Typical Cell-penetrating Peptide Research Articles

Effect of helicity and hydrophobicity on cell-penetrating ability of arginine-rich peptides

Arginine (Arg)-rich peptides are one of the typical cell-penetrating peptides (CPPs), which can deliver membrane-impermeable compounds into intracellular compartments. Guanidino groups in Arg-rich peptides are critical for their high cell-penetrating ability, although it remains unclear whether peptide secondary structures contribute to this ability. In the current study, we designed four Arg-rich peptides containing α,α-disubstituted α-amino acids (dAAs), which prefer to adopt a helical structure. The four dAA-containing peptides adopted slightly different peptide secondary structures, from a random structure to a helical structure, with different hydrophobicities. In these peptides, dipropylglycine-containing peptide exhibited the highest helicity and hydrophobicity, and showed the best cell-penetrating ability. These findings suggested that the helicity and hydrophobicity of Arg-rich peptides contributes to their high cell-penetrating ability.

Performance of Cell-Penetrating Peptides Anchored to Polysaccharide Platforms Applied via Various Mucosal Routes as an Absorption Enhancer.

We have been investigating the potential of cell-penetrating peptides anchored to polymeric platforms as a novel absorption enhancer which delivers biologics into systemic circulation via mucosal routes. Our previous mouse experiments demonstrated that hyaluronic acid modified with l-octaarginine, a typical cell-penetrating peptide, via a tetraglycine spacer significantly enhanced the mucosal absorption of protein drugs applied into the nasal cavities, irrespective of the molecular weights (Mw) of the drugs. The present study evaluated the performance of tetraglycine-l-octaarginine-linked hyaluronic acid applied via various mucosal routes. Somatropin (Mw: ca. 22.1 kDa) was moderately absorbed from the lung mucosa, and the mean absolute bioavailability (BA) reached 19% under enhancer-free conditions; nevertheless, its BA under intranasal administration was approximately 1% or less. Its BA significantly elevated to 46% on average through intrapulmonary coadministration with tetraglycine-l-octaarginine-linked hyaluronic acid. When the administration site was replaced with the oral cavities, an extreme reduction in somatropin absorption was observed with a mean BA of 0.056% under enhancer-free conditions. Intraoral coadministration with tetraglycine-l-octaarginine-linked hyaluronic acid resulted in a 6.3-fold elevation of somatropin absorption with statistical significance. A similar enhancement was observed under intrarectal administration with a further reduction in BA. On the other hand, the hyaluronic acid derivative did not exhibit the absorption-enhancing ability under intragastric administration, probably due to the lack of stabilization effects against enzyme-susceptible biologics. The results indicated that the intrapulmonary route was suitable for maximizing the mucosal absorption of biologics, and that there was a likelihood of the intraoral route with user convenience. When somatropin was substituted with fluorescein isothiocyanate-conjugated dextran with an average Mw range of 4-70 kDa, similar phenomena were observed under intrapulmonary and intranasal administration. BA decreased with an increase in the Mw of dextran; however, the ratio of BA under enhancer-present conditions to that under enhancer-free conditions was consistently around 3, indicating that the performance of the hyaluronic acid derivative was Mw-independent, irrespective of the administration route.

Nasal absorption enhancement of protein drugs independent to their chemical properties in the presence of hyaluronic acid modified with tetraglycine-L-octaarginine

Our previous mouse studies demonstrated that mean bioavailability of exendin-4, which is an injectable glucagon-like peptide-1 (GLP-1) analogue whose molecular weight (Mw) and isoelectric point (pI) are ca. 4.2 kDa and 4.5, respectively, administered nasally with poly(N-vinylacetamide-co-acrylic acid) (PNVA-co-AA) bearing D-octaarginine, which is a typical cell-penetrating peptide, was 20% relative to subcutaneous administration even though it was less than 1% when exendin-4 alone was given nasally. The studies also revealed that the absorption-enhancing ability of D-octaarginine-linked PNVA-co-AA for exendin-4 was statistically equivalent to that of sodium salcaprozate (SNAC), which is an absorption enhancer formulated in tablets of semaglutide approved recently as an orally available GLP-1 analogue. From a perspective of clinical application of our technology, we have separately developed hyaluronic acid modified with L-octaarginine via a tetraglycine spacer which would be degraded in biological conditions. The present study revealed that tetraglycine-L-octaarginine-linked hyaluronic acid enhanced nasal absorption of exendin-4 in mice, as did D-octaarginine-linked PNVA-co-AA. There was no significant difference in absorption-enhancing abilities between the hyaluronic acid derivative and SNAC when octreotide (Mw: ca. 1.0 kDa, pI: 8.3) and lixisenatide (Mw: ca. 4.9 kDa, pI: 9.5) were used as a model protein drug. On the other hand, SNAC did not significantly enhance nasal absorption of somatropin (Mw: ca. 22.1 kDa, pI: 5.3) when compared with absorption enhancer-free conditions. Substitution of SNAC with tetraglycine-L-octaarginine-linked hyaluronic acid resulted in a 5-fold increase in absolute bioavailability of somatropin with statistical significance. It appeared that pI hardly ever influenced absorption-enhancing abilities of both enhancers. Results indicated that our polysaccharide derivative would be a promising absorption enhancer which delivers biologics applied on the nasal mucosa into systemic circulation and was of greater advantage than SNAC for enhancing nasal absorption of protein drugs with a larger Mw.

SiRNA delivery using amphipathic cell-penetrating peptides into human hepatoma cells

Cell-penetrating peptides (CPPs) are an attractive tool for delivering membrane-impermeable compounds, including anionic biomacromolecules such as DNA and RNA, into living cells. Amphipathic helical peptides composed of hydrophobic amino acids and cationic amino acids are typical CPPs. In the current study, we designed amphipathic helical 12-mer peptides containing α,α-disubstituted α-amino acids (dAAs), which are known to stabilize peptide secondary structures. The dominant secondary structures of peptides in aqueous solution differed according to the introduced dAAs. Peptides containing hydrophobic dAAs and adopting a helical structure exhibited a good cell-penetrating ability. As an application of amphipathic helical peptides, small interfering RNA (siRNA) delivery into living human hepatoma cells was investigated. One of the peptides containing dAAs dipropylglycine formed stable complexes with siRNA at appropriate zeta-potential and size for intracellular siRNA delivery. This peptide showed effective RNA interference efficiency at short peptide length and low concentrations of peptide and siRNA. These findings will be helpful for the design of amphipathic helical CPPs as intracellular siRNA delivery.

Using a Membrane-Penetrating-Peptide to Anchor Ligands in the Liposome Membrane Facilitates Targeted Drug Delivery.

Antimicrobial peptides (AMPs) are typical cell penetrating peptides (CPPs) that intercalate into biomembranes and exhibit broad activities. We designed a triple fusion protein consisting of an AMP, Ib-AMP4 at the N-terminus, a fluorescent GFP probe in the center, and the tumor-targeting peptide P1c at the other terminus. After purification from E. coli, the interaction between the Ib-AMP4-GFP-P1c fusion protein (IGP) and the lipid membrane was characterized. Experiments using isothermal titration calorimetry (ITC) and quartz crystal microbalance with dissipation (QCM-D) demonstrated that IGP proteins spontaneously bound the lipid bilayer with a maximal molar ratio of 1:52 (protein:lipid). Furthermore, transmission electron microscopy (TEM) confirmed that the IGP protein was present in the liposome membrane. After decoration with IGP proteins, the DOPC:DOPG liposomes were applied to cancer cells. Microscopy and flow cytometry reveal that the decorated liposomes selectively bound integrin αvβ3-positive A549 cells. In addition, compared with the common chemical conjugation method, the reported method seemed to be superior in certain aspects, such as simple sample preparation and cost-effectiveness. Next, the IGP protein was applied to decorate red blood cell (RBC) liposomes for targeted delivery in both in vitro and in vivo applications. The IGP-decorated RBC liposomes preferentially targeted integrin αvβ3 expressing A549 cancer cells. The in vivo imaging showed that IGP-decorated RBC liposomes were concentrated in tumor tissue and were primarily metabolized by the liver and kidney.

Biodegradable Hyaluronic Acid Modified with Tetraglycine-l-octaarginine as a Safe Adjuvant for Mucosal Vaccination.

We have been investigating the potential use of polymers modified with cell-penetrating peptides as an adjuvant for mucosal vaccination and have already developed nondegradable poly( N-vinylacetamide- co-acrylic acid) (PNVA- co-AA) with which d-octaarginine, a typical cell-penetrating peptide, was grafted. Our previous murine infection experiments demonstrated that immunoglobulin G (IgG) and immunoglobulin A (IgA) were induced in systemic circulation and secreted on nasal mucosa, respectively, through 4-time nasal inoculations with a mixture of influenza viral antigens and d-octaarginine-linked PNVA- co-AA at 7-day intervals, and that immunized mice were perfectly protected from homologous virus infection. In the present study, we designed novel biodegradable polymers bearing cell-penetrating peptides from a perspective of clinical application. Hyaluronic acid whose glucuronic acid was modified with tetraglycine-l-octaarginine at a monosaccharide unit ratio of 30% was successfully developed. The hyaluronic acid derivative exhibited adjuvant activities identical to PNVA- co-AA bearing either d-octaarginine or tetraglycine-d-octaarginine under the above-mentioned inoculation schedule. We further found that there was no difference in humoral immunity between the 4-time inoculations at 7-day intervals and the 2-time inoculations at 28-day intervals. Intranasal IgA induced through the latter schedule with a smaller number of inoculations, which is clinically practical, exhibited cross-reactivity beyond the subtype of viral strains. In vitro toxicity studies demonstrated that the hyaluronic acid derivative was much less toxic than the corresponding PNVA- co-AA derivatives, and that both the polymers and their metabolites did not exhibit genotoxicity. Our results suggested that tetraglycine-l-octaarginine-linked hyaluronic acid would be a clinically valuable and safe adjuvant for mucosal vaccination.

Biocompatible Polymers Modified with d-Octaarginine as an Absorption Enhancer for Nasal Peptide Delivery.

Peptide and protein drugs, which are categorized as biologics, exhibit poor membrane permeability. This pharmacokinetic disadvantage has largely restricted the development of noninvasive dosage forms of biologics that deliver into systemic circulation. We have been investigating the potential use of cell-penetrating peptide-linked polymers as a novel absorption enhancer to overcome this challenge. Since our previous study revealed that biocompatible poly( N-vinylacetamide- co-acrylic acid) modified with d-octaarginine, a typical cell-penetrating peptide, enhanced in vitro permeation of biomolecules such as plasmid DNA and bovine serum albumin through cell membranes, the present study evaluated whether the polymers enhanced in vivo absorption of biologics applied on the mucosa. Mouse experiments demonstrated that d-octaarginine-linked polymers drastically enhanced nasal absorption of exendin-4, whose injection is clinically used. The mean bioavailability was 20% relative to subcutaneous administration, even though it fell short of 1% when exendin-4 alone was administered nasally. The absorption-enhancing function of the polymers was superior to that of sodium caprate and sodium N-(8-(2-hydroxybenzoyl)amino) caprylate, which have been used for humans as an absorption enhancer. In vitro experiments using several biologics with different characteristics revealed that biologics interacted with d-octaarginine-linked polymers and were taken up into cells when incubated with the polymers. The interaction and cellular uptake were enhanced as molecular weights of the biologics increased; however, their charge-dependent in vitro performance was not clearly observed. The current data suggested that biologics formulated with our polymers became an alternative to their conventional invasive parenteral formulations.

Oligohistidine and targeting peptide functionalized TAT-NLS for enhancing cellular uptake and promoting angiogenesis in vivo

BackgroundGene therapy has been developed and used in medical treatment for many years, especially for the enhancement of endothelialization and angiogenesis. But slow endosomal escape rate is still one of the major barriers to successful gene delivery. In order to evaluate whether introducing oligohistidine (Hn) sequence into gene carriers can promote endosomal escape and gene transfection or not, we designed and synthesized Arg-Glu-Asp-Val (REDV) peptide functionalized TAT-NLS-Hn (TAT: typical cell-penetrating peptide, NLS: nuclear localization signals, Hn: oligohistidine sequence, n: 4, 8 and 12) peptides with different Hn sequence lengths. pEGFP-ZNF580 (pZNF580) was condensed by these peptides to form gene complexes, which were used to transfect human umbilical vein endothelial cells (HUVECs).ResultsMTT assay showed that the gene complexes exhibited low cytotoxicity for HUVECs. The results of cellular uptake and co-localization ratio demonstrated that the gene complexes prepared from TAT-NLS-Hn with long Hn sequence (n = 12) benefited for high internalization efficiency of pZNF580. In addition, the results of western blot analysis and PCR assay of REDV-TAT-NLS-H12/pZNF580 complexes showed significantly enhanced gene expression at protein and mRNA level. Wound healing assay and transwell migration assay also confirmed the improved proliferation and migration ability of the transfected HUVECs by these complexes. Furthermore, the in vitro and in vivo angiogenesis assay illustrated that these complexes could promote the tube formation ability of HUVECs.ConclusionThe above results indicated that the delivery efficiency of pZNF580 and its expression could be enhanced by introducing Hn sequence into gene carriers. The Hn sequence in REDV-TAT-NLS-Hn is beneficial for high gene transfection. These REDV and Hn functionalized TAT-NLS peptides are promising gene carriers in gene therapy.

Self-Assembled DNA Dendrimer Nanoparticle for Efficient Delivery of Immunostimulatory CpG Motifs.

Dendrimer-like DNA nanostructures have attractive properties such as mechanical stability, highly branched nanostructure, customized sizes, and biocompatibility. In this study, we construct programmable DNA dendrimeric nanoparticles as efficient vehicles to deliver immunostimulatory cytosine-phosphate-guanosine (CpG) sequences for activation of the immune response. DNA dendrimers decorated with CpG-containing hairpin-loops triggered stronger immune response characterized by pro-inflammatory cytokines production, in contrast to DNA dendrimers loading linear CpG. After further modification with TAT peptide, a typical cell-penetrating peptide, on the surface of the nanocarriers, CpG loops-loaded DNA dendrimers showed the enhanced cell internalization and cytokines production. The TAT-DNA dendrimer-CpG loops constructs did not affect the viability of immune cells and no detectable cytotoxicity was observed. Our results demonstrate that the DNA dendrimers can serve as designable and safe vehicles for delivery of immune modulators and anticancer drugs.

Efficient Cellular Protein Transduction Using a Coiled-coil Protein Carrier

Cell-penetrating peptides (CPPs) as carriers for the intracellular delivery of various proteins and macromolecules have been studied extensively. Nonetheless, the delivery efficiency is relatively low. We showed previously that a rigid and fibrous cationic coiled-coil protein, CCPC 140, has cell-penetrating activity superior to that of reported CPPs. In this report, we exploited CCPC 140 for the intracellular delivery of the green fluorescent protein (GFP). The activity of CCPC 140 for intracellular delivery of GFP was compared with an octa-arginine peptide, a typical CPP that is widely used. The cellular delivery of proteins by CCPC 140 was >20-fold more efficient than that of octa-arginine. These results indicate that CCPC 140 has significant potential for delivery of proteins to cells.

The Telomerase-Derived Anticancer Peptide Vaccine GV1001 as an Extracellular Heat Shock Protein-Mediated Cell-Penetrating Peptide.

Cell-penetrating peptides (CPPs), which can facilitate the transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular delivery of macromolecules. GV1001, a peptide derived from a reverse-transcriptase subunit of telomerase (hTERT) and developed as a vaccine against various cancers, reportedly has unexpected CPP properties. Unlike typical CPPs, such as the HIV-1 TAT peptide, GV1001 enabled the cytosolic delivery of macromolecules such as proteins, DNA and siRNA via extracellular heat shock protein 90 (eHSP90) and 70 (eHSP70) complexes. The eHSP-GV1001 interaction may have biological effects in addition to its cytosolic delivery function. GV1001 was originally designed as a major histocompatibility complex (MHC) class II-binding cancer epitope, but its CPP properties may contribute to its strong anti-cancer immune response relative to other telomerase peptide-based vaccines. Cell signaling via eHSP-GV1001 binding may lead to unexpected biological effects, such as direct anticancer or antiviral effects. In this review, we focus on the CPP effects of GV1001 bound to eHSP90 and eHSP70.

The heparin-binding domain of HB-EGF as an efficient cell-penetrating peptide for drug delivery.

Cell-penetrating peptides (CPPs) have been shown to be potential drug carriers for cancer therapy. The inherently low immunogenicity and cytotoxicity of human-derived CPPs make them more suitable for intracellular drug delivery compared to other delivery vehicles. In this work, the protein transduction ability of a novel CPP (termed HBP) derived from the heparin-binding domain of HB-EGF was evaluated. Our data shows, for the first time, that HBP possesses similar properties to typical CPPs and is a potent drug delivery vector for improving the antitumor activity of impermeable MAP30. The intrinsic bioactivities of recombinant MAP30-HBP were well preserved compared to those of free MAP30. Furthermore, HBP conjugated to the C-terminus of MAP30 promoted the cellular uptake of recombinant MAP30-HBP. Moreover, the fusion of HBP to MAP30 gave rise to significantly enhanced cytotoxic effects in all of the tumor cell lines tested. In HeLa cells, this cytotoxicity was mainly caused by the induction of cell apoptosis. Further investigation revealed that HBP enhanced MAP30-induced apoptosis through the activation of the mitochondrial- and death receptor-mediated signaling pathways. In addition, the MAP30-HBP fusion protein caused more HeLa cells to become arrested in S phase compared to MAP30 alone. These results highlight the MAP30-HBP fusion protein as a promising drug candidate for cancer therapy and demonstrate HBP, a novel CPP derived from human HB-EGF, as a new potential vector for antitumor drug delivery. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Multitargeting Gene Delivery Systems for Enhancing the Transfection of Endothelial Cells.

Gene therapy demonstrates promising prospects on cardiovascular diseases. However, nonviral gene delivery system has relatively low transfection efficiency, especially for endothelial cells (ECs). Herein, typical cell-penetrating peptide (TAT), nuclear localization signals (NLSs), and REDV functional peptide have been used to prepare multitargeting complexes. These complexes exhibit higher transfection efficiency owing to the targeting sequences of REDV and NLSs as well as the cell-penetrating function of TAT. The multifunction of the complexes provides high cell uptake, endo/lysosomal escape, and nucleus accumulation of the encapsulated DNA. Thus these multitargeting complexes can provide a potential platform for gene delivery, especially for EC transfection.

Potential of D-Octaarginine-Linked Polymers as an in Vitro Transfection Tool for Biomolecules.

We have been investigating the potential use of cell-penetrating peptide-linked polymers as a novel penetration enhancer. Since previous in vivo studies demonstrated that poly(N-vinylacetamide-co-acrylic acid) bearing D-octaarginine, a typical cell-penetrating peptide, enhanced membrane permeation of biomolecules, its potential as an in vitro transfection tool was evaluated in this study. A plasmid DNA encoding green fluorescent protein (pGFP-C1), β-galactosidase, and bovine serum albumin (BSA) were used as model biomolecules. Anionic pGFP-C1 interacted electrostatically with cationic d-octaarginine-linked polymers. When the ratio of mass concentration of polymers to that of pGFP-C1 reached 2.5, complexes whose size and zeta potential were approximately 200 nm and 15 mV, respectively, were obtained. GFP expression was observed in cells incubated with complexes prepared under conditions in which the polymer/pDNA concentration ratio exceeded 2.5. The expression level elevated with an increase in the concentration ratio, but physicochemical properties of the complexes remained unchanged. Results suggested that free polymers contributed to pGFP-C1 internalization. Another cell study demonstrated that β-galactosidase premixed with polymers was taken up into cells in its active tetrameric form. Similar electrostatic interaction-driven complex formation was observed for BSA charged negatively in neutral solution. However, it appeared that the internalization processes of BSA differed from those of pGFP-C1. A mass concentration-dependent increase in internalized BSA was observed, irrespective of the polymer/protein concentration ratio. Due to frail interactions, polymers that were released from the complexes and subsequently immobilized on cell membranes might also contribute to membrane permeation of BSA.

Noninvasive Insulin Delivery: the Great Potential of cell-penetrating Peptides

Insulin, a potent therapeutic peptide used in the treatment of diabetes, is administered to patients via subcutaneous injections because of the poor pharmacokinetics associated with alternative routes of administration such as oral, nasal and pulmonary delivery. Noninvasive nasal and oral formulations are appealing to patients who need consecutive daily treatments of insulin. However, to achieve mucosal absorption of insulin via oral or nasal administration, two barriers must be overcome: the impermeability of insulin through the epithelial membranes and local digestion and enzymatic degradation. Cell-penetrating peptides (CPPs), which efficiently bring exogenous proteins and nucleic acids into cells, have great potential to facilitate insulin permeation from the intestinal lumen or nasal cavity into systemic circulation via efficient uptake by epithelial cells. In fact, the coadministration of insulin with the peptide penetratin, a typical CPP, increased intestinal and nasal insulin bioavailability to 35 and 50%, respectively. In this review, the authors describe recent findings using this novel CPP-based formulation for noninvasive delivery of insulin.

Cell-Penetrating Peptide-Linked Polymers as Carriers for Mucosal Vaccine Delivery

We evaluated the potential of poly(N-vinylacetamide-co-acrylic acid) modified with d-octaarginine, which is a typical cell-penetrating peptide, as a carrier for mucosal vaccine delivery. Mice were nasally inoculated four times every seventh day with PBS containing ovalbumin with or without the d-octaarginine-linked polymer. The polymer enhanced the production of ovalbumin-specific immunoglobulin G (IgG) and secreted immunoglobulin A (IgA) in the serum and the nasal cavity, respectively. Ovalbumin internalized into nasal epithelial cells appeared to stimulate IgA production. Ovalbumin transferred to systemic circulation possibly enhanced IgG production. An equivalent dose of the cholera toxin B subunit (CTB), which was used as a positive control, was superior to the polymer in enhancing antibody production; however, dose escalation of the polymer overcame this disadvantage. A similar immunization profile was also observed when ovalbumin was replaced with influenza virus HA vaccines. The polymer induced a vaccine-specific immune response identical to that induced by CTB, irrespective of the antibody type, when its dose was 10 times that of CTB. Our cell-penetrating peptide-linked polymer is a potential candidate for antigen carriers that induce humoral immunity on the mucosal surface and in systemic circulation when nasally coadministered with antigens.

Performance of cell-penetrating peptide-linked polymers physically mixed with poorly membrane-permeable molecules on cell membranes

We are investigating a new class of penetration enhancers that enable poorly membrane-permeable molecules physically mixed with them to effectively penetrate cell membranes without their concomitant cellular uptake. Since we previously revealed that poly(N-vinylacetamide-co-acrylic acid) modified with d-octaarginine, which is a typical cell-penetrating peptide, significantly enhanced the nasal absorption of insulin, we examined the performance of the polymers on cell membranes. When Caco-2 cells were incubated with 5(6)-carboxyfluorescein (CF) for 30min, approximately 0.1% of applied CF was internalized into the cells. This poor membrane permeability was dramatically enhanced by d-octaarginine-linked polymers; a 25-fold increase in the cellular uptake of CF was observed when the polymer concentration was adjusted to 0.2mg/mL. None of the individual components, for example, d-octaarginine, had any influence on CF uptake, demonstrating that only d-octaarginine anchored chemically to the polymeric platform enhanced the membrane permeation of CF. The polymer-induced CF uptake was consistently high even when the incubation time was extended to 120min. Confocal laser scanning microphotographs of cells incubated with d-octaarginine-linked polymers bearing rhodamine red demonstrated that the cell outline was stained with red fluorescence. The polymer-induced CF uptake was significantly suppressed by 5-(N-ethyl-N-isopropyl)amiloride, which is an inhibitor of macropinocytosis. Results indicated that d-octaarginine-linked polymers remained on the cell membrane and poorly membrane-permeable CF was continuously internalized into cells mainly via macropinocytosis repeated for the individual peptidyl branches in the polymer backbone.

Importance of intermolecular interaction on the improvement of intestinal therapeutic peptide/protein absorption using cell-penetrating peptides

Our previous reports showed that the absorption of therapeutic peptides and proteins was significantly improved by coadministration of cell-penetrating peptides (CPPs) as the physical mixture. However, the mechanisms for this improvement are not clear. In the present study, we verified the hypothesis that the electrostatic interaction between drug and CPP is related to the enhancing effect of the CPP on the intestinal absorption of therapeutic peptides and proteins. In this study, the intermolecular binding was analyzed by surface plasmon resonance (SPR)-based binding assay, and the effect of CPPs on the intestinal absorption of peptide drugs was examined by in situ absorption study using a rat intestinal loop. Among the 16 peptide drugs possessing different isoelectric points, it was observed that only gastrin, insulin and glucagon-like peptide-1 (GLP-1) bound to D-R8 (D-form arginine octamer, a typical CPP), and subsequently their intestinal absorption increased by coadministration of D-R8. In contrast, the intestinal absorption of other peptide drugs that did not bind to D-R8 was not affected in the presence of D-R8. Thus, this study suggests that intermolecular binding between drug and CPP is an important factor governing the enhancing effect of the CPP on the intestinal absorption of therapeutic peptides and proteins.

Usefulness of cell-penetrating peptides to improve intestinal insulin absorption

Cell-penetrating peptides (CPPs) are a useful tool for delivering therapeutic macromolecules across cell membranes. We previously devised an approach using CPPs without intermolecular cross-linking and showed the efficient delivery of insulin from the intestine to the systemic circulation using a typical CPP, oligoarginine. However, this approach required relatively high doses of the CPP. Therefore, this study aimed to identify CPPs that are more effective for the delivery of insulin and do not induce toxic effects on the intestine. In this study, we examined the effects of various types of CPPs including arginine-rich peptides and amphipathic peptides that aid insulin absorption from rat ileal segments. Among these peptides, coadministration of insulin with R8, penetratin, pVEC, and RRL helix significantly increased ileal insulin absorption compared with insulin administration alone. In the case of R8, the d-form of the peptide had stronger absorption enhancing ability than the l-form. In contrast, the other three peptides exerted a more significant effect when the l-forms were applied, and l-penetratin had the strongest ability to enhance intestinal insulin absorption. Meanwhile, in a physical mixture of CPP and insulin, aggregates formed in the solution when high concentrations of CPPs were present. l-penetratin enhanced insulin absorption even when administered in an aggregated solution. We then showed that aggregates of l-penetratin and insulin were broken down in the presence of intestinal degradation enzymes. Thus, among CPPs used in this study, l-penetratin had the strongest ability to improve insulin intestinal absorption.