Cell Penetrating Research Articles

Correction: Treating ICB-resistant cancer by inhibiting PD-L1 via DHHC3 degradation induced by cell penetrating peptide-induced chimera conjugates

Correction: Treating ICB-resistant cancer by inhibiting PD-L1 via DHHC3 degradation induced by cell penetrating peptide-induced chimera conjugates

Translational implications of a novel combination of iPRF and collagen scaffold for proliferation of gingival mesenchymal stem cells.

"Tissue engineering," is a concept that involves the use of scaffolds, cells, and growth factors/signaling molecules in the field of regenerative medicine. The present study was carried out to evaluate the attachment and depth of penetration of the Gingival Mesenchymal Stem Cells (GMSCs) onto the collagen scaffold preconditioned with Fetal Bovine Serum (FBS), Injectable Platelet Rich Fibrin (i-PRF) and a combination of FBS with i-PRF using histological analysis and environmental scanning electron microscopy (ESEM) respectively. In the present study, commercially available collagen membranes were used as scaffolds and divided into 3 groups where Group I was preconditioned with FBS, Group II was preconditioned with i-PRF, and Group III with a combination of FBS and i-PRF. Scaffolds were then seeded with GMSCs and incubated in a CO2 incubator at 37 0C for 7 days. Both histological and ESEM analysis showed proliferation, attachment, and depth of penetration along with the combination of cell morphological changes in all the three groups. In group I cells showed mild depth of penetration along with a round morphological appearance. In group II the cells showed moderate depth of penetration and rounded morphology. In group III maximum depth of penetration was seen along with the round and spheroidal morphology of cells. The combination of the growth media showed the best effect on cell attachment, depth of penetration, and cell morphology. This is the first report demonstrating the effect of the combination of collagen and i-PRF supports the proliferation of GMSCs. Since FBS alone and i-PRF alone exhibited similar cell proliferation it is recommended that i-PRF could be used in clinical scenarios making it xenofree.

Abstract 4134456: Hybrid Cardiac Targeting Peptide Ameliorates HFpEF Phenotype in Mice

Introduction: Heart failure with preserved ejection fraction (HFpEF) is a form of heart failure which is rapidly rising in incidence in the US today. Despite this, no therapies with mortality benefit exist for this syndrome. Previous work with our novel hybrid cardiac targeting peptide (hCTP), a cardiomyocyte-specific cell penetrating peptide, has shown multiple beneficial biological effects (improved calcium handling, anti-inflammatory properties) beyond its transduction abilities. Therefore, we hypothesized that treatment with hCTP in a mouse model of HFpEF would ameliorate the pathophysiology of this disease. Methods: To establish a HFpEF model, 6-week-old, C57BL/6NJ mice were placed on a high-fat diet (HFD), 60% kCal from lard, and water with 0.5 g/L of an iNOS inhibitor (N-Nitro-L-arginine methylester) to induce hypertension. After 5 weeks of the diet, HFD mice and their littermate controls underwent baseline exercise testing and echocardiograms. Exercise capacity was measured after 2 days of exercise training with 2 days of rest in between; mice were placed on a treadmill at a 10-degree angle and made to run at increasingly fast speeds (5-30 m/min) until exhaustion. Echocardiograms were performed under 1.5% inhalational isoflurane. After baseline testing, the mice were treated with vehicle, or either 2.5 or 5 mg/Kg hCTP, either 3 or 5 days a week (five groups, n = 3 per group). After four weeks of treatment, the mice were again subjected to the same battery of tests and euthanized. Data was compared using two way ANOVA with Sidak’s test to compare measurements pre- and post-treatment. Results: Treatment with hCTP at either concentration for five days/week led to an increase in running distance from 39.8 m to 116.3 m (p = 0.03, n = 6), compared to changes in control distance from 208.4 m to 157.8 m (p = 0.399). Running distance did not correlate with weight (R 2 < .001). Treatment with 5 mg/Kg hCTP for 5 days/week led to a normalization of the E/e’ ratio, from 43.3 to 26.9 (p = 0.004, n = 3) compared to a change in the control of 34.7 to 26.1 (p = 0.312). Ejection fractions remained consistent (>55%) across all groups. Conclusions: Our data suggests that hCTP appears to be a promising treatment in ameliorating some of the physiological effects in a murine model of HFpEF. Gene and protein expression studies in heart tissue are underway. Further work is warranted with a larger sample size.

Abstract 4136448: A Novel Cardiomyocyte Targeting Peptide Enhances Calcium Handling

Introduction: Intracellular calcium handling plays a central role in cardiac contractility and relaxation. Genes such as the Ca 2+ -ATPase pump (SERCA2a) and the Dihydropteridine reductase (DHPR) enzyme are especially important in the cycling of intracellular calcium. As prior research has established a link between treatment with our cardiac targeting peptide (CTP: a 12-amino acid, cardiomyocyte-specific cell penetrating peptide) and upregulation of calcium-handling genes such as SERCA2a and DHPR, we hypothesized that treatment with our novel hybrid cardiac targeting peptide (hCTP: positions #3 and 11 replaced with the D-enantiomer form of the amino acids) will improve intracellular calcium handling in human cardiomyocytes (CMCs) by increasing expression of key calcium handling genes instrumental in sequestering calcium in diastole and lowering cytosolic levels. Methods: To test hCTP’s calcium handling abilities, CMCs were plated on day 1 and allowed to settle for 24 hours, after which they received daily treatment with various concentrations of hCTP (1-25µM) for three days. On day 5, cells underwent either intracellular calcium measurements using the FLIPR 6 Calcium assay or were prepared for western blotting. Western blotting was performed to determine the protein concentrations of SERCA2a and DHPR, as compared to a GAPDH control. To test hCTP’s intrinsic calcium handling abilities, various concentrations of hCTP were mixed with a constant amount of elemental calcium dissolved in water. After 20 min at 37 o C, the concentration of free Ca 2+ was measured with an ABL90 Flex Plus radiometer. Results: In CMCs, treatment with hCTP led to a decrease in cytosolic calcium in a dose dependent fashion; concentrations as low as 1µM led to a significant decrease in cytosolic calcium (p< 0.0001 for all groups). Western blots performed on the treated CMCs revealed an increase in expression of both DHPR and SERCA2a, normalized to GAPDH control. Outside of the cellular environment, hCTP did not appear to display inherent calcium sequestring properties, suggesting that improvements in calcium handling are due to upregulation of gene expression. Conclusion: hCTP appears to lower cytosolic calcium and improve calcium handling through upregulation of SERCA2a and DHPR. Our data indicates that hCTP is worth pursuing as a stand-alone, cardio-selective therapy for cardiomyopathies involving calcium mishandling.

DDEL-09. AS1411 APTAMER-CONJUGATED NANOSPHERES FOR TARGETING GLIOBLASTOMA THERAPY

Abstract Postoperative chemotherapy remains essential for treating glioblastoma (GBM), with nanocarrier-based drug delivery emerging as the most efficient method. However, non-specific delivery across the blood-brain barrier (BBB) can limit drug accumulation in tumors and cause damage to nearby tissues. As cancer progresses, angiogenic processes lead to the formation of an irregular blood-tumor barrier (BTB), necessitating nanodrug platforms for precise targeting and efficient drug delivery to cancer cells. In this study, we aimed to demonstrate targeted cancer therapy by selectively identifying tumor cells overexpressing nucleolin (NCL) protein using Dox-loaded AS1411 aptamer-conjugated nanospheres. Drug nanocarriers were synthesized from small molecules with sizes ranging from 100 to 300 nm to facilitate drug delivery. Selective drug delivery and cellular uptake were demonstrated for NCL-positive GBM cells (U87 and U251) compared to NCL-negative normal human astrocyte (NHA) cells. Flow cytometric analysis using FAM dye-labeled AS1411 aptamer-conjugated nanospheres confirmed that drug delivery to GBM U87 and U251 cells was actively targeted by specific AS1411-NCL interactions. Confocal laser scanning microscopy and cytotoxicity analysis confirmed that inoculation of Dox-loaded AS1411 aptamer-conjugated nanospheres induced selective internalization in GBM U87 and U251 cells. Moreover, to evaluate cytotoxicity and anti-tumor effects after treatment with PBS, Apt-Nanospheres, Free Dox, and Dox-Apt-Nanospheres, the results were analyzed through cell counting kit (CCK-8) analysis, evaluation of differences in 3D tumor sphere radii, and an in vivo GBM xenograft tumor model. The AS1411 aptamer-conjugated nanosphere drug delivery system was developed for dual-target treatment of GBM. AS1411-mediated specific recognition and drug accumulation significantly enhanced binding to NCL-positive U87 and U251 cells and improved cytotoxicity efficiency compared to Free Dox. Additionally, this study demonstrated that the active targeting model using aptamer-mediated drug delivery was more effective in improving drug penetration and retention in tumor cells than non-specific enhanced permeability and retention (EPR)-mediated delivery and passive drug delivery.

IMMU-28. LOW-INTENSITY PULSED ULTRASOUND COMBINED WITH POLY-ICLC AND INTERLEUKIN-2 UNLOCKS THE IMMUNE PRIVILEGE OF THE BRAIN BY PROMOTING EPITOPE SPREADING AND ACTIVATION OF CNS-SPECIFIC NAÏVE T CELLS

Abstract Immunotherapies targeting glioma, such as chimeric antigen receptor (CAR) T cell therapies, offer new therapeutic avenues; however, their application has yielded limited success in clinical trials so far. Due to the antigenic heterogeneity of gliomas, targeting a few or several antigens still results in recurrence with “antigen-loss” tumors, indicating the need to engage the endogenous immune system to eliminate these therapy-resistant tumor cells. However, natural anatomical barriers and an immunosuppressive environment allow brain tumors to evade detection by the endogenous immune system. Low-intensity pulsed ultrasound combined with microbubbles (LIPU/MB) has recently been shown to transiently open the blood-brain barrier (BBB), enabling penetration of cells and drugs into the CNS. Using intravital two-photon imaging, two-photon microscopy, multiparametric spectral flow cytometry, and bone marrow chimera mice, alongside blood samples from glioblastoma patients treated with LIPU/MB, we observed that LIPU/MB promotes migration of CNS antigen-presenting cells from the perivascular space into the periphery. To assess the immunological relevance of LIPU/MB-mediated CNS antigen presentation to the peripheral immune system, we have developed new transgenic mice (GFAP-minigene mice) with an intact BBB expressing four immunogenic peptides under the CNS-specific GFAP promoter. In GFAP-minigene mice, LIPU/MB combined with toll-like receptor 3 agonist poly-ICLC and Interleukin-2 promoted priming, activation, and homing of naïve CNS-specific T cells into the brain parenchyma. Cytotoxic T cell activity in the brain was accompanied by MHC-I and MHC-II epitope spreading, mirrored by the occurrence of endogenous T cells in the brain, deep cervical lymph nodes, and spleen harboring other CNS-(minigene) antigen-specific T cell receptors. Collectively, our findings suggest that LIPU/MB combined with poly-ICLC can overcome inherent limitations of immunotherapy by enhancing CNS epitope spreading and activation of naïve CNS-antigen-specific T cells within the context of an intact blood-brain barrier.

Role of Peptide Associations in Enhancing the Antimicrobial Activity of Adepantins: Comparative Molecular Dynamics Simulations and Design Assessments

Adepantins are peptides designed to optimize antimicrobial biological activity through the choice of specific amino acid residues, resulting in helical and amphipathic structures. This paper focuses on revealing the atomistic details of the mechanism of action of Adepantins and aligning design concepts with peptide behavior through simulation results. Notably, Adepantin-1a exhibits a broad spectrum of activity against both Gram-positive and Gram-negative bacteria, while Adepantin-1 has a narrow spectrum of activity against Gram-negative bacteria. The simulation results showed that one of the main differences is the extent of aggregation. Both peptides exhibit a strong tendency to cluster due to the amphipathicity embedded during design process. However, the more potent Adepantin-1a forms smaller aggregates than Adepantin-1, confirming the idea that the optimal aggregations, not the strongest aggregations, favor activity. Additionally, we show that incorporation of the cell penetration region affects the mechanisms of action of Adepantin-1a and promotes stronger binding to anionic and neutral membranes.

Decoding core genes and intercellular communication in osteosarcoma: bioinformatic investigation and immune cell profiling for diagnostic and therapeutic insights

The study focusing on developing an artificial neural network (ANN) model in accordance with genetic characteristics of osteosarcoma (OS) to accurately speculate OS cases. In the present study, we identified 467 DEGs through differentially acting gene investigation and that 345 exist suppressed and 122 exist stimulated. The resultant of GO enrichment analysis displayed the functions mainly included T cell activation, secretory granule lumen, antioxidant property etc. The pathways identified in the differentially acting genes (DAGs) were greatly interacted with Phagosome, Staphylococcus aureus infection, Human T − cell leukemia virus 1 infection, etc. Next, we found out top ten hub DEGs (HDEGs) by PPI network analysis. In addition, through the validation of ANN itself and Test set samples, it was proved that the prediction performance of our constructed ANN model is accurate and reliable. Finally, the penetration of immune cells and its interaction with target CDEGs were examined, and variations in penetration of 22 types of immune cells amongst different classes were found, additionally correlation amongst immune cells and between immune cells and target CDEGs. Furthermore, we analyzed the expression of the top two CDEGs (YES1 and MFNG) in OS tissues and normal tissues, also the interrelationship among the activity of YES1 and MFNG in OS tissues and clinicopathological properties of OS cases. Furthermore, the correlation analysis between the top two CDEGs and immune infiltrating cells was performed in OS tissues. Our research results revealed that CDEGs-based ANN model is effective at predicting OS patients, which facilitates early diagnosis and treatment of OS.

Functional interplay between short antimicrobial peptides and model lipid membranes

Antimicrobial peptides (AMPs) are considered an attractive generation of novel antibiotics due to their advantageous properties such as a broad spectrum of antimicrobial activity against pathogens, low cytotoxicity, and drug resistance. Although they have common structural features and it has been widely demonstrated that bacterial membranes represent the main target of the peptide activity, the exact mechanism underlying the membrane perturbation by AMPs is not fully understood. Nevertheless, all the proposed modes of action implicate the preliminary interaction of AMPs with the negatively charged lipids in bacterial membranes. Recently, the structural and functional characterization of two AMPs, RiLK1 and RiLK3, was reported. Specifically, both peptides were revealed to be multitalented compounds capable of binding Gram-positive and Gram-negative liposome models with high affinity, but their mechanism of action remains elusive. In this paper, the effects of RiLK1 and RiLK3 on vesicles mimicking prokaryotic and eukaryotic cell membranes were further examined by using different approaches. Fluorescence and quenching assays either by acrylamide or lipophilic probes suggested that the peptides were mainly located at the interface of the negatively charged membranes that mimicked those of Salmonella Typhimurium and Staphylococcus aureus, possibly oriented in a parallel manner. Furthermore, RiLK1 and RiLK3 caused a significant leakage of carboxyfluorescein from bacterial liposomes, demonstrating that they can permeabilize the target membranes at high doses. Conversely, both peptides appear to behave like cell penetrating peptides (CPPs) at concentrations near their MIC values evaluated against the bacterial targets. Moreover, Dynamic Light Scattering provided further insights on the mechanisms of antimicrobial peptide against the bacterial liposomes. Conclusively, in vitro experiments indicated that RiLK1 and RiLK3 displayed potent bacteriostatic efficacy at low micromolar concentrations against an antibiotic-resistant ESKAPE pathogen, making them a valuable tool in preventing and treating infections caused by such bacteria.

Modeling and Evaluation of Penetration Process Based on 3D Mechanical Simulation.

In biological micromanipulation, cell penetration is a typical procedure that precedes cell injection or oocyte enucleation. During this procedure, cells usually undergo significant deformation, which leads to cell damage. In this paper, we focus on modeling and evaluating the cell penetration process to reduce cell deformation and stress, thereby reducing cell damage. Initially, a finite element model (FEM) is established to simulate the cell penetration process. The effectiveness of the model is then verified through visual detection and comparison of cell deformation with experimental data. Next, various mechanical responses are analyzed, considering the influence of parameters, such as the radius and shape of the injection micropipettes, material properties, and size of the cells. Finally, the relationship between the intracellular stress and the cell penetration depth of biological cells is obtained. The evaluation results will be applied to develop optimized operation plans, enhancing the efficiency and safety of the cell penetration process.

The Molecular Design and Electroluminescent Performance of Near-Infrared (NIR) Iridium(III) Complexes Bearing Isoquinoline-, Phthalazine- and Phenazine-Based Ligands.

Near-infrared (NIR) light has characteristics of invisibility to human eyes, less background interference, low light scattering, and strong cell penetration. Therefore, NIR luminescent materials have significant applications in imaging, sensing, energy, information storage and display. The development of NIR luminescent materials thus has emerged as a highly dynamic area of research in the realm of contemporary materials. To date, NIR luminescent materials are roughly divided into inorganic materials and organic materials. Compared with inorganic materials, organic NIR luminescent materials have become a hot research topic in recent years due to their rich sources, easy control of structure, simple preparation process, low cost, and good film-forming properties. Among them, iridium(III) [Ir(III)] complexes exhibit excellent properties such as thermal stability, simple synthesis, easy color modulation, short excited state lifetimes, and high brightness, thus becoming one of the ideal luminescent material systems for preparing high-quality organic light-emitting diodes. Therefore, how to obtain Ir(III) complexes with NIR emission and high efficiency through molecular design is a necessary and promising research topic. This work reviews the research progress of representative NIR Ir(III) complexes bearing isoquinoline-, phenazine-, and phthalazine-based ligands reported in recent years and introduces the design strategies and electroluminescent performances of NIR Ir(III) complexes.

Development of an Intracellular Nitric Oxide-Donating Cell-Penetrating Polypeptide as an Immunogenic Cell Death Inducer.

Recently, nitric oxide (NO) has been shown to induce immunogenic cell death (ICD) in tumor cells through endoplasmic reticulum (ER) stress and mitochondrial outer membrane permeabilization (MOMP). However, NO is unstable, making direct delivery difficult. In this study, we developed a cell-penetrating polypeptide-based NO donor, poly(l-guanidine) (PLG). Given that the guanidine structure can be catalyzed by reactive oxygen species (ROS) to produce NO, helical PLG plays three roles: spontaneous cell penetration, intracellular ROS generation to produce NO, and induction of ICD. The results revealed that helical PLG generates NO inside the cell by self-inducible guanidine oxidation and that NO effectively elicits ICD by ER stress- and MOMP-dependent intertwined mechanisms.

Improved solubility and bioavailability of cyclolinopeptides by diacylglycerol in the β-cyclodextrin Pickering emulsions

Cyclolinopeptides (CLs) have anti-inflammatory, anti-osteoporosis, and anti-tumor effects, however, low water and oil solubility greatly limit their application. Herein, CLs-loaded β-cyclodextrin (β-CD) emulsions were prepared with different oil phases. The in vitro digestibility, cellular absorption, and anti-inflammatory effects were evaluated. Camellia oil diacylglycerol (CO DAG) showed enhanced dissolving ability for CLs due to high polarity. β-CD formed inclusion complexes with DAG through hydrogen bond and the emulsions showed smaller size and higher physical stability with 50 % (w/w) oil. The in vitro digestibility of the DAG emulsion was increased and the CLs' bioavailability was 13.6–fold higher than CLs in oil. The CLs–loaded Pickering emulsion digesta exhibited a higher nitric oxides (NO) inhibition rate (58.62 %) and Caco-2 cell penetration (3.09 × 10−6 cm/s). Therefore, emulsion formulated with β-cyclodextrin and DAG can effectively improve the solubility and bioavailability of CLs, which has significant potential for application in functional foods and pharmaceutical industry.

Turn-On Fluorescence Probe for Cancer-Related γ-Glutamyltranspeptidase Detection.

The design and development of fluorescent materials for detecting cancer-related enzymes are crucial for cancer diagnosis and treatment. Herein, we present a substituted rhodamine derivative for the chromogenic and fluorogenic detection of the cancer-relevant enzyme γ-glutamyltranspeptidase (GGT). Initially, the probe is non-chromic and non-emissive due to its spirolactam form, which hinders extensive electronic delocalization over broader pathway. However, selective enzymatic cleavage of the side-coupled group triggers spirolactam ring opening, resulting in electronic flow across the rhodamine skeleton, and reduces the band gap for low-energy electronic transitions. This transformation turns the reaction mixture from colorless to intense pink, with prominent UV and fluorescence bands. The sensor's selectivity was tested against various human enzymes, including urease, alkaline phosphatase, acetylcholinesterase, tyrosinase, and cyclooxygenase, and showed no response. Absorption and fluorescence titration analyses of the probe upon incremental addition of GGT into the probe solution revealed a consistent increase in both absorption and emission spectra, along with intensified pink coloration. The cellular toxicity of the receptor was evaluated using the MTT assay, and bioimaging analysis was performed on BHK-21 cells, which produced bright red fluorescence, demonstrating the probe's excellent cell penetration and digestion capabilities for intracellular analytical detection. Molecular docking results supported the fact that probe-4 made stable interactions with the GGT active site residues.

Rapid precision targeting of nanoparticles to lung via caveolae pumping system in endothelium.

Modern medicine seeks precision targeting, imaging and therapy to maximize efficacy and avoid toxicities. Nanoparticles (NPs) have tremendous yet unmet clinical potential to carry and deliver imaging and therapeutic agents systemically with tissue precision. But their size contributes to rapid scavenging by the reticuloendothelial system and poor penetration of key endothelial cell (EC) barriers, limiting target tissue uptake, safety and efficacy. Here we discover the ability of the EC caveolae pumping system to outpace scavenging and deliver NPs rapidly and specifically into the lungs. Gold and dendritic NPs are conjugated to antibodies targeting caveolae of the lung microvascular endothelium. SPECT-CT imaging and biodistribution analyses reveal that rat lungs extract most of the intravenous dose within minutes to achieve precision lung imaging and targeting with high lung concentrations exceeding peak blood levels. These results reveal how much ECs can both limit and promote tissue penetration of NPs and the power and size-dependent limitations of the caveolae pumping system. This study provides a new retargeting paradigm for NPs to avoid reticuloendothelial system uptake and achieve rapid precision nanodelivery for future diagnostic and therapeutic applications.

Brassinosteroids mediate arbuscular mycorrhizal symbiosis through multiple potential pathways and partial identification in tomato

Currently, little is known regarding the specific processes through which brassinosteroids (BR) affect arbuscular mycorrhizal (AM) symbiosis. Understanding this relationship is vital for advancing plant physiology and agricultural applications. In this study, we aimed to elucidate the regulatory mechanisms of BR in AM symbiosis. According to the log2 fold change-value and adjP-value, we integrated the common differentially expressed genes (DEGs) in maize (Zea mays L.) treated with BR and AM, Arabidopsis (Arabidopsis thaliana) mutants deficient in BR receptors, and tomato (Solanum lycopersicum) plants inoculated with AM fungi. In addition, we characterized the symbiotic performance of tomato plants with BR receptor defects and overexpression. The results indicated that the common differential genes induced by BR and AM were involved in metabolic processes, such as cell wall modification, cytoskeleton remodeling, auxin and ethylene signaling, photosynthesis, mineral nutrient transport, and stress defense. Specifically, these include the BR1 gene, which modifies the cell wall. However, the fungal colonization rate of BR receptor-deficient tomato plants was significantly reduced, and the total phosphorus concentration was increased. Conversely, the performance of the overexpressing tomato transformation plants demonstrated a significant contrast. Additionally, the mild rescue of mycorrhizal attenuation in mutants treated with exogenous BR suggests the possibility of direct feedback from BR synthesis to AM. Notably, the cell wall modification gene (SlBR1) and calcium spike gene (SlIPD3) were induced by both BR and AM, suggesting that BR may influence cell penetration during the early stages of AM colonization. Synthesis: Our results demonstrated that BR positively regulates AM symbiosis through multiple pathways. These findings pave the way for future research, including isolation of the individual contributions of each pathway to this complex process and exploration of possible agricultural applications.

Next-generation cell-penetrating antibodies for tumor targeting and RAD51 inhibition.

Monoclonal antibody therapies for cancer have demonstrated extraordinary clinical success in recent years. However, these strategies are thus far mostly limited to specific cell surface antigens, even though many disease targets are found intracellularly. Here we report studies on the humanization of a full-length, nucleic acid binding, monoclonal lupus-derived autoantibody, 3E10, which exhibits a novel mechanism of cell penetration and tumor specific targeting. Comparing humanized variants of 3E10, we demonstrate that cell uptake depends on the nucleoside transporter ENT2, and that faster cell uptake and superior in vivo tumor targeting are associated with higher affinity nucleic acid binding. We show that one human variant retains the ability of the parental 3E10 to bind RAD51, serving as a synthetically lethal inhibitor of homology-directed repair in vitro. These results provide the basis for the rational design of a novel antibody platform for therapeutic tumor targeting with high specificity following systemic administration.

Evaluation of the safety and efficacy of skin penetration enhancer Putocrin®.

Substances that can efficiently enhance skin penetration while exerting no adverse effect are useful for drug and cosmetics formulation. To investigate the safety and enhance skin penetration efficacy of Putocrin®, a combination containing 2% isosorbide dimethyl ether, 1% pentanediol, and 0.5% inositol. An invitro keratinocyte cell assay using 3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide (MTT), and an invitro EpiKutis® skin study adopted hematoxylin and eosin staining, immunostaining, and liquid chromatography-mass spectrometry (LC-MS) analysis were carried out to investigate the safety of Putocrin®. A pigskin-Franz cell system experiment applied high-performance liquid chromatography (HPLC) to compare the skin penetration efficiency of fluorescein isothiocyanate (Fitc)-labeled tranexamic acid with or without the assistance of Putocrin®. The safety and efficacy of Putocrin® was further evaluated on zebrafish embryos. The MTT assay showed that Putocrin® at concentration ≤2.5% did not significantly affect cell viability. The invitro EpiKutis® skin study revealed that 2.5% Putocrin® did not affect skin morphology, filaggrin content, ceramide/protein, or fatty acid/protein ratios, but significantly increased loricrin content by 86.00% (p < 0.001). The pigskin-Franz cell penetration experiment demonstrated that Fitc-labeled tranexamic acid could barely penetrate the skin (with penetration rate of 1.121%), while Putocrin® significantly enhanced the penetration rate up to 83.983%, which was close to unlabeled tranexamic acid (90.013%). The zebrafish embryo study showed that 2.5% Putocrin® did not exert observable toxicity and obviously assisted the skin penetration of Fitc-labeled tranexamic acid into fish embryos. These results indicate the strong enhancing skin penetration potency of Putocrin®. This study demonstrated the safety as well as the strong enhancing skin penetration potency of Putocrin® for cosmetics formulation use.

Investigating the Impact of Pore Size and Specification on Soft Tissue Ingrowth in 3D-Printed PEEK Material.

Bone pelvis tumor resection and reconstruction is a complex surgical procedure that poses challenges in soft tissue reconstruction despite advancements in stabilizing pelvic structure. This study aims to investigate the potential of using Polyetheretherketone (PEEK) material in repairing and reconstructing soft tissues surrounding pelvic implants. Specifically, the study focuses on exploring the effectiveness of 3D printed porous PEEK material in promoting cell growth and adhesion. The interaction between PEEK materials with different pore sizes (200, 400, 600µm) and different specifications (through-hole (T)/non-through-hole (C)) is evaluated by cell experiments and animal experiments. The soft tissue ingrowth potential of PEEK materials is evaluated by cell growth and adhesion observation. The findings indicate that PEEK material, particularly the T400 variant, exhibits stronger interaction with muscle tissue compared to its interaction with bone and fibrous tissue. The moderately sized pores present in the T400 material facilitate enhanced cell adhesion and penetration, thereby promoting cell growth and differentiation. PEEK materials with through-hole structures show promise for applications involving the repair and reconstruction of soft tissues and muscle tissue. The study provides valuable insights into the development and application of biomedical materials, specifically PEEK, contributing to the advancement of pelvic tumor resection and reconstruction techniques.

Amphiphilic shuttle peptide delivers base editor ribonucleoprotein to correct the CFTR R553X mutation in well-differentiated airway epithelial cells.

Base editing could correct nonsense mutations that cause cystic fibrosis (CF), but clinical development is limited by the lack of delivery methods that efficiently breach the barriers presented by airway epithelia. Here, we present a novel amphiphilic shuttle peptide based on the previously reported S10 peptide that substantially improved base editor ribonucleoprotein (RNP) delivery. Studies of the S10 secondary structure revealed that the alpha-helix formed by the endosomal leakage domain (ELD), but not the cell penetrating peptide (CPP), was functionally important for delivery. By isolating and extending the ELD, we created a novel shuttle peptide, termed S237. While S237 achieved lower delivery of green fluorescent protein, it outperformed S10 at Cas9 RNP delivery to cultured human airway epithelial cells and to pig airway epithelia in vivo, possibly due to its lower net charge. In well-differentiated primary human airway epithelial cell cultures, S237 achieved a 4.6-fold increase in base editor RNP delivery, correcting up to 9.4% of the cystic fibrosis transmembrane conductance regulator (CFTR) R553X allele and restoring CFTR channel function close to non-CF levels. These findings deepen the understanding of peptide-mediated delivery and offer a translational approach for base editor RNP delivery for CF airway disease.