Retinoblastoma (RB) is a rare pediatric retinal cancer. Due to financial and medical limitations, many children with retinablastoma (RB) only receive therapy when the disease has progressed to the metaphase and advanced clinical stages, which increases the risk of blindness and impairment. Despite the fact that there are numerous methods for treating RB, some children do not respond well to treatment for a variety of reasons. Natural medicines produced from plants are frequently employed in the research of RB and have demonstrated clear therapeutic effects in the treatment of a variety of malignancies. In order to offer suggestions for the clinical usage of these medications as well as the creation of novel therapeutic pharmaceuticals, we examine natural compounds produced from plants that are employed in the investigation of anti-RB.

RB research makes substantial use of natural chemicals derived from plants, which have shown promising therapeutic advantages in the treatment of many cancers. To provide recommendations for the clinical application of these drugs and the development of new therapeutic drugs, we analyze natural chemicals derived from plants that are used in anti-RB research. It has also been demonstrated that certain naturally occurring chemicals, in addition to conventional anticancer medications, are useful in treating RB. These consist of sterol derivatives (such as ursolic and oleanolic acid), naphthoquinones (such as β-lapachone), and catechol derivatives (such as curcumin). Our main objective is to explore natural novel therapeutics for RB and make use of these natural active moiety in the formulation of the development of a nano drug delivery system.

As one of the principal etiological determinants of irreversible progressive dementia, the pathogenesis of Alzheimer's disease (AD) is predominantly ascribed to the intracellular accumulation of neurofibrillary aggregates composed of hyperphosphorylated tau proteins within limbic and cortical regions, alongside the extracellular deposition of dense/diffuse β-amyloid neuritic plaques. AD represents a multifactorial, conformational, neurodegenerative disorder with a predominant proteinopathic character, exhibiting an insidious onset and an irreversible progression.

The selected experimental model, the 5xFAD mouse, exhibits severe amyloidosis, harboring five pathogenic mutations within the human transgenes APP (amyloid precursor protein) and PSEN1 (presenilin-1), leading to a fulminant disease progression. The experimental cohorts comprised a negative control, a positive control treated with galantamine (3mg/kg), and two groups per tested extract, corresponding to the two predefined concentrations (50mg/kg and 100mg/kg). The impact on cognitive performance was assessed through a battery of behavioral tests: Y-maze test (YMT), open-field test (OFT), novel object recognition test (NORT), elevated plus maze test (EPM), forced swimming test (FST), and radial arm maze test (RAM). The pharmacokinetic profiles of the investigated compounds were analyzed in silico using computational tools such as SwissADME and pKCSM.

The study highlights the neuroprotective potential of Rubus fruticosus extracts, demonstrating a significant enhancement in cognitive performance and exploratory activity across behavioral tests, alongside a marked reduction in stress levels, indicative of anxiolytic and antidepressant properties. In silico analyses revealed favorable pharmacokinetic attributes conducive to ameliorating memory deficits, including the capacity of the compounds to cross the blood–brain barrier, promising interactions with key molecular targets, and a bioactivity profile suggestive of drug-like potential.

The findings suggest that Rubus fruticosus extracts exhibit promising pharmacokinetic profiles and neuroprotective pharmacological potential, underscoring the imperative for further multifaceted, multidisciplinary investigations to validate their underlying mechanisms and clinical applicability.

Introduction

Recent advancements in molecular profiling technologies based on liquid media (so-called liquid biopsy) have achieved a significant increase in detection sensitivity, enhancing our power of investigation for biofluids and suggesting their potential integration into pivotal diagnostic and predictive molecular analysis, such in non-small-cell lung cancer. Pre-analytical variables play a crucial role in the accuracy of analysis results. Evidence suggests that supernatant fluid obtained from cytological sample centrifugation is enriched with nucleic acids and less susceptible to nucleic acid degradation during processing compared to cytoblock samples. This study reports preliminary results from a recent workflow implementation of liquid biopsy in a routine molecular biology laboratory within the context of a validation study.

Methods

Eleven cytological samples of lung adenocarcinoma and corresponding cytoblock slides underwent a morphological analysis and review in terms of cellularity, cell integrity, necrosis/debris, and the presence of artifacts. Following NSG analysis of both the effusion fluid and cytoblock, data analysis and a comparison of the results were performed, focusing on concordance and the pre-analytical and analytical variables observed in the two groups.

Results

Molecular profiling led to the detection of an actionable alteration in all patients, with more robust results, evaluated in terms of variant allele frequency (VAF) in the supernatant group (53,3%) in comparison with the cytoblock group (26,49 %). This study resulted in a 100% concordance in molecular profiling between the cytoblock and supernatant analyses, with the latter showing higher yields in nucleic acid extraction, suggesting the possibility of achieving clinically useful results for running molecular profiling on biofluid materials.

Conclusions

Liquid biopsy technology has gained enough robustness to be a useful tool in routine analysis.

Biofluid applications in tumor molecular profiling is a promising field of exploration with outstanding potential in the case of insufficient cytologic material, contributing to improved patient management and thus avoiding repetitive procedures and optimizing the overall efficiency and cost-effectiveness of diagnostic practices.

Introduction: Chronic inflammation and mitochondrial dysfunction are key drivers of endothelial aging and cardiovascular diseases. Sodium-glucose co-transporter 2 (SGLT2) inhibitors are known for their cardiovascular protective effects. However, the underlying molecular mechanisms, especially their impact on autophagy and mitochondrial function, remain unclear. This study aims to investigate the effects of SGLT2 inhibitors on mitochondrial function and autophagy in endothelial cells.

Methods: Human endothelial cells derived from induced pluripotent stem cells (iPS-ECs) (n = 12) were activated with 10 ng/ml TNF-α for 24 hours to create a pro-inflammatory environment and were then treated with 1 µM Empagliflozin for an additional 24 hours. Western blot analyses were performed to assess the expression and activity of key mitochondrial and autophagy markers, e.g., AMPK activity, SOD2 expression and autophagy flux.

Results: Pro-inflammatory treatment with TNF-α resulted in mitochondrial dysfunction and a disruption of autophagy. AMPK, a key cellular energy sensor and regulator of mitochondrial homeostasis, showed no significant differences in pAMPK/AMPK ratios among the investigated groups. SOD2, an anti-oxidative enzyme critical for mitochondrial antioxidant defense, exhibited lower expression in TNF-α-treated cells compared to untreated cells. Empagliflozin treatment partially recovered the expression of SOD2. Conversely, the autophagy flux (LC3II/I ratio) was elevated after pro-inflammatory treatment but subsequently decreased following Empagliflozin treatment.

Conclusion: Empagliflozin exhibits protective effects on endothelial inflammation by modulating mitochondrial function and autophagy activity. While Empagliflozin partially restored SOD2 expression, the AMPK activity was not changed. Additionally, SGLT2 inhibition recovered the autophagy flux to the basal levels, suggesting a regulatory effect of Empagliflozin on autophagy. These findings indicate that SGLT2 inhibitors may play a role in mitigating inflammation-induced mitochondrial dysfunction and autophagy disruption, offering potential therapeutic benefits for endothelial aging and cardiovascular diseases.

Introduction: Gene therapy offers a promising approach for treating various diseases, yet its success hinges on efficient and safe gene delivery systems. Poly(β-amino esters) (PBAE) is an excellent non-viral gene carrier which has the advantages of easy synthesis and various chemical structure. However, there are some obvious shortcomings, such as cytotoxicity and limited penetration in tissues. To address these issues, we developed PEGylated PBAE (PBAE-E) with tailored chain lengths and contents to enhance both transfection and biocompatibility.

Methods: We synthesized PBAE-E by incorporating short-chain PEG into the PBAE structure via Michael addition polymerization. The materials were characterized using 1H-NMR and FT-IR. PBAE-E nanoparticles were formed by complexing with the GFP plasmid and were analyzed for size, morphology (DLS, TEM), and DNA encapsulation (agarose gel electrophoresis). In vitro transfection was assessed in multiple cancer cell lines, and cytotoxicity was evaluated using the MTT assay. In vivo imaging of mouse vaginas was used to investigate whether the introduction of PEG chains could increase the mucosal permeability of nanoparticles.

Results: PBAE-E was synthesized successfully, as confirmed by 1H-NMR and FT-IR. PBAE-E could compact the plasmid into spherical nanoparticles with a diameter of about 250 nm. PBAE-E nanoparticles demonstrated improved transfection efficiency and reduced cytotoxicity compared to unmodified PBAE. Notably, PBAE-E4-5 and PBAE-E9-5 exhibited the best performance. In vivo imaging in mice revealed an enhanced mucosal permeability of PBAE-E, suggesting improved tissue penetration.

Conclusion: Our study demonstrates that PEG-modified PBAEs can significantly enhance plasmid transfection efficiency and biocompatibility compared to unmodified PBAEs. The PEGylation strategy provides a promising avenue for improving non-viral gene therapy.

Introduction: Sarcomas are mesenchymal tumors often associated with therapeutical resistance and recurrence. Immunotoxins (ITs) represent a promising strategy, joining the specificity of carriers to the cytotoxicity of plant toxins (Ribosome-Inactivating Protein, RIP). Three-dimensional models represent a ground-breaking advance in cancer therapy, especially for predictive high-throughput drug screening. Our study aimed to comparatively evaluate the IT efficacy to TfR1-, EGFR1- and Her2-expressing sarcoma cells, in 2D (adherent cells) and 3D (spheroids) models. Methods Three different ITs were obtained by chemical conjugation of the RIP Saporin to Transferrin (Tf-IT) and of the RIP Ocymoidine to α-EGFR1 (α-EGFR1-IT) and to α-Her2 (α-Her2-IT). The IT efficacy was evaluated on rhabdomyosarcoma (RD18) and osteosarcoma (U2OS), in dose–response viability experiments (MTS colorimetric assay for the 2D model and ATP luminometric assay for the 3D model) and using a Caspase 3/7 activation assay. Results: In the RD18 and U2OS 2D and 3D models, the RIP cytotoxicity was significantly enhanced after chemical conjugation to carriers. The ITs strongly reduced cell viability, showing EC₅₀ values 1.3-2.3 logs lower than the corresponding unconjugated RIP and carrier. Despite the increased complexity and penetration difficulty characterizing 3D models, ITs were also effective in 3D models, showing similar EC₅₀ compared to 2D ones. All the ITs were able to strongly activate Caspase 3/7. Conclusions: Our data showed the strong efficacy of all tested ITs. The strategy of targeting different antigens on tumour-cells' surfaces may also allow us to kill clones expressing mutated target antigens. RIPs trigger different cell-death mechanisms, avoiding the selection of drug-resistant neoplastic clones. Thus, our results support the potential use of ITs as a therapeutic strategy in the treatment of sarcomas. Further experiments will be necessary to evaluate "in vivo" the tolerable doses of ITs and to compare their efficacy with the drugs approved by the FDA for sarcoma therapy.

Antibodies are extensively used in medicine for therapeutic and diagnostic purposes. They are traditionally purified by 2-3 chromatographic steps, where the first exploits a protein–ligand called Protein A. The latter is used by most, if not all, pharmaceutical companies since it binds to diverse antibody types with high affinity and specificity and leads to high process yields (>90%) and purity (>95%) within a single chromatographic step. However, as the antibody concentration increases, antibody purification becomes challenging, since a single industrial-scale Protein A column cannot capture the entire antibody population. Therefore, we developed an alternative, non-chromatographic antibody purification platform that quantitatively captures antibodies regardless of their concentration. The studied strategy relied on aromatic [metal/chelator] complexes rather than on resins conjugated to Protein A. Two aromatic complexes were evaluated and were composed of a commercially available chelator called bathophenanthroline (batho) bound to either Fe²⁺ or Zn²⁺. Such water-insoluble complexes (i.e., [(batho)₃:Fe²⁺] or [(batho)₃:Zn²⁺]) allow for (a) an efficient separation of IgG antibodies from their mixture with IgM antibodies or (b) the purification of an Fc-fusion protein composed of the Fc-domain of an IgG1 bound to the enzyme acetylcholinesterase. The process yield (>85%, by densitometry) and purity (>95%, by SDS-PAGE) values are encouraging. The recovered targets were monomeric (by dynamic light scattering and Native-PAGE) and preserved their secondary structure (by circular dichroism) and catalytic activity. Purification was achieved at pH~7, thereby circumventing the exposure of antibodies to harsh acidic conditions and, hence, antibody aggregation. The relevance of the developed platform for industrial-scale purification of therapeutic-grade monoclonal antibodies (mAbs) will be further discussed.

ABC transporters are fascinating proteins responsible for transporting a variety of substrates through the hydrolysis of ATP. Some ABC transporters are multidrug-resistant (MDR), commonly associated with human cancers and pathogenic microbes. Their ability to transport toxic substances and drugs across membranes, even against the concentration gradient, leads to a reduced concentration of drugs inside cells, which diminishes the drug's effectiveness. Natural compounds, such as polyphenols and flavonoids, have anticancer properties. By inhibiting ATP hydrolysis, they can potentially inhibit MDRs in cancer cells, reduce the activity of these proteins, and enhance the therapeutic effects of anticancer drugs. In this study, we investigate the inhibitory effects of 77 of these compounds on the nucleotide-binding domains (NBDs) of ABCC1 (MRP1) by using molecular docking and molecular dynamics (MD) simulation. The results indicate that five compounds, (-)-catechingallat, limonin, naringin, rhoifolin, and robinin with -7.8,-8.5, -8.3, -8.3, and -8.5 binding affinity in NBD1 and -7.1, -7.9, -8.2, -7.9, and -7.7 in NBD2, respectively, in comparison with ATP with -6.8 and -7.1 in NBD1 and NBD 2, respectively, had high binding affinities and occupied the same binding site, namely Asp793 and Tyr831 in NBD1 and Arg1445 in NBD2, with ATP. A molecular dynamics trajectory analysis of the NBDs and ligands revealed these domains were stable throughout 200ns MD simulations. The MD simulations confirm the stability of the complex formed by the interaction of five ligands with NBD, characterized by structural compactness and minimal to no fluctuations. This in silico study offers key information for developing potential ATP inhibitors that NBDs could be the suitable binding site for the flavonoid family. The discovery of novel MDR-inhibiting compounds has the potential to make cancer treatment more effective for all types of cancers, making it a comprehensive solution to drug resistance.

Introduction: Ribosome-inactivating proteins (RIPs) are toxic plant enzymes. Several RIPs have been conjugated to specific carriers to obtain immunotoxins, which gave promising results in pre-clinical and clinical studies. However, some adverse effects were reported, mainly immunogenicity. Recently, a new RIP from the edible plant Salsola soda, named sodin-5, has been identified and characterized. The aim of this work was to evaluate the sodin-5 cytotoxic profile on intestinal cell lines, comparing it with saporin, one of the best known and most studied RIPs for the construction of immunotoxins.

Methods: Different endpoints were considered to evaluate RIP cytotoxicity on colon adenocarcinoma Caco-2 and HT29 cell lines: cell viability experiments through tetrazolium salt reduction and cell death through cytofluorimetric analysis of AnnexinV/PI positivity. The effect of the RIPs on the membrane integrity was monitored by Trans-Epithelial- Electrical Resistance (TEER) measurements on Caco-2 monoculture and/or Caco-2/HT29 co-culture. RIP immunological cross-reactivity was evaluated through ELISA.

Results: Our results showed that sodin-5 had high cytotoxicity, very similar to that of saporin. Both RIPs reduced cell viability after 72 h, with EC₅₀s in the nM range. Apoptosis was the main cell death pathway triggered by RIPs, and no necrosis involvement was observed. The lack of necrosis is very important as apoptosis does not cause inflammation. Sodin-5 and saporin significantly reduced TEER values both in Caco-2 cell mono- and Caco-2/HT29 co-culture in comparison to untreated controls. Finally, no immunological cross-reactivity was observed with saporin anti-serum.

Conclusions: Since sodin-5 and saporin had similar cytotoxic activities, sodin-5 could be proposed for further study as a potential toxic payload in immunotoxin construction to overcome some adverse effects of saporin and other RIPs. Theoretically, RIPs from edible sources should be less immunogenic and better tolerated by humans; therefore, it could represent a better candidate for immunotoxin-based experimental therapy.

Introduction: Platinum-based drug resistance is intricately associated with a disordered tumor metabolic–immune microenvironment (TMIME). This study sought to develop a combined strategy for TMIME reprogramming to enhance cisplatin sensitivity.

Methods: A chemo-gene co-delivery nanoparticle (NP) was engineered using poly(β-amino ester) (PBAE) to encapsulate the cisplatin prodrug (Pt^IV) and CRISPR/Cas9-PKM2 plasmid. The morphology and elemental composition of PPPt^IV NPs were visualized using a transmission electron microscope (TEM) and field-emission TEM. Hydrodynamic size, polydispersity index, and zeta potential were measured using dynamic light scattering. The viabilities and apoptosis effects were investigated in SCC7 and Cal27 cells using CCK-8 and Annexin V/PI double-staining assays. In vivo biodistribution and antitumor efficacy were investigated in SCC7 tumor-bearing C57BL/6 mice.

Results: PPPt^IV NPs exhibited a uniform, near-spherical morphology with a positively charged surface and an average diameter of approximately 150 nm. PPPt^IV NPs possessed good stability and pH-responsive release. Mechanically, PPPt^IV NPs were efficiently internalized through endocytosis and then escaped from lysosomes and released their components. Pt^IV was reduced to cisplatin (Pt^II) via GSH depletion, leading to increased DNA damage and ROS levels to induce apoptosis. CRISPR/Cas9-mediated PKM2 knockdown significantly reduced lactic acid production via inhibiting the Warburg effect while downregulating PD-L1 and HIF-1α levels. These metabolic alterations remodeled the tumor immune microenvironment by promoting dendritic cell maturation, polarizing macrophages to the M1 phenotype, and modulating cytokine release (IFN-γ, TNF-α, IL-12, and IL-10), thereby initiating T cell-mediated antitumor immunity. As a result, PPPt^IV NPs achieved the chemo-immunotherapy goal. Compared to cisplatin alone, PPPt^IV NPs achieved superior antitumor efficacy against both in situ and recurrent tumors with less nephrotoxicity in vivo.

Conclusions: The combined chemo-immunotherapy nanohybrids address the limitations of cisplatin, including resistance and adverse effects, and demonstrate significant potential for clinical application in patients resistant to cisplatin.