methoxy poly(ethylene glycol) (mPEG) or 10 kDa two-arm-mPEG to non-toxic low molecular weight (2.6 and 4.6 kDa) linear polyethylenimine (lPEI) at different molar ratios (mPEG-lPEI copolymers). All of the copolymers condensed pEGFP-N1 pDNA to form nanoparticles with hydrodynamic diameters between 150 and 420 nm - sizes that were maintained for the entire duration of measurement. PEGylated complexes exhibited a reduced particle stability in comparison to the unmodified lPEI-pDNA polyplexes, determined by gel retardation assays and DNase I experiments. Copolymer-pDNA complexes exhibited a zeta potential between -4 and 6 mV, strongly depending on the dispersion medium applied (0.15M NaCl or 5% glucose supplemented with serum-free cell culture medium). The transfection efficacy, determined in CHO-K1 (between 0.28+/-0.08% and 1.92+/-0.46%) and HeLa (between 1.02+/-0.19% and 3.53+/-0.30%) cells, was significantly reduced compared to lPEI-pDNA particles (between 3.2+/-1.3% and 38.8+/-5.5%). The architecture of the copolymer, the molecular weight of the lPEI residue, and the supplementation of endosomolytic agents (saccharose, chloroquine) all failed to impact the efficacy of gene transfer. Uptake studies, based on Confocal Laser Scanning Microscopy (CLSM) imaging and flow cytometry analysis, suggest that the use of mPEG5/3-lPEI2.6, mPEG10/2-lPEI2.6, and mPEG20-lPEI4.6 lowers unspecific internalization of the corresponding transfection complexes. This provides an ideal basis for the development of transfection vehicles for targeted gene transfer.

Cell-penetrating-peptide-based delivery of oligonucleotides: an overview

Cationic CPPs (cell-penetrating peptides) have been used largely for intracellular delivery of low-molecular-mass drugs, biomolecules and particles. Most cationic CPPs bind to cell-associated glycosaminoglycans and are internalized by endocytosis, although the detailed mechanisms involved remain controversial. Sequestration and degradation in endocytic vesicles severely limits the efficiency of cytoplasmic and/or nuclear delivery of CPP-conjugated material. Re-routing the splicing machinery by using steric-block ON (oligonucleotide) analogues, such as PNAs (peptide nucleic acids) or PMOs (phosphorodiamidate morpholino oligomers), has consequently been inefficient when ONs are conjugated with standard CPPs such as Tat (transactivator of transcription), R(9) (nona-arginine), K(8) (octalysine) or penetratin in the absence of endosomolytic agents. New arginine-rich CPPs such as (R-Ahx-R)(4) (6-aminohexanoic acid-spaced oligo-arginine) or R(6) (hexa-arginine)-penetratin conjugated to PMO or PNA resulted in efficient splicing correction at non-cytotoxic doses in the absence of chloroquine. SAR (structure-activity relationship) analyses are underway to optimize these peptide delivery vectors and to understand their mechanisms of cellular internalization.

Synthesis, characterization, and biomedical applications of core–shell-type stimuli-responsive nanogels – Nanogel composed of poly[2-( N, N-diethylamino)ethyl methacrylate] core and PEG tethered chains

We present a review of stimuli-responsive smart PEGylated nanogels and their biomedical applications. We describe the synthesis and characterization of stimuli-responsive PEGylated nanogels composed of a cross-linked poly[2-( N, N-diethylamino)ethyl methacrylate] (PDEAMA) core and PEG tethered chains that bear a carboxylic acid group as a platform moiety for the installation of bio-tags. In addition, PEGylated nanogels show unique properties and functions in synchronizing with the reversible volume phase transition of the PDEAMA core in response to various stimuli, such as pH, ionic strength and temperature. We list some of the biomedical applications of the nanogels, including endosomolytic agents for nonviral gene delivery, drug delivery carriers, nanoreactors, and skin-specific nanocatalysts for reactive oxygen species (ROS).

Efficient splicing correction by PNA conjugation to an R6-Penetratin delivery peptide.

Sequence-specific interference with the nuclear pre-mRNA splicing machinery has received increased attention as an analytical tool and for development of therapeutics. It requires sequence-specific and high affinity binding of RNaseH-incompetent DNA mimics to pre-mRNA. Peptide nucleic acids (PNA) or phosphoramidate morpholino oligonucleotides (PMO) are particularly suited as steric block oligonucleotides in this respect. However, splicing correction by PNA or PMO conjugated to cell penetrating peptides (CPP), such as Tat or Penetratin, has required high concentrations (5–10 μM) of such conjugates, unless an endosomolytic agent was added to increase escape from endocytic vesicles. We have focused on the modification of existing CPPs to search for peptides able to deliver more efficiently splice correcting PNA or PMO to the nucleus in the absence of endosomolytic agents. We describe here R6-Penetratin (in which arginine-residues were added to the N-terminus of Penetratin) as the most active of all CPPs tested so far in a splicing correction assay in which masking of a cryptic splice site allows expression of a luciferase reporter gene. Efficient and sequence-specific correction occurs at 1 μM concentration of the R6Pen–PNA705 conjugate as monitored by luciferase luminescence and by RT-PCR. Some aspects of the R6Pen–PNA705 structure–function relationship have also been evaluated.

Lipid Encapsulation Enables the Effective Systemic Delivery of Polyplex Plasmid DNA

Using a new controlled mixing process, highly transfection-competent polyplexes were formed and subsequently encapsulated within a lipid bilayer. The resulting "pre-condensed stable plasmid lipid particles" (pSPLPs) have small size (104+/-3 nm) and low surface charge characteristics. The formulation process equally enabled lipid encapsulation of either poly-L-lysine or poly(ethyleneimine) (PEI) condensed DNA, and the endosomolytic benefits of PEI were demonstrated in in vitro gene expression studies. The clearance properties of pSPLP were compared to similar formulations with an uncondensed payload (SPLP) in A/J mice bearing subcutaneous Neuro-2a tumors. Plasma clearance of pSPLP (t(1/2)=6.6 h) was similar to SPLP (t(1/2)=7.1 h), allowing significant accumulation at distal tumor target sites. Gene expression profiles were evaluated in vivo using the Neuro-2a model, and PEI-pSPLP formulations demonstrated a sixfold increase in reporter gene expression in tumors compared to SPLP. No significant gene expression was observed in the liver, lung, or spleen when mice were treated with either SPLP or pSPLP, and both formulations were equally well tolerated. The results support the lipid encapsulation of polyplex plasmid DNA as a means of changing its pharmacologic properties and enabling systemic delivery. The inclusion of endosomolytic DNA-condensing agents such as PEI greatly improves the potency of SPLP.

PH-Responsive PEGylated nanogels as targetable and low invasive endosomolytic agents to induce the enhanced transfection efficiency of nonviral gene vectors

A pH-responsive PEGylated nanogel was successfully prepared by means of emulsion copolymerization of 2-(N,N-diethylamino)ethyl methacrylate (AMA) with heterobifunctional poly(ethylene glycol) (PEG) bearing a 4-vinylbenzyl group at the α-end and a lactose moiety at the ω-end in the presence of potassium persulfate and ethyleneglycol dimethacrylate as a cross-linker. Polyplex micelle composed of PEG-block-poly(l-lysine) copolymer and plasmid DNA (PEG-b-PLL/pDNA) exhibited a far more efficient transfection ability in the presence of lac-nanogel-8k-1.0% (PEG, Mn = 8000; cross-linking density, 1.0%) than the PEG-b-PLL/pDNA polyplex micelle alone (in the absence of lac-nanogel-8k-1.0%), suggesting that an appreciable fraction of lac-nanogel-8k-1.0% along with the PEG-b-PLL/pDNA polyplex micelle is taken up into the HuH-7 cells through the asialoglycoprotein receptor-mediated endocytosis process mediated by the cluster of a large number of lactose moieties on the surface of lac-nanogel-8k-1.0%, followed by the effective disruption of the endosome by the buffer effect of the unprotonated PAMA core in lac-nanogel-8k-1.0%.

Peptide-based delivery of nucleic acids: design, mechanism of uptake and applications to splice-correcting oligonucleotides

CPPs (cell-penetrating peptides) have given rise to much interest for the delivery of biomolecules such as peptides, proteins or ONs (oligonucleotides). CPPs and their conjugates were initially thought to translocate through the cell membrane by a non-endocytotic mechanism which has recently been re-evaluated. Basic-amino-acid-rich CPPs first interact with cell-surface proteoglycans before being internalized by endocytosis. Sequestration and degradation in endocytotic vesicles severely limits the cytoplasmic and nuclear delivery of the conjugated biomolecules. Accordingly, splicing correction by CPP-conjugated steric-block ON analogues is inefficient in the absence of endosomolytic agents. New arginine-rich CPPs allowing efficient splicing correction by conjugated PNAs (peptide nucleic acids) or PMO (phosphorodiamidate morpholino oligomer) steric blockers in the absence of endosomolytic agents have recently been defined in our group and are currently being characterized. They offer promising leads for the development of efficient cellular delivery vectors for therapeutic steric-block ON analogues.