Cell Penetrating Research Articles

A water-soluble preparation for intravenous administration of isorhamnetin and its pharmacokinetics in rats

Flavonoids are considered as health-protecting food constituents. The testing of their biological effects is however hampered by their low oral absorption and complex metabolism. In order to investigate the direct effect(s) of unmetabolized flavonoid, a preparation in a biologically friendly solvent for intravenous administration is needed. Isorhamnetin, a natural flavonoid and a human metabolite of the most frequently tested flavonoid quercetin, has very low water solubility (<3.5 μg/mL). The aim of this study was to improve its solubility to enable intravenous administration and to test its pharmacokinetics in an animal model. By using polyvinylpyrrolidone (PVP10) and benzalkonium chloride, we were able to improve the solubility approximately 600 times to 2.1 mg/mL. This solution was then administered intravenously at a dose of 0.5 mg/kg of isorhamnetin to rats and its pharmacokinetics was analyzed. The pharmacokinetics of isorhamnetin corresponded to two compartmental model with a rapid initial distribution phase (t1/2α: 5.7 ± 4.3 min) and a slower elimination phase (t1/2β: 61 ± 47.5 min). Two sulfate metabolites were also identified. PVP10 and benzalkonium did not modify the properties of isorhamnetin (iron chelation and reduction, and cell penetration) substantially. In conclusion, the novel preparation reported in this study is suitable for future testing of isorhamnetin effects under in vivo conditions.

Fingolimod phosphoramidate prodrugs: Synthesis, photophysical characterisation and lipid bilayer interaction of fluorescent tagged Prodrug

FTY720 (Fingolimod, Gilenya, 1), a structural analog of sphingosine (2) was the first orally administered drug approved for the treatment of multiple sclerosis (MS). However, the phosphate derivative of Fingolimod, namely Fingolimod-1-phosphate (FTY720-1P, 3) is the biologically active molecule in MS as well as in various lysosomal storage diseases such as mucolipidosis IV and Nieman-Pick. In all cases, Fingolimod requires to be phosphorylated, resembling the natural sphingosine phosphate (4), to accomplish its therapeutic/biological effects. Here, we report the synthesis of a small library of prodrugs that could provide the efficient delivery of 3, the bioactive species. Moreover, to gain insight about the biological behaviour of these novel candidates within human body such as cell and brain blood barrier penetration we have prepared and spectroscopically characterised a fluorescent tagged version of one of the new prodrugs. The behavior of our prodrugs with respect to biological membranes was evaluated by studying the interaction between the fluorescent prodrug 18 and DOPC/DPPC liposome models.

Conditional Cell Penetration of Masked CPPs by an ADEPT-like Approach.

The intracellular delivery of cargos via cell penetrating peptides (CPPs) holds significant promise as a drug delivery vehicle, but a major issue is their lack of cell type specificity, which can lead to detrimental off-target effects. We use an ADEPT-like concept to introduce conditional and selective activation of cellular uptake by using the lysine-rich, cationic, and amphiphilic L17E peptide as a model CPP. By masking the lysine residues of the L17E peptide with enzyme-cleavable acetyl protecting groups, the delivery of the covalently conjugated fluorophore TAMRA to HeLa cells was diminished. Recovery of cellular uptake could be achieved by deacetylation of the masked acetylated L17E peptide using the NAD-dependent sirtuin 2 (SirT2) deacetylase in vitro. Finally, trastuzumab-SirT2 and anti-B7H3-SirT2 antibody-enzyme conjugates were generated for the conditional and selective delivery of a cryptophycin cytotoxin by the L17E peptide. While the masked peptide still demonstrated some cytotoxicity, selective cell killing mediated by the antibody-enzyme conjugates was observed.

Blockade of histamine receptor H1 augments immune checkpoint therapy by enhancing MHC-I expression in pancreatic cancer cells

BackgroundAlthough immune checkpoint blockade (ICB) therapy has proven to be extremely effective at managing certain cancers, its efficacy in treating pancreatic ductal adenocarcinoma (PDAC) has been limited. Therefore, enhancing the effect of ICB could improve the prognosis of PDAC. In this study, we focused on the histamine receptor H1 (HRH1) and investigated its impact on ICB therapy for PDAC.MethodsWe assessed HRH1 expression in pancreatic cancer cell (PCC) specimens from PDAC patients through public data analysis and immunohistochemical (IHC) staining. The impact of HRH1 in PCCs was evaluated using HRH1 antagonists and small hairpin RNA (shRNA). Techniques including Western blot, flow cytometry, quantitative reverse transcription polymerase chain reaction (RT-PCR), and microarray analyses were performed to identify the relationships between HRH1 and major histocompatibility complex class I (MHC-I) expression in cancer cells. We combined HRH1 antagonism or knockdown with anti-programmed death receptor 1 (αPD-1) therapy in orthotopic models, employing IHC, immunofluorescence, and hematoxylin and eosin staining for assessment.ResultsHRH1 expression in cancer cells was negatively correlated with HLA-ABC expression, CD8+ T cells, and cytotoxic CD8+ T cells. Our findings indicate that HRH1 blockade upregulates MHC-I expression in PCCs via cholesterol biosynthesis signaling. In the orthotopic model, the combined inhibition of HRH1 and αPD-1 blockade enhanced cytotoxic CD8+ T cell penetration and efficacy, overcoming resistance to ICB therapy.ConclusionsHRH1 plays an immunosuppressive role in cancer cells. Consequently, HRH1 intervention may be a promising method to amplify the responsiveness of PDAC to immunotherapy.

Blood pressure reduction through brain delivery of nanoparticles loaded with plasmid DNA encoding angiotensin receptor shRNA

Elevated brain angiotensin II activity plays a key role in the development of neurogenic hypertension. While blood pressure control in neurogenic hypertension has been successfully demonstrated by regulating central angiotensin II activity, current techniques involving cerebrovascular injections of potential therapeutic agents are not suitable for clinical translation. To address this gap, we present the synthesis of dual-functionalized liposomes functionalized with targeting ligand and cell penetrating peptide. Functionalized liposomes were synthesized using the thin film hydration technique and loaded with plasmid DNA encoding shRNA targeted towards Angiotensin II receptors (PEAS), via the post-insertion method. The synthesized liposomes had a cationic surface charge, an average size of 150 nm, and effectively entrapped over 89% of loaded PEAS. These liposomes loaded with PEAS demonstrated biocompatibility and efficient delivery to brain-derived cell lines, resulting in a remarkable reduction of over 70% in receptor expression within 7 days. To assess the therapeutic potential, spontaneously hypertensive rats were administered intravenous injections of functionalized liposomes loaded with PEAS, and the changes in mean arterial pressure were monitored for 45 days. Remarkably, this treatment led to a significant (p < 0.001) reduction in blood pressure of over 30 mmHg compared to saline-treated rats.

Effects of PNA Sequence and Target Site Selection on Function of a 4.5S Non-Coding RNA.

Peptide nucleic acid (PNA) based antisense strategy is a promising therapeutic approach to specifically inhibit target gene expression. However, unlike protein coding genes, identification of an ideal PNA binding site for non-coding RNA is not straightforward. Here, we compare the inhibitory activities of PNA molecules that bind a non-coding 4.5S RNA called SRP RNA, a key component of the bacterial signal recognition particle (SRP). A 9-mer PNA (PNA9) complementary to the tetraloop region of the RNA was more potent in inhibiting its interaction with the SRP protein, compared to an 8-mer PNA (PNA8) targeting a stem-loop. PNA9, which contained a homo-pyrimidine sequence could form a triplex with the complementary stretch of RNA in vitro as confirmed using a fluorescent derivative of PNA9 (F-PNA13). The RNA-PNA complex formation resulted in inhibition of SRP function with PNA9 and F-PNA13, but not PNA8 highlighting the importance of target site selection. Surprisingly, F-PNA13 which was more potent in inhibiting SRP function in vitro, showed weaker antibacterial activity compared to PNA9 likely due to poor cell penetration of the longer PNA. Our results underscore the importance of suitable target site selection and optimum PNA length to develop better antisense molecules against non-coding RNA.

A Catalytic Approach for the Synthesis of Peptide-Oligonucleotides Conjugates in Aqueous Solution or On-Column.

Peptide-oligonucleotide conjugates (POCs) are covalent architectures composed of a DNA or RNA molecules linked to a peptide. These constructs have found widespread applications ranging from hybrid nanomaterials to gene-targeted therapies. Considering the important role of POCs, a new catalytic approach for their preparation is reported here, that could be applied either on solid support in anhydrous media, or post-synthetically in aqueous buffer. Single amino acids, peptides and cell penetrating peptides (CPPs) were conjugated to various oligo(ribo)nucleotides with high conversions and good isolated yields. The applicability of the method was demonstrated on more than 35 examples including an analogue of a commercial therapeutic oligonucleotide. Other conjugation partners, such as deoxycholic acid and biotin were also successfully conjugated to oligonucleotides. To highlight the potential of this catalytic approach, these conditions have been applied to iterative processes, which is of high interest for the development of DNA-Encoded Libraries.

LAMC2 regulates the proliferation, invasion, and metastasis of gastric cancer via PI3K/Akt signaling pathway

ObjectivesGastric cancer (GC) is a prevalent malignant tumor widely distributed globally, exhibiting elevated incidence and fatality rates. The gene LAMC2 encodes the laminin subunit gamma-2 chain and is found specifically in the basement membrane of epithelial cells. Its expression is aberrant in multiple types of malignant tumors. This research elucidated a link between LAMC2 and the clinical characteristics of GC and investigated the potential involvement of LAMC2 in GC proliferation and advancement.Materials and methodsLAMC2 expressions were detected in GC cell lines and normal gastric epithelial cell lines via qRT-PCR. Silencing and overexpression of the LAMC2 were conducted by lentiviral transfection. A xenograft mouse model was also developed for in vivo analysis. Cell functional assays were conducted to elucidate the involvement of LAMC2 in cell growth, migration, and penetration. Further, immunoblotting was conducted to investigate the impact of LAMC2 on the activation of signal pathways after lentiviral transfection.ResultsIn the findings, LAMC2 expression was markedly upregulated in GC cell lines as opposed to normal gastric epithelial cells. In vitro analysis showed that sh-LAMC2 substantially inhibited GC cell growth, migration, and invasion, while oe-LAMC2 displayed a contrasting effect. Xenograft tumor models demonstrated that oe-LAMC2 accelerated tumor growth via high expression of Ki-67. Immunoblotting analysis revealed a substantial decrease in various signaling pathway proteins, PI3K, p-Akt, and Vimentin levels upon LAMC2 knockdown, followed by increased E-cadherin expression. Conversely, its overexpression exhibited contrasting effects. Besides, epithelial-mesenchymal transition (EMT) was accelerated by LAMC2.ConclusionThis study provides evidence indicating that LAMC2, by stimulating signaling pathways, facilitated EMT and stimulated the progression of GC cells in laboratory settings and mouse models. Research also explored that the abnormal LAMC2 expression acts as a biomarker for GC.

Heat-inducible CAR-T overcomes adverse mechanical tumor microenvironment in a 3D bioprinted glioblastoma model

Glioblastoma (GBM) presents a significant therapeutic challenge due to the limited efficacy of existing treatments. Chimeric antigen receptor (CAR) T-cell therapy offers promise, but its potential in solid tumors like GBM is undermined by the physical barrier posed by the extracellular matrix (ECM). To address the inadequacies of traditional 2D cell culture, animal models, and Matrigel-based 3D culture in mimicking the mechanical characteristics of tumor tissues, we employed biomaterials and digital light processing-based 3D bioprinting to fabricate biomimetic tumor models with finely tunable ECM stiffness independent of ECM composition. Our results demonstrated that increased material stiffness markedly impeded CAR-T cell penetration and tumor cell cytotoxicity in GBM models. The 3D bioprinted models enabled us to examine the influence of ECM stiffness on CAR-T cell therapy effectiveness, providing a clinically pertinent evaluation tool for CAR-T cell development in stiff solid tumors. Furthermore, we developed an innovative heat-inducible CAR-T cell therapy, effectively overcoming the challenges posed by the stiff tumor microenvironment.

A Conditionally Activated Cytosol-Penetrating Antibody for TME-Dependent Intracellular Cargo Delivery.

Currently, therapeutic and diagnostic applications of antibodies are primarily limited to cell surface-exposed and extracellular proteins. However, research has been conducted on cell-penetrating peptides (CPP), as well as cytosol-penetrating antibodies, to overcome these limitations. In this context, a heparin sulfate proteoglycan (HSPG)-binding antibody was serendipitously discovered, which eventually localizes to the cytosol of target cells. Functional characterization revealed that the tested antibody has beneficial cytosol-penetrating capabilities and can deliver cargo proteins (up to 70 kDa) to the cytosol. To achieve tumor-specific cell targeting and cargo delivery through conditional activation of the cell-penetrating antibody in the tumor microenvironment, a single-chain Fc fragment (scFv) and a VL domain were isolated as masking units. Several in vitro assays demonstrated that fusing the masking protein with a cleavable linker to the cell penetration antibody results in the inactivation of antibody cell binding and internalization. Removal of the mask via MMP-9 protease cleavage, a protease that is frequently overexpressed in the tumor microenvironment (TME), led to complete regeneration of binding and cytosol-penetrating capabilities. Masked and conditionally activated cytosol-penetrating antibodies have the potential to serve as a modular platform for delivering protein cargoes addressing intracellular targets in tumor cells.

Abstract PO2-24-04: Exploring the role and therapeutic potential of Ubiquitin Specific Peptidase 11 (USP11) in estrogen receptor positive breast cancer

Abstract Dysregulated estrogen receptor (ER) function is a key feature of 70% of breast cancers. In this setting ER primarily functions as a growth-controlling transcription factor driving pro-tumorigenic properties, thus there is a need to identify novel modulators of ER transcriptional activity as alternative therapeutic options. Previously, we showed a significant association between high ubiquitin specific protease 11 (USP11) expression and poor survival in ER+ breast cancer patients, but not in ER- patients, indicating a role for USP11 in ER+ subtypes. However, the precise role of USP11 in controlling ER function remains undescribed. To interrogate USP11 function in ER+ breast cancer we generated a CRISPR USP11 knockout MCF7 breast cancer cell line. Our results showed for the first time that USP11 knockout significantly reduces proliferation, induces G2 cell cycle arrest and apoptosis, abrogates ERα transcriptional activity, significantly impairs cell migration and invasion, whilst also impairing MCF7 cancer stem-cell like properties and malignant transformation in vitro. In addition, we also observed significant dysregulation of epithelial-mesenchymal transition-related markers including E-cadherin, N-cadherin, Snail and Slug. Using RNA-seq of MCF7 cells +/- estradiol, +/- stable USP11 knockdown we identified dysregulated pathways in protein binding, cell adhesion molecule binding and cell cycle regulation following USP11 modulation. Of note, gene expression analysis indicated NCOA3 as the most differentially expressed coding gene. Further validation confirmed a significant reduction in NCOA3- encoded Steroid receptor coactivator- 3 (SRC-3) protein expression following USP11 modulation with immunocytochemical analysis also indicating co-localisation within the nucleus. Frequently upregulated in breast cancer and associated with poor outcome, SRC-3 promotes ER transcriptional activity and co-activates a wide range of additional pro-tumorigenic transcription factors. Thus, SRC-3 modulation offers an attractive therapeutic target in breast cancer. This finding may present a novel role for USP11 in controlling SRC-3 and therefore associated ER transcriptional activity. We then aimed to explore the therapeutic potential of USP11. Although rare in primary breast cancer, activating mutations in the ER ligand-binding domain have recently been linked to recurrent, anti-endocrine resistant disease and metastasis. As such cancers remain dependent on ER-signalling for proliferation, but are resistant to ER targeted therapies, we identified USP11 as a potential novel therapeutic target. Using CRISPR knock-in ER mutant MCF7 cell lines expressing the four most common ER activating mutations, we demonstrated that USP11 knockdown continues to significantly reduce proliferation and abrogates mutant ERα transcriptional activity. We then explored amphipathic cell penetrating peptide RALA encapsulation as a delivery method for siRNA targeting USP11. Optimisation of nanoparticle delivery in the Y537S ER-mutant MCF7 breast cancer cell line achieved significant knockdown of USP11 and ER- related target genes including TFF1, PgR, GREB1 and PKIB in 2D with minimal associated cellular toxicity. To overcome the siRNA-related issues of rapid clearance upon systemic delivery, we then explored the use of a freeze-dried collagen-based scaffold model as an in vitro 3D RALA-encapsulated siRNA delivery system. Here we observed successful delivery of nanoparticle to MCF7 breast cancer cells, with a significant reduction in USP11 mRNA expression. Bio-compatible scaffold- facilitated delivery may therefore offer a more feasible translation of this siRNA-based therapeutic. Overall, this study highlights a novel role for USP11 in various oncogenic pathways. USP11 may therefore offer a novel therapeutic target to be exploited for the management of ER-positive breast cancer, including ER mutant breast cancer. Citation Format: Rachel Moore, Anna Blümel, Elspeth Ward, Elizabeth Sainsbury, Emer Conroy, Fergal O’Brien, Caroline Curtin, Simak Ali, Helen McCarthy, William Gallagher, Darran O'Connor. Exploring the role and therapeutic potential of Ubiquitin Specific Peptidase 11 (USP11) in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-04.

Lactobacillus plantarum 299v administration prevents high-sodium diet-induced glycocalyx degradation and arterial dysfunction

A central feature of cardiovascular disease (CVD) is arterial dysfunction (i.e., elevated systolic blood pressure [BP], aortic stiffening, and endothelial dysfunction), which may be initiated by a degraded endothelial glycocalyx. The glycocalyx is a negatively charged structure bound to the vascular endothelium that appears to play a critical role in cardiovascular health, particularly by buffering sodium in blood, although excessive sodium degrades the glycocalyx. In the United States, a high-sodium (HS) diet is consumed by ~90% of the population with average sodium intake ~2.3x greater than recommended by the American Heart Association. We have previously shown that HS diet induces glycocalyx degradation and arterial dysfunction in outbred, genetically diverse UM-HET3 mice. Others have shown that HS diet lowers Lactobacillus abundance in mouse gut microbiome. However, it is unknown whether maintaining Lactobacillus would prevent HS diet-induced glycocalyx degradation and arterial dysfunction. To test this concept, male and female UM-HET3 mice (n=21-35/group) were randomized into a low-sodium (LS) diet (1% NaCl), HS diet (4% NaCl), or HS diet with Lactobacillus plantarum 299v (Lp299v) administration (60 million CFU/ml in drinking water) for 12 weeks. Glycocalyx barrier function was determined by assessing perfused boundary region (PBR), which represents red blood cell penetration into the glycocalyx. At week 12, we observed higher PBR in HS mice (2.36±0.04 μm) compared to LS and HS+Lp299v mice (2.09±0.03 and 2.20±0.04 μm, respectively; p&lt;0.05), indicating a worse glycocalyx barrier function in HS mice. PBR was similar between LS and HS+Lp299v mice (P&gt;0.05), indicating a preserved glycocalyx barrier function in HS+Lp299v mice. Systolic BP was higher in HS (127±1 mmHg) compared to LS and HS+Lp299v mice (113±1 and 118±1 mmHg, respectively; p&lt;0.05). Aortic stiffness, determined by aortic pulse wave velocity (PWV), was also higher in HS (345±5 cm/s) compared to LS and HS+Lp299v mice (268±5 and 300±3 cm/s, respectively; p&lt;0.05). However, systolic BP and aortic PWV were higher in HS+Lp299v compared to LS mice (p&lt;0.05). Ex vivo endothelial function, determined by the maximal vasodilatory response to acetylcholine in the carotid artery, was lower in HS (83.9±1.3%) compared to LS and HS+Lp299v mice (89.8±1.3 and 90.1±1.0%, respectively; p&lt;0.05). Endothelial function was similar between LS and HS+Lp299v mice (P&gt;0.05). In summary, Lp299v administration prevents HS diet-induced glycocalyx degradation and endothelial dysfunction, while mitigating HS diet-induced elevations in systolic BP and aortic stiffness. These findings suggest that Lp299v administration may be a novel strategy to lower the risk of CVD in humans that consume a HS diet. This study was funded in part by a grant from the National Institutes of Health (R00 AT010017). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identifying the time course and contributors for Western diet-induced increase in glycocalyx barrier function

The endothelial glycocalyx is a dynamic gel-like structure bound to the vascular endothelium composed of glycosaminoglycans, such as hyaluronan. The glycocalyx plays a critical role in cardiovascular health, acting as a physical barrier to the contents of flowing blood, including red blood cells. Interestingly, we have shown that 12 weeks of Western diet (WD), characterized by excess saturated fat and sugar content, results in greater glycocalyx barrier function in the mesenteric microcirculation that is strongly related to blood glucose values. Currently, the time course that WD induces an increase in glycocalyx barrier function is unknown, as are the contributors to that increase. Thus, we first examined glycocalyx barrier function 1, 2, 4, and 12 weeks after initiating WD in non-fasted male and female UM-HET3 mice (n=9-14/group). Glycocalyx barrier function was assessed in the mesenteric microcirculation by determining perfused boundary region (PBR), which represents red blood cell penetration into the glycocalyx. A higher PBR indicates worse glycocalyx barrier function. Compared to age-matched normal chow (NC) control mice, PBR was lower in WD at week 1 (NC: 2.35±0.05 vs. WD: 1.94±0.04 μm, p&lt;0.05), 2 (NC: 2.36±0.04 vs. WD: 2.01±0.07 μm, p&lt;0.05), 4 (NC: 2.38±0.04 vs. WD: 1.91±0.1 μm, p&lt;0.05), and 12 (NC: 2.41±0.03 vs. WD 1.99±0.04 μm, p&lt;0.05). While blood glucose was higher in WD compared to NC mice at week 12 (NC: 166±3 vs. WD 179±4 mg/dL, p&lt;0.05), it was similar between NC and WD at week 1, 2, and 4 (P&gt;0.05). These data indicate that the WD-induced increase in glycocalyx barrier function occurs as early as 1 week after initiating WD and appears to be independent of blood glucose. Because glycocalyx barrier function was assessed in the mesenteric microcirculation in non-fasted mice, we then sought to determine if the WD-induced increase in glycocalyx barrier function was due to postprandial state. Thus, we determined PBR in non-fasted and fasted (6h) male and female UM-HET3 mice 1 week after initiating WD (n=14-16/group). Fasting status did not affect PBR in age-matched NC (non-fasted 2.32±0.05 vs. fasted: 2.25±0.04 μm, P&gt;0.05) or WD mice (non-fasted 1.94±0.04 vs. fasted: 2.06±0.05 μm, P&gt;0.05), though WD had lower PBR than NC mice in non-fasted and fasted states (p&lt;0.05). These data indicate that glycocalyx barrier function is unaffected by fasting status. Given the role of hyaluronan in the glycocalyx, we then sought to determine if the WD-induced increase in glycocalyx barrier function was due to higher hyaluronan content in the glycocalyx. Thus, we examined PBR pre- and 30 min post-hyaluronidase administration (35 U) in male and female UM-HET3 mice 1 week after initiating WD (n=5/group). There was a tendency for hyaluronidase administration to increase PBR in age-matched NC mice (Pre: 2.37±0.07 vs. Post: 2.65±0.12 μm, P=0.11), whereas PBR increased in WD mice after hyaluronidase administration (Pre: 2.13±0.04 vs. Post: 2.53±0.09 μm, p&lt;0.05). Additionally, the difference in PBR between NC and WD mice was no longer present post-hyaluronidase administration (P&gt;0.05). Taken together, these data indicate that glycocalyx barrier function is increased as early as 1 week after initiating WD and appears to be independent of blood glucose or fasting status but is at least partially due to increased hyaluronan content in the glycocalyx. This study was partly funded by a grant from the National Institutes of Health (R00 AT010017). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A cleavable peptide adapter augments the activity of targeted toxins in combination with the glycosidic endosomal escape enhancer SO1861

BackgroundTreatment with tumor-targeted toxins attempts to overcome the disadvantages of conventional cancer therapies by directing a drug’s cytotoxic effect specifically towards cancer cells. However, success with targeted toxins has been hampered as the constructs commonly remain bound to the outside of the cell or, after receptor-mediated endocytosis, are either transported back to the cell surface or undergo degradation in lysosomes. Hence, solutions to ensure endosomal escape are an urgent need in treatment with targeted toxins. In this work, a molecular adapter that consists of a cell penetrating peptide and two cleavable peptides was inserted into a targeted toxin between the ribosome-inactivating protein dianthin and the epidermal growth factor. Applying cell viability assays, this study examined whether the addition of the adapter further augments the endosomal escape enhancement of the glycosylated triterpenoid SO1861, which has shown up to more than 1000-fold enhancement in the past.ResultsIntroducing the peptide adapter into the targeted toxin led to an about 12-fold enhancement in the cytotoxicity on target cells while SO1861 caused a 430-fold increase. However, the combination of adapter and glycosylated triterpenoid resulted in a more than 4300-fold enhancement and in addition to a 51-fold gain in specificity.ConclusionsOur results demonstrated that the cleavable peptide augments the endosomal escape mediated by glycosylated triterpenoids while maintaining specificity. Thus, the adapter is a promising addition to glycosylated triterpenoids to further increase the efficacy and therapeutic window of targeted toxins.

Topically applied GHK as an anti-wrinkle peptide: Advantages, problems and prospective

Introduction: Peptides are promising and attractive anti-wrinkle active ingredients, amongst which glycyl-histidyl-lysine peptide (GHK) is one of the most broadly promoted peptide for topical application. This simple sequence of amino acid residues not only has the capability of tissue regeneration and the enhancement of collagen and glycosaminoglycans synthesis but also is able to increase nerve outgrowth and angiogenesis. Consequently, GHK has several properties, from wound healing to prevention/reduction wrinkles. GHK-Cu and Pal-GHK are metal complex and palmitoylated derivatives of GHK, respectively. Although GHK-Cu and Pal-GHK are widely used in anti-wrinkle products available on the cosmetic market, the published information on their skin permeability, effectiveness, physicochemical properties and so on is insufficient. Methods: This review aims to highlight whether GHK is sufficiently effective on wrinkle prevention/reduction. Apart from the effectiveness, another question that is tried to be answered is whether skin permeability of GHK allows it to act as an anti-wrinkle peptide at its site of action? Skin permeation enhancement methods employed so far are also reviewed. Results: Based on cellular studies, undoubtedly, GHK can be considered as an anti-wrinkle ingredient. Although GHK-Cu and Pal-GHK have been of interest as effective peptides to be incorporated in the anti-wrinkle products, there is a surprising absence of clinical studies using them. Metal complexation and chemical modification with a hydrophobic moiety increase permeability of this peptide. Besides, cell penetrating peptides seem promising to increase skin permeation of GHK and its derivatives. Skin pretreatment with microneedles also has the potential to be further studied for permeation enhancement of such peptides. As peptide ingredients, their formulation may encounter some challenges, mainly due to their hydrophilic (high aqueous solubility and low partition coefficient) and unstable nature. Conclusion: Although GHK-Cu and Pal-GHK are effective and relatively skin permeable, their permeability could be successfully increased using permeation enhancement methodologies.

Fabrication of magnesium-doped biphasic calcium phosphate granules with sea urchin spine-derived porous structure

In bone regeneration, synthetic materials are required to replace autogenous bone grafts. The shape, composition, surface, and porous structure of the material are key factors in bone formation and material resorption. In this study, we describe the fabrication of Mg-doped biphasic calcium phosphate (Mg-BCP) granules comprising hydroxyapatite (HAp) and beta-tricalcium phosphate (β-TCP) by exploiting the structural and chemical characteristics of sea urchin spines. The Mg-BCP granules were cylinders with a 1.2–1.8 mm diameter and height of 1.4–1.7 mm. The β-TCP/HAp ratio and Mg content in the Mg-BCP granules were controlled within the ranges of 0.36–0.45 and 2.40–6.21 mol%, respectively. Controlling the β-TCP/HAp ratio and Mg content may contribute to controlling the material resorption rate and promoting osteogenesis and angiogenesis. The Mg-BCP granules maintained the structural characteristics of the sea urchin spines. The granules comprised radial wedges termed septa, and a meshwork termed stereom. The interseptal gaps, or micropores, decreased from the granular exterior (24–29 μm) to the interior (14–28 μm). The septa in the outermost layer provide concavities on the sides of the cylindrical granules. The stereom meshworks in the core and outer layers involved micropores in the ranges of 3–15 and 3–10 μm, respectively. These multiscale pores and surface undulations may improve cell penetration and attachment, bodily fluid permeability, and protein adsorption. The compressive strengths of the Mg-BCP granules were in the range of 45.2–46.1 MPa, which are useful as bone grafts. Due to their rounded shapes, the Mg-BCP granules filled a mold more densely than the clinically used and irregularly shaped granules. These findings demonstrated that Mg-BCP granules are promising materials for bone regeneration.

Enhancing the Efficacy and Safety of Methotrexate Treatment: A Focus on Drug Interactions (Review)

INTRODUCTION. Methotrexate (MTX) is the main disease-modifying antirheumatic drug (DMARD) and the gold standard for the safety and efficacy evaluation of biologicals and targeted small molecules. However, its narrow therapeutic range, interpatient variability in pharmacokinetics and pharmacodynamics, and potential clinically relevant drug–drug interactions (DDIs) may lead to treatment failure and increase the risk of adverse drug reactions (ADRs).AIM. The study aimed to describe the main clinically significant DDIs associated with MTX used in rheumatic disease therapy and determine possible approaches to addressing this issue based on a literature review.DISCUSSION. MTX is characterised by pharmacokinetic DDIs during absorption, cell penetration, and elimination. Some non-steroidal anti-inflammatory drugs (NSAIDs), theophylline, sulfasalazine, antibacterial agents, and proton pump inhibitors (PPIs) affect MTX elimination and therapeutic effects. The main ADRs associated with MTX include haematotoxicity, hepatotoxicity, lung tissue damage (interstitial pneumonitis and pulmonary fibrosis), and renal dysfunction. The severity of these ADRs depends on the dose, comorbidities, and concomitant therapy. The toxicity of MTX may be increased by the concomitant administration of medicinal products that exhibit haematotoxicity and affect renal function (impair the elimination of medicines). When co-administering MTX and medicines having clinically significant DDIs described in the literature, healthcare providers should consider the risk factors for each individual patient. The most significant risk factors include moderate to severe renal and hepatic impairment, older age, polypharmacy, and hypoalbuminemia.CONCLUSIONS. This article describes potential clinically significant interactions between MTX and certain NSAIDs, antibacterial agents, and PPIs that depend on individual patient characteristics and may increase the toxicity or decrease the effectiveness of MTX. MTX deprescribing, short-term withdrawal, and dosing optimisation may be considered as approaches to DDI risk mitigation.

Design and Expression of Recombinant cell Penetrating Protein Based on Tat and pertussis-like Toxin A and Evaluation of its Effects on the lung cancer

Design and Expression of Recombinant cell Penetrating Protein Based on Tat and pertussis-like Toxin A and Evaluation of its Effects on the lung cancer

Development of a Decafluorobiphenyl Cyclized Peptide Targeting the NEMO-IKKα/β Interaction that Enhances Cell Penetration and Attenuates Lipopolysaccharide-Induced Acute Lung Injury.

Aberrant canonical NF-κB signaling has been implicated in diseases, such as autoimmune disorders and cancer. Direct disruption of the interaction of NEMO and IKKα/β has been developed as a novel way to inhibit the overactivation of NF-κB. Peptides are a potential solution for disrupting protein-protein interactions (PPIs); however, they typically suffer from poor stability in vivo and limited tissue penetration permeability, hampering their widespread use as new chemical biology tools and potential therapeutics. In this work, decafluorobiphenyl-cysteine SNAr chemistry, molecular modeling, and biological validation allowed the development of peptide PPI inhibitors. The resulting cyclic peptide specifically inhibited canonical NF-κB signaling in vitro and in vivo, and presented positive metabolic stability, anti-inflammatory effects, and low cytotoxicity. Importantly, our results also revealed that cyclic peptides had huge potential in acute lung injury (ALI) treatment, and confirmed the role of the decafluorobiphenyl-based cyclization strategy in enhancing the biological activity of peptide NEMO-IKKα/β inhibitors. Moreover, it provided a promising method for the development of peptide-PPI inhibitors.

Multilayer woven scaffolds with gradient degradation and nano guidance properties for tendon reconstruction: Continuously promoting tissue infiltration and inducing tissue alignment

Limited tissue infiltration and disordered collagen alignment are primary factors in tendon repair failure. This study introduces a multilayer-induced scaffold (IS@DN) for tendon repair, combining gradient-degraded layers (DLs) and nano-guided layers (NLs). DL degradation creates 3-D pore channels for cell infiltration, while NLs offer continuous contact guidance for cell alignment. Enhanced cell penetration and alignment within the scaffold promote orderly collagen regeneration. IS@DN demonstrates suitable mechanical properties and degradation-enhanced porosity, promoting cell and collagen infiltration. Sequential exposure of NLs facilitates continuous alignment of cells and collagen fibers. At a macro level, IS@DN effectively regenerates the rat Achilles tendon, restoring 91.37 % of its native failure load. This integrative approach, incorporating gradient degradation and nano guidance, holds considerable promise for clinical tissue regeneration.