Cell-penetrating Peptide Penetratin Research Articles

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.

Composite of KLVFF-Transthyretin-Penetratin and Manganese Dioxide Nanoclusters: A Multifunctional Agent against Alzheimer's β-Amyloid Fibrillogenesis.

Design of amyloid β-protein (Aβ) inhibitors is considered an effective strategy for the prevention and treatment of Alzheimer's disease (AD). However, the limited blood-brain barrier (BBB) penetration and poor Aβ-targeting capability restricts the therapeutic efficiency of candidate drugs. Herein, we have proposed to engineer transthyretin (TTR) by fusion of the Aβ-targeting peptide KLVFF and cell-penetrating peptide Penetratin to TTR, and derived a fusion protein, KLVFF-TTR-Penetratin (KTP). Moreover, to introduce the scavenging activity for reactive oxygen species (ROS), a nanocomposite of KTP and manganese dioxide nanoclusters (KTP@MnO2) was fabricated by biomineralization. Results revealed that KTP@MnO2 demonstrated significantly enhanced inhibition on Aβ aggregation as compared to TTR. The inhibitory effect was increased from 18%, 33%, and 49% (10, 25, and 50 μg/mL TTR, respectively) to 52%, 81%, and 100% (10, 25, and 50 μg/mL KTP@MnO2). In addition, KTP@MnO2 could penetrate the BBB and target amyloid plaques. Moreover, multiple ROS, including hydroxyl radicals, superoxide radicals, hydrogen peroxide, and Aβ-induced-ROS, which cannot be scavenged by TTR, were scavenged by KTP@MnO2, thus resulting in the mitigation of cellular oxidative damages. More importantly, cell culture and in vivo experiments with AD nematodes indicated that KTP@MnO2 at 50 μg/mL increased the viability of Aβ-treated cells from 66% to more than 95%, and completely cleared amyloid plaques in AD nematodes and extended their lifespan by 7 d. Overall, despite critical aspects such as the stability, metabolic distribution, long-term biotoxicity, and immunogenicity of the nanocomposites in mammalian models remaining to be investigated, this work has demonstrated the multifunctionality of KTP@MnO2 for targeting Aβ in vivo, and provided new insights into the design of multifunctional nanocomposites of protein-metal clusters against AD.

Investigating the Insertion Mechanism of Cell-Penetrating Peptide Penetratin into Cell Membranes: Implications for Targeted Drug Delivery

The cell-penetrating peptide (CPP) penetratin (PEN) has garnered attention for its potential to enter tumor cells. However, its translocation mechanism and lack of selectivity remain debated. This study investigated PEN’s insertion into healthy cells (H-) and cancer cells (C-) using micromolar concentrations and various techniques. Raman spectroscopy was used to determine PEN’s location in the lipid bilayer at different lipid-to-peptide ratios. Dynamic light scattering (DLS) and zeta potential analysis were used to measure the lipid–PEN complex’s size and charge. The results showed helical PEN particles directly inserted into C- membranes at a ratio of 110, while aggregated particles stayed on H- surfaces. Raman spectroscopy and scanning electron microscopy confirmed PEN insertion in C- membranes. Zeta potential studies revealed highly negative charges for PEN–C- complexes and neutral charges for PEN–H- complexes at pH 6.8. C- integrity remained unchanged at a ratio of 110. Specific lipid-to-peptide ratios with dipalmitoylphosphatidylserine (DPPS) were crucial for direct insertion. These results provide valuable insights into CPP efficacy for targeted drug delivery in cancer cells, considering membrane composition and lipid-to-peptide ratios.

Carrier peptide interactions with liposome membranes induce reversible clustering by surface adsorption and shape deformation

The cell-penetrating peptide penetratin and its analogues shuffle and penetramax have been used as carrier peptides for oral delivery of therapeutic peptides such as insulin. Their mechanism of action for this purpose is not fully understood but is believed to depend on the interactions of the peptide with the cell membrane. In the present study, peptide-liposome interactions were investigated using advanced biophysical techniques including small-angle neutron scattering and fluorescence lifetime imaging microscopy. Liposomes were used as a model system for the cell membrane. All the investigated carrier peptides induced liposome clustering at a specific peptide/lipid ratio. However, distinctively different types of membrane interactions were observed, as the liposome clustering was irreversible for penetratin, but fully or partly reversible for shuffle and penetramax, respectively. All three peptides were found to adsorb to the surface of the lipid bilayers, while only shuffle and penetramax led to shape deformation of the liposomes. Importantly, the peptide interactions did not disrupt the liposomes under any of the investigated conditions, which is advantageous for their application in drug delivery. This detailed insight on peptide-membrane interactions is important for understanding the mechanism of peptide-based excipients and the influence of peptide sequence modifications.

Stereochemistry and Intermolecular Interactions Influence Carrier Peptide-Mediated Insulin Delivery

The inherent low oral bioavailability of therapeutic peptides can be enhanced by the cell-penetrating peptide penetratin and its analogues shuffle and penetramax applied as carriers for delivery of insulin. In this study, the objective was to gain mechanistic insights on the effect of the carrier peptide stereochemistry on their interactions with insulin and on insulin delivery. Insulin-carrier peptide interactions were investigated using small-angle X-ray scattering and cryogenic transmission electron microscopy, while the insulin and peptide stability and transepithelial insulin permeation were evaluated in the Caco-2 cell culture model along with the carrier peptide-induced effects on epithelial integrity and cellular metabolic activity. Interestingly, the insulin transepithelial permeation was influenced by the degree of insulin-carrier peptide complexation and depended on the stereochemistry of penetramax but not of penetratin and shuffle. The l-form of the peptides initially decreased the epithelial integrity comparable to that induced by the d-peptides, suggesting a comparable mechanism of action. The immediate decrease was reversible during exposure of the Caco-2 epithelium to the l-peptides but not during exposure to the d-peptides, likely a result of their higher stability. Overall, exploration of the stereochemistry showed to be an interesting strategy for carrier peptide-mediated insulin delivery.

Conjugation to a cell-penetrating peptide drives the tumour accumulation of the GLP1R antagonist exendin(9-39)

PurposeExendin, an analogue of the glucagon-like peptide 1 (GLP1), is an excellent tracer for molecular imaging of pancreatic beta cells and beta cell-derived tumours. The commonly used form, exendin-4, activates the GLP1 receptor and causes internalisation of the peptide-receptor complex. As a consequence, injection of exendin-4 can lead to adverse effects such as nausea, vomiting and hypoglycaemia and thus requires close monitoring during application. By comparison, the antagonist exendin(9-39) does not activate the receptor, but its lack of internalisation has precluded its use as a tracer. Improving the cellular uptake of exendin(9-39) could turn it into a useful alternative tracer with less side-effects than exendin-4.MethodsWe conjugated exendin-4 and exendin(9-39) to the well-known cell-penetrating peptide (CPP) penetratin. We evaluated cell binding and internalisation of the radiolabelled peptides in vitro and their biodistribution in vivo.ResultsExendin-4 showed internalisation irrespective of the presence of the CPP, whereas for exendin(9-39) only the penetratin conjugate internalised. Conjugation to the CPP also enhanced the in vivo tumour uptake and retention of exendin(9-39).ConclusionWe demonstrate that penetratin robustly improves internalisation and tumour retention of exendin(9-39), opening new avenues for antagonist-based in vivo imaging of GLP1R.

A penetratin-derived peptide reduces the membrane permeabilization and cell toxicity of α-synuclein oligomers

Parkinson's disease is a neurodegenerative movement disorder associated with the intracellular aggregation of α-synuclein (α-syn). Cytotoxicity is mainly associated with the oligomeric species (αSOs) formed at early stages in α-syn aggregation. Consequently, there is an intense focus on the discovery of novel inhibitors such as peptides to inhibit oligomer formation and toxicity. Here, using peptide arrays, we identified nine peptides with high specificity and affinity for αSOs. Of these, peptides p194, p235, and p249 diverted α-syn aggregation from fibrils to amorphous aggregates with reduced β-structures and increased random coil content. However, they did not reduce αSO cytotoxicity and permeabilization of large anionic unilamellar vesicles. In parallel, we identified a non-self-aggregating peptide (p216), derived from the cell-penetrating peptide penetratin, which showed 12-fold higher binding affinity to αSOs than to α-syn monomers (Kdapp 2.7 and 31.2μM, respectively). p216 reduced αSOs-induced large anionic unilamellar vesicle membrane permeability at 10-1 to 10-3mg/ml by almost 100%, was not toxic to SH-SY5Y cells, and reduced αSOs cytotoxicity by about 20%. We conclude that p216 is a promising starting point from which to develop peptides targeting toxic αSOs in Parkinson's disease.

Control of Enzyme Reaction Initiation inside Giant Unilamellar Vesicles by the Cell-Penetrating Peptide-Mediated Translocation of Cargo Proteins.

Cell-penetrating peptides (CPPs) play important roles in directly delivering biomolecules, such as DNA, proteins, and peptides, into living cells. In artificial lipid membranes, such as planar lipid bilayers, the direct membrane translocation of β-galactosidase via Pep-1 (one of the CPPs) is dependent upon a voltage gradient between the inner and outer leaflets of the lipid membranes. Giant unilamellar vesicles (GUVs) with asymmetric lipid distributions, which are recently generated using microfluidic technologies, can be observed by optical microscopy. Therefore, interactions between CPPs and asymmetric lipid bilayers in different kinds of lipids and the translocation mechanism of proteins via CPPs into GUVs can be investigated at the level of a single asymmetric GUV. This CPP-based system for transporting proteins into GUVs will be applied to control the start of enzyme reactions in GUVs. This study aimed to explore efficient protein translocation into GUVs via CPP and demonstrate that enzymatic reactions start in GUVs using a CPP-mediated direct translocation. The interactions and the enzyme reactions between the CPP (Pep-1 or penetratin)-DNase I complexes and the asymmetric or symmetric GUV membranes containing the negatively or neutrally charged lipids were observed by confocal laser-scanning microscopy. The asymmetric GUVs containing phosphatidylserine (PS) in the inner leaflet showed efficient DNase I translocation into GUVs via penetratin. Finally, the formation of a cross-linked actin network was observed in asymmetric PS GUVs incubated with Pep-1-streptavidin complexes. The CPP-mediated direct translocation can contribute to developing artificial cell models with the capacity to control the initiation of enzymatic reactions.

Structural Bases for the Involvement of Phosphatidylinositol-4,5-bisphosphate in the Internalization of the Cell-Penetrating Peptide Penetratin.

Cell-penetrating peptides cross cell membranes through various parallel internalization pathways. Herein, we analyze the role of the negatively charged lipid phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the internalization of Penetratin. Contributions of both inner leaflet and outer leaflet pools of PI(4,5)P2 were revealed by quantifying the internalization of Penetratin in cells treated with PI(4,5)P2 binders. Studies on model systems showed that Penetratin has a strong affinity for PI(4,5)P2 and interacts selectively with this lipid, even in the presence of other negatively charged lipids, as demonstrated by affinity photo-crosslinking experiments. Differential scanning calorimetry experiments showed that Penetratin induces lateral segregation in PI(4,5)P2-containing liposomes, which was confirmed by coarse-grained molecular dynamics simulations. NMR experiments indicated that Penetratin adopts a stabilized helical conformation in the presence of PI(4,5)P2-containing membranes, with an orientation parallel to the bilayer plane, which was also confirmed by all-atom simulations. NMR and photo-crosslinking experiments also suggest a rather shallow insertion of the peptide in the membrane. Put together, our findings suggest that PI(4,5)P2 is a privileged interaction partner for Penetratin and that it plays an important role in Penetratin internalization.

Revealing the importance of carrier-cargo association in delivery of insulin and lipidated insulin

Delivery of therapeutic peptides upon oral administration is highly desired and investigations report that the cell-penetrating peptide (CPP) penetratin and its analogues shuffle and penetramax show potential as carriers to enhance insulin delivery. Exploring this, the specific aim of the present study was to understand the impact that their complexation with a lipidated or non-lipidated therapeutic cargo would have on the delivery, to evaluate the effect of differences in membrane interactions in vitro and in vivo, as well as to deduce the mode of action leading to enhanced delivery. Fundamental biophysical aspects were studied by a range of orthogonal methods. Transepithelial permeation of therapeutic peptide was evaluated using the Caco-2 cell culture model supplemented with epithelial integrity measurements, real-time assessment of the carrier peptide effects on cell viability and on mode of action. Pharmacokinetic and pharmacodynamic (PK/PD) parameters were evaluated following intestinal administration to rats and tissue effects were investigated by histology. The biophysical studies revealed complexation of insulin with shuffle and penetramax, but not with penetratin. This corresponded to enhanced transepithelial permeation of insulin, but not of lipidated insulin, when in physical mixture with shuffle or penetramax. The addition of shuffle and penetramax was associated with a lowering of Caco-2 cell monolayer integrity and viability, where the lowering of cell viability was immediate, but reversible. Insulin delivery in rats was enhanced by shuffle and penetramax and accompanied by a 10–20-fold decrease in blood glucose with immediate effect on the intestinal mucosa. In conclusion, shuffle and penetramax, but not penetratin, demonstrated to be potential candidates as carriers for transmucosal delivery of insulin upon oral administration, and their effect depended on association with both cargo and cell membrane. Interestingly, the present study provides novel mechanistic insight that peptide carrier-induced cargo permeation points towards enhancement via the paracellular route in the tight epithelium. This is different from the anticipated belief being that it is the cell-penetrating capability that facilitate transepithelial cargo permeation via a transcellular route.

CPPs to the Test: Effects on Binding, Uptake and Biodistribution of a Tumor Targeting Nanobody.

Nanobodies are well-established targeting ligands for molecular imaging and therapy. Their short circulation time enables early imaging and reduces systemic radiation exposure. However, shorter circulation time leads to lower tracer accumulation in the target tissue. Cell-penetrating peptides (CPPs) improve cellular uptake of various cargoes, including nanobodies. CPPs could enhance tissue retention without compromising rapid clearance. However, systematic investigations on how the functionalities of nanobody and CPP combine with each other at the level of 2D and 3D cell cultures and in vivo are lacking. Here, we demonstrate that conjugates of the epidermal growth factor receptor (EGFR)-binding nanobody 7D12 with different CPPs (nonaarginine, penetratin, Tat and hLF) differ with respect to cell binding and induction of endocytosis. For nonaarginine and penetratin we compared the competition of EGF binding and performance of L- and D-peptide stereoisomers, and tested the D-peptide conjugates in tumor cell spheroids and in vivo. The D-peptide conjugates showed better penetration into spheroids than the unconjugated 7D12. Both in vivo and in vitro, the behavior of the agent reflects the combination of both functionalities. Although CPPs cause promising increases in in vitro uptake and 3D penetration, the dominant effect of the CPP in the control of biodistribution warrants further investigation.

Increased Carrier Peptide Stability through pH Adjustment Improves Insulin and PTH(1-34) Delivery In Vitro and In Vivo Rather than by Enforced Carrier Peptide-Cargo Complexation.

Oral delivery of therapeutic peptides is hampered by their large molecular size and labile nature, thus limiting their permeation across the intestinal epithelium. Promising approaches to overcome the latter include co-administration with carrier peptides. In this study, the cell-penetrating peptide penetratin was employed to investigate effects of co-administration with insulin and the pharmacologically active part of parathyroid hormone (PTH(1-34)) at pH 5, 6.5, and 7.4 with respect to complexation, enzymatic stability, and transepithelial permeation of the therapeutic peptide in vitro and in vivo. Complex formation between insulin or PTH(1-34) and penetratin was pH-dependent. Micron-sized complexes dominated in the samples prepared at pH-values at which penetratin interacts electrostatically with the therapeutic peptide. The association efficiency was more pronounced between insulin and penetratin than between PTH(1-34) and penetratin. Despite the high degree of complexation, penetratin retained its membrane activity when applied to liposomal structures. The enzymatic stability of penetratin during incubation on polarized Caco-2 cell monolayers was pH-dependent with a prolonged half-live determined at pH 5 when compared to pH 6.5 and 7.4. Also, the penetratin-mediated transepithelial permeation of insulin and PTH(1-34) was increased in vitro and in vivo upon lowering the sample pH from 7.4 or 6.5 to 5. Thus, the formation of penetratin-cargo complexes with several molecular entities is not prerequisite for penetratin-mediated transepithelial permeation a therapeutic peptide. Rather, a sample pH, which improves the penetratin stability, appears to optimize the penetratin-mediated transepithelial permeation of insulin and PTH(1-34).

Inhibition of post-trabeculectomy fibrosis via topically instilled antisense oligonucleotide complexes co-loaded with fluorouracil

Trabeculectomy is the mainstay of surgical glaucoma treatment, while the success rate was unsatisfying due to postoperative scarring of the filtering blebs. Clinical countermeasures for scar prevention are intraoperative intervention or repeated subconjunctival injections. Herein, we designed a co-delivery system capable of transporting fluorouracil and anti-TGF-β2 oligonucleotide to synergistically inhibit fibroblast proliferation via topical instillation. This co-delivery system was built based on a cationic dendrimer core (PAMAM), which encapsulated fluorouracil within hydrophobic cavity and condensed oligonucleotide with surface amino groups, and was further modified with hyaluronic acid and cell-penetrating peptide penetratin. The co-delivery system was self-assembled into nanoscale complexes with increased cellular uptake and enabled efficient inhibition on proliferation of fibroblast cells. In vivo studies on rabbit trabeculectomy models further confirmed the anti-fibrosis efficiency of the complexes, which prolonged survival time of filtering blebs and maintained their height and extent during wound healing process, exhibiting an equivalent effect on scar prevention compared to intraoperative infiltration with fluorouracil. Qualitative observation by immunohistochemistry staining and quantitative analysis by Western blotting both suggested that TGF-β2 expression was inhibited by the co-delivery complexes. Our study provided a potential approach promising to guarantee success rate of trabeculectomy and prolong survival time of filtering blebs.

Translocation of the Cell Penetrating Peptide Penetratin through Asymmetric Model Membranes Formed by a Microfluidic Device: Role of the Lipids and Transmembrane Potential

Translocation of the Cell Penetrating Peptide Penetratin through Asymmetric Model Membranes Formed by a Microfluidic Device: Role of the Lipids and Transmembrane Potential

Nanoscopic Structure of Complexes Formed between DNA and the Cell-Penetrating Peptide Penetratin.

One of the most remarkable examples of cell-penetrating peptides (CPPs) is Penetratin, a 16-mer fragment derived from the Drosophila Antennapedia homeobox. Understanding the structure of Penetratin/DNA complexes is a key factor for the successful design of new vectors for gene delivery and may assist in optimizing molecular carriers based on CPPs. Herein, we present a comprehensive study on the nanoscale structure of noncovalent complexes formed between Penetratin and DNA. The strong cationic nature of the peptide makes it a very efficient agent for condensing DNA strands via electrostatic attraction, and we show for the first time that DNAcondensation is accompanied by random-to-β-sheet transitions of Penetratin secondary structure, demonstrating that nucleic acids behave as a structuring agent upon complexation. For the first time, nanoscale-resolved spectroscopy is used to provide single-particle infrared data from DNA carriers based on CPPs, and they show that the structures are stabilized by Penetratin β-sheet cores, whereas larger DNA fractions are preferentially located in the periphery of aggregates. In-solution infrared assays indicate that phosphate diester groups are strongly affected upon DNA condensation, presumably as a consequence of charge delocalization induced by the proximity of cationic amide groups in Penetratin. The morphology is characterized by nanoassemblies with surface fractal features, and short-range order is found in the inner structure of the scaffolds. Interestingly, the formation of beads-on-a-string arrays is found, producing nanoscale architectures that resemble structures observed in early steps of chromatin condensation. A complexation pathway where DNA condensation and peptide pairing into β-sheets are key steps for organization is proposed.

Dual functionalized liposomes for efficient co-delivery of anti-cancer chemotherapeutics for the treatment of glioblastoma

Glioblastoma is a hostile brain tumor associated with high infiltration leading to poor prognosis. Anti-cancer chemotherapeutic agents have limited access into the brain due to the presence of the blood brain barrier (BBB). In this study, we designed a dual functionalized liposomal delivery system, surface modified with transferrin (Tf) for receptor mediated transcytosis and a cell penetrating peptide-penetratin (Pen) for enhanced cell penetration. We loaded doxorubicin and erlotinib into liposomes to enhance their translocation across the BBB to glioblastoma tumor. In vitro cytotoxicity and hemocompatibility studies demonstrated excellent biocompatibility for in vivo administration. Co-delivery of doxorubicin and erlotinib loaded Tf-Pen liposomes revealed significantly (p < 0.05) higher translocation (~15%) across the co-culture endothelial barrier resulting in regression of tumor in the in vitro brain tumor model. The biodistribution of Tf-Pen liposomes demonstrated ~12 and 3.3 fold increase in doxorubicin and erlotinib accumulation in mice brain, respectively compared to free drugs. In addition, Tf-Pen liposomes showed excellent antitumor efficacy by regressing ~90% of tumor in mice brain with significant increase in the median survival time (36 days) along with no toxicity. Thus, we believe that this study would have high impact for treating patients with glioblastoma.

19F-substituted amino acids as an alternative to fluorophore labels: monitoring of degradation and cellular uptake of analogues of penetratin by 19F NMR.

Current methods for assessment of cellular uptake of cell-penetrating peptides (CPPs) often rely on detection of fluorophore-labeled CPPs. However, introduction of the fluorescent probe often confers changed physicochemical properties, so that the fluorophore-CPP conjugate may exhibit cytotoxic effects and membrane damage not exerted by the native CPP. In the present study, introduction of fluorine probes was investigated as an alternative to fluorophore labeling of a CPP, since this only confers minor changes to its overall physicochemical properties. The high sensitivity of 19F NMR spectroscopy and the absence of background signals from naturally occurring fluorine enabled detection of internalized CPP. Also, degradation of fluorine-labeled peptides during exposure to Caco-2 cells could be followed by using 19F NMR spectroscopy. In total, five fluorinated analogues of the model CPP penetratin were synthesized by using commercially available fluorinated amino acids as labels, including one analogue also carrying an N-terminal fluorophore. The apparent cellular uptake was considerably higher for the fluorophore-penetratin conjugate indicating that the fluorophore moiety promoted uptake of the peptide. The use of 19F NMR spectroscopy enabled monitoring of the fate of the CPPs over time by establishing molar balances, and by verifying CPP integrity upon uptake. Thus, the NMR-based method offers several advantages over currently widespread methods relying on fluorescence detection. The present findings provide guidelines for improved labeling strategies for CPPs, thereby expanding the repertoire of analytical techniques available for studying degradation and uptake of CPPs.

Inhibition of Inflammatory Changes in Human Myometrial Cells by Cell Penetrating Peptide and Small Molecule Inhibitors of NFκB.

Complications arising from Preterm Birth are the leading causes of neonatal death globally. Current therapeutic strategies to prevent Preterm Birth are yet to demonstrate success in terms of reducing this neonatal disease burden. Upregulation of intracellular inflammatory pathways in uterine cells, including those involving nuclear factor kappa-B (NFκB), have been causally linked to both human term and preterm labor, but the barrier presented by the cell membrane presents an obstacle to interventions aimed at dampening these inflammatory responses. Cell penetrating peptides (CPPs) are novel vectors that can traverse cell membranes without the need for recognition by cell surface receptors and offer the ability to deliver therapeutic cargo internal to cell membranes. Using a human uterine cell culture inflammatory model, this study aimed to test the effectiveness of CPP-cargo delivery to inhibit inflammatory responses, comparing this effect with a small molecule inhibitor (Sc514) that has a similar intracellular target of action within the NFκB pathway (the IKK complex). The CPP Penetratin, conjugated to rhodamine, was able to enter uterine cells within a 60 min timeframe as assessed by live confocal microscopy, this phenomena was not observed with the use of a rhodamine-conjugated inert control peptide (GC(GS)4). Penetratin CPP conjugated to an IKK-inhibitory peptide (Pen-NBD) demonstrated ability to inhibit both the IL1β-induced expression of the inflammatory protein COX2 and dampen the expression of a bespoke array of inflammatory genes. Truncation of the CPP vector rendered the CPP-cargo conjugate much less effective, demonstrating the importance of careful vector selection. The small molecule inhibitor Sc514 also demonstrated ability to inhibit COX2 protein responses and a broad down-regulatory effect on uterine cell inflammatory gene expression. These results support the further exploration of either CPP-based or small molecular treatment strategies to dampen gestational cell inflammatory responses in the context of preterm birth. The work underlines both the importance of careful selection of CPP vector-cargo combinations and basic testing over a broad time and concentration range to ensure effective responses. Further work should demonstrate the effectiveness of CPP-linked cargos to dampen alternative pathways of inflammation linked to Preterm Birth such as MAP Kinase or AP1.

Effective nose-to-brain delivery of exendin-4 via coadministration with cell-penetrating peptides for improving progressive cognitive dysfunction

In a recent study, we demonstrated the potential of a cell-penetrating peptide (CPP) penetratin to deliver the peptide drug insulin to the brain via nasal administration, and its pharmacological effect on the mild cognitive dysfunction in senescence-accelerated mouse (SAMP8). However, the therapeutic potential of intranasal insulin administration was attenuated when applied to the aged SAMP8 with severe cognitive dysfunction. The present study, therefore, aimed to overcome the difficulty in treating severe cognitive dysfunction using insulin by investigating potential alternatives, glucagon-like peptide-1 (GLP-1) receptor agonists such as exendin-4. Examination using normal ddY mice demonstrated that the distribution of exendin-4 throughout the brain was dramatically increased by intranasal coadministration with the L-form of penetratin. The activation of hippocampal insulin signaling after the simultaneous nose-to-brain delivery of exendin-4 and an adequate level of insulin were confirmed by analyzing the phosphorylation of Akt. Furthermore, spatial learning ability, evaluated in the Morris water maze test after daily administration of exendin-4 with L-penetratin and supplemental insulin for 4 weeks, suggested therapeutic efficacy against severe cognitive dysfunction. The present study suggests that nose-to-brain delivery of exendin-4 with supplemental insulin, mediated by CPP coadministration, shows promise for the treatment of progressive cognitive dysfunction in SAMP8.

Dual Functionalized 5-Fluorouracil Liposomes as Highly Efficient Nanomedicine for Glioblastoma Treatment as Assessed in an In Vitro Brain Tumor Model

Drug delivery to the brain has been a major challenge due to the presence of the blood-brain barrier, which limits the uptake of most chemotherapeutics into brain. We developed a dual-functionalized liposomal delivery system, conjugating cell penetrating peptide penetratin to transferrin-liposomes (Tf-Pen–conjugated liposomes) to enhance the transport of an anticancer chemotherapeutic drug, 5-fluorouracil (5-FU), across the blood-brain barrier into the tumor cells. The in vitro cellular uptake study showed that the dual-functionalized liposomes are capable of higher cellular uptake in glioblastoma (U87) and brain endothelial (bEnd.3) cells monolayer. In addition, dual-functionalized liposomes demonstrated significantly higher apoptosis in U87 cells. The liposomal nanoparticles showed excellent blood compatibility and in vitro cell viability, as studied by hemolysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. The 5-FU–loaded dual-functionalized liposomes demonstrated higher transport across the brain endothelial barrier and delivered 5-FU to tumor cells inside poly(lactic-co-glycolic acid)-chitosan scaffold (an in vitro brain tumor model), resulting in significant tumor regression.