Search Results (419)

Obesity is well-known as a major risk factor for metabolic disorders, and natural compounds are being explored as alternatives to conventional therapies. While Centella asiatica is well known for its medicinal and dietary benefits, the biological activities of Giant Centella asiatica (GCA), especially when extracted with mineral-rich lava seawater, remain poorly characterized. This study aimed to evaluate the anti-adipogenic and lipid-metabolism-regulating effects of a novel GCA extract (GCA-LS-90) and its ability to stimulate GLP-1 secretion in vitro. GCA-LS-90 significantly inhibited lipid accumulation in 3T3-L1 adipocytes by up to 24.3% at 200 µg/mL (p < 0.001). It downregulated adipogenic transcription factors (C/EBPβ, C/EBPα, PPARγ) and lipogenic regulators (SREBP1c, FAS, G6PD, ME), while upregulating KLF2 (all p < 0.001). Western blotting confirmed reduced SREBP1c and SREBP2 protein expression, increased phosphorylation of AMPKα/ACC, and enhanced HSL activity (p < 0.05–0.001). In STC-1 cells, GCA-LS-90 increased GLP-1 secretion (53.5 pmol/L at 90 µg/mL vs. 41.3 pmol/L in control, p < 0.001). The major compounds, 3,5- and 4,5-di-O-caffeoylquinic acids, reproduced these effects. In conclusion, GCA-LS-90 modulated adipogenesis-, lipid-metabolism-, and GLP-1 secretion-related pathways in vitro, suggesting its potential as a functional ingredient for obesity management. Further in vivo studies are needed to confirm efficacy and translational relevance. Full article

Mechanosensitive Piezo1 channels participate in regulating pain sensitivity, insulin secretion, and vascular tension; however, their expression in the autonomic paraventricular nucleus (PVN) and role in modulating sympathetic outflow and cardiovascular function remain unstudied. In this study, unilateral PVN microinjection of the Piezo1 channel blocker Dooku1 (0.1, 1, 10, 100, and 200 pmol) administered to anesthetized male rats increased renal sympathetic nerve activity (RSNA) and mean artery pressure (MAP) in a dose-dependent manner, with maximum increases of 93 ± 30% (p < 0.0001) and 21 ± 5 mmHg (p < 0.0001), respectively, elicited by Dooku1 at 100 pmol. Similarly, PVN microinjection of the peptide Piezo1 channel blocker GsMTx4 (1 nmol) significantly increased RSNA (p < 0.001) and MAP (p < 0.0001). Conversely, PVN-microinjected Piezo1 channel activators Yoda1 (5 nmol) and Jedi2 (5 nmol) did not significantly alter RSNA or MAP. Western blot and qRT-PCR analyses of the hypothalamic PVN showed abundant Piezo1 mRNA and protein expression. Immunofluorescence detection showed that Piezo1 was expressed in pre-sympathetic PVN neurons with axons projecting to the rostral ventrolateral medulla. We conclude that Piezo1 channels expressed in the autonomic PVN neurons play an important role in regulating sympathetic outflow and cardiovascular function. Full article

Introduction: Recurrent hypoglycemia occurring prior to the initiation of insulin therapy is an uncommon finding at the onset of pediatric type 1 diabetes mellitus (T1D). Aim: The aim of this study was to describe an unusual presentation of T1D onset characterized by recurrent hypoglycemia in a pediatric patient and to provide an updated review of the literature on hypoglycemic episodes occurring before insulin initiation in children with new-onset T1D. Case report: We present the case of a child who exhibited recurrent fasting hypoglycemia and postprandial hyperglycemia at the onset of T1D. Clinical findings also included persistently elevated glucagon levels (66–93 pmol/L; normal values [n.v.] 3–60); markedly elevated glycated hemoglobin (98 mmol/moL); low C-peptide levels (0.05 nmol/L); and a slight positivity for antibodies to glutamic acid decarboxylase (38.3 KUI/L; n.v. < 10). Review of the literature: A narrative review of the literature was conducted using the PubMed (MEDLINE) database to identify studies reporting hypoglycemia at the onset of T1D in pediatric patients prior to insulin initiation. The search included combinations of the terms hypoglycemia, new-onset, type 1 diabetes, child, and adolescent, with an exclusion of insulin-treated cases. We also explore potential pathogenetic mechanisms underlying this unusual presentation, including alpha-cell acute damage or dysregulation and loss of intra-islet paracrine signaling. Conclusions: Among individuals with T1D onset, episodes of recurrent hypoglycemia before the initiation of insulin therapy have been reported in a few studies. The presented case expands the clinical spectrum of early T1D and suggests that early alpha-cell dysfunction may contribute to atypical glycemic patterns during disease onset. Full article

Background/Objectives: Vitamin B₁₂ (cobalamin) and folate (vitamin B₉) are essential cofactors in one-carbon metabolism required for DNA synthesis, methylation, and genomic stability. Deficiencies in these nutrients can cause megaloblastic anemia, neurological dysfunction, and hyperhomocysteinemia, linking micronutrient imbalance to cardiovascular and neurocognitive outcomes. Population-based surveillance of these biomarkers provides insight into nutritional trends and supports analytical standardization. Methods: This retrospective study included all routine plasma (P) vitamin B₁₂ and folate measurements performed at Uppsala University Hospital from 2005 to 2024 (n = 647,302 and 578,509, respectively). Data were extracted from the laboratory information system and summarized using annual medians, percentile distributions, and coefficients of variation (CV). Linear regression was used to validate the method comparison and assess the impact of the 2021 transition from the Abbott Architect to the Roche cobas platform. Descriptive statistics summarized the temporal and seasonal patterns of P-vitamin B₁₂ and P-folate. Results: Median P-vitamin B₁₂ concentrations remained stable (340–370 pmol/L; median CV = 4.6%), while P-folate increased from 10.5 to 15.5 nmol/L (median CV = 12.9%) from 2005 to 2024. Low P-folate (<7 nmol/L) was observed in 7.1% of measurements and low or borderline P-vitamin B₁₂ (<250 pmol/L) in 22.6%. Females exhibited slightly higher concentrations of both analytes. Although no clear seasonal pattern was observed, small biological effects cannot be excluded. Sample volumes decreased during the summer. The transition to Roche assays introduced measurable methodological shifts, particularly for P-folate. Conclusions: Levels of P-vitamin B₁₂ remained stable over two decades, while P-folate status increased modestly. This reflects both dietary influences and assay-related differences following the 2021 platform transition. Continuous surveillance of biomarker medians provides a sensitive tool for detecting analytical drift and for monitoring long-term nutritional trends in clinical populations. Full article

To help assess the relationship between N-terminal pro-B-type natriuretic peptide (NT-proBNP) and feline cardiogenic arterial thromboembolism (ATE), the objective of this retrospective study was to compare plasma NT-proBNP concentrations between cats with cardiomyopathy that developed ATE (ATE group) and cats with occult cardiomyopathy that did not develop ATE within 1 year of testing (occult cardiomyopathy [OCM] group). Cats with cardiomyopathy and congestive heart failure (CHF) but no ATE (CHF group) were included for comparison. Cats with cardiomyopathy that had NT-proBNP testing were classified into ATE, OCM, or CHF groups. Clinical, echocardiographic, treatment, and NT-proBNP data from medical records were reviewed and compared among groups. A receiver operating characteristic curve was generated to develop a cutoff point for NT-proBNP. Cats were then compared based on this cutoff point. The ATE group (n = 25) had significantly higher NT-proBNP concentrations than the OCM group (n = 31; p < 0.001); there was no significant difference in NT-proBNP concentrations between the ATE and CHF groups (p = 0.92). The estimated optimal NT-proBNP cutoff point to separate OCM and ATE groups was 491 pmol/L (sensitivity = 96.0%, specificity = 93.5%). Cats with NT-proBNP > 491 pmol/L had a larger left atrium, thicker left ventricle, lower fractional shortening, and higher prevalence of spontaneous echogenic contrast and left atrial thrombi on echocardiography. These preliminary, hypothesis-generating findings suggest that NT-proBNP concentrations > 491 pmol/L may help detect cats with OCM at risk for ATE, but given the limitations of this retrospective study, prospective studies are needed to evaluate the potential utility of this measurement. Full article

Background/Objectives: Hypoaldosteronism is classified into “aldosterone deficit” (Aldo-D) and “aldosterone/mineralocorticoid resistance” (Aldo-R) based on etiopathogenic mechanisms. This distinction could be useful for guiding the treatment. However, no reliable methods have been established to differentiate these subtypes. We first aimed to assess whether aldosterone levels could help identify them when assessed in the setting of hyperkalemia or hyperreninemia. Methods: We conducted a retrospective analysis of eighty-four cases of hypoaldosteronism. Aldo-D and Aldo-R classification was based on the presence of clinical factors associated with aldosterone deficit and mineralocorticoid resistance, respectively. The accuracy of plasma aldosterone (PAC) to identify each type of hypoaldosteronism individually was evaluated using AUC-ROC analysis. Results: Aldo-D was identified in 66 (78.6%), and Aldo-R in 41 (48.8%) cases. Factors related to both subtypes were observed in forty-seven (56%) cases. AUC-ROC analysis of PAC measured during hyperkalemia showed low accuracy for detecting either subtype. During hyperreninemia, PAC accuracy was adequate for identifying Aldo-D but unsatisfactory for Aldo-R. Nevertheless, a PAC ≤ 60 pg/mL (6 ng/dL, ~166 pmol/L) during hyperkalemia and hyperreninemia yielded positive predictive values (PPV) of 94% and 100%, respectively, for Aldo-D, while a PAC value > 160 pg/mL (~443 pmol/L), particularly ≥ 200 pg/mL (20 ng/dL, ~550 pmol/L) in either condition had a PPV of 100% for Aldo-R. Conclusions: Although overall diagnostic accuracy was limited, extreme low and high PAC values (≤ 60 pg/mL or ≥ 200 pg/mL) may be suggestive of Aldo-D or Aldo-R, respectively, while intermediate values remain difficult to interpret due to substantial overlap. Full article

The oxidative potential (OP) of airborne particulate matter (PM) has emerged as a promising metric to assess the capacity of particles to induce oxidative stress and related health effects. Thus, ensuring the reliability and comparability of OP measurements is essential for accurate environmental and toxicological assessment. This study aimed to develop and evaluate a quality control approach for the dithiothreitol (DTT) assay used in OP determination. The DTT assay provides an estimation on how harmful PM can be to human health through oxidative stress, relating it to the consumption of DTT during the test period. Two experiments were conducted using the Standard Reference Material (SRM) 1648–Urban Particulate Matter (NIST, USA). The first assessed the effect of trichloroacetic acid (TCA) addition order and the feasibility of using SRM 1648 as a reference material. The second evaluated the stability of the SRM solution over a 63-day period. Statistical analysis (Mann–Whitney test) indicated that the order of TCA addition did not significantly affect OP values (p > 0.05). SRM 1648 solution determination showed high reproducibility (mean DTTₘ = 14.6 ± 2.4 pmol·min⁻¹·µg⁻¹), although a gradual increase in DTT metrics was observed over time, consistent with progressive dissolution. The results support the application of SRM 1648 as a reference material for DTT assay quality control, supporting methodological harmonization in OP determination, provided that a freshly prepared solution is used. Full article

Black crusts are degradation features found on stone buildings, offering valuable insights into local pollution sources. Their composition and structure reflect environmental conditions, making them important indicators for environmental and conservation studies. In this study, black crusts collected from funerary monuments in the Monumental Cemetery of Milan were comprehensively characterized using SEM-EDX, Raman spectroscopy, LIBS, and oxidative potential (OP) assays. SEM-EDX and Raman spectroscopy revealed extensive degradation of the substrate and the incorporation of pollutant-derived particles, with heavy metals such as Fe, Zn, and Pb detected in more than 90% of the samples. Correlation analysis proved effective in distinguishing major pollution sources, primarily vehicular and railway traffic, indicated by strong associations such as Zn–Mn (r = 0.896), Fe–Zn (r = 0.734), and Fe–Mn (r = 0.655), from minor sources linked to industrial emissions, reflected in correlations including Ti–Pb (r = 0.589), Pb–Cl (r = 0.702), and S–Pb (r = 0.661). Instead, LIBS analysis confirmed stratigraphic penetration of these elements beyond the surface layers, suggesting long-term accumulation. OP assays, applied here for the first time to black crusts, showed values between 0.5 and 3.0 pmol min⁻¹ µg⁻¹, indicating moderate oxidative reactivity linked to metal content. Overall, the findings contribute to a deeper understanding of pollution-driven stone decay and support the development of more effective diagnostic and conservation strategies. Full article

Background: Vitamin D is increasingly recognized as a key immunomodulatory nutrient, influencing innate and adaptive immune responses. While 25-hydroxyvitamin D [25(OH)D] is widely used to assess vitamin D status, the active form, 1,25-dihydroxyvitamin D [1,25(OH)₂D], may exert distinct effects on immune function. This study investigates the concentration-dependent and sex-specific relationships of both vitamin D metabolites with systemic inflammatory markers in a large clinical cohort. Objectives: To characterize the associations of 25(OH)D and 1,25(OH)₂D with total white blood cell (WBC) count and leukocyte subtypes, including neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Methods: We conducted a retrospective cross-sectional analysis of 125,537 adults (37.9% male; age 18–89 years) from routine laboratory diagnostics collected between 2014 and 2020 in Germany. Serum 25(OH)D and 1,25(OH)₂D were measured using standardized chemiluminescent immunoassays. Inflammatory markers were assessed via automated hematology. Multivariable-adjusted linear and non-linear regression models were used to assess associations, adjusting for age, sex, and season. Results: Neutrophils and Monocytes: Displayed U-shaped associations with both 25(OH)D and 1,25(OH)₂D. Neutrophil counts were lowest at 25(OH)D levels of ~40–60 nmol/L and increased significantly at both lower and higher extremes (p < 0.001). Lymphocytes: Inverse relationship with 25(OH)D (p < 0.001), and an inverse U-shaped relationship with 1,25(OH)₂D, peaking at ~90 pmol/L, with counts decreasing at both lower and higher levels (p < 0.001). Sex-specific analysis revealed that the relationship between 1,25(OH)₂D and lymphocyte count remained independent only in men, Eosinophils and Basophils: Demonstrated consistently negative correlations with both forms of vitamin D across all concentration ranges (p < 0.001). Conclusions: Our findings reveal distinct, concentration-dependent associations between vitamin D metabolites and leukocyte profiles, with evidence for nonlinear and sex-specific immunological effects. Both low and high levels of 25(OH)D and 1,25(OH)₂D were linked to increased neutrophil and monocyte counts, suggesting that vitamin D excess, like deficiency, may be linked to low-grade inflammation. These data are hypothesis-generating and suggest that personalized monitoring of vitamin D status may be relevant for future research on immune health, particularly in populations at risk for inflammatory or metabolic disease, but they do not provide a basis for clinical decision-making. Full article

The C1858T PTPN22 (R620W) variant has been implicated in the pathogenesis of several autoimmune disorders and represents a promising immunotherapeutic target for Type 1 diabetes. We have been implementing a novel immunotherapeutic approach based on the use of lipoplexes that deliver siRNA duplexes. The efficacy and safety of lipoplexes was previously demonstrated in vitro in halting variant expression in the peripheral blood of patients. Preclinical safety and efficacy must be ascertained in vivo in appropriate animal models before clinical investigations can be undertaken, according to regulatory authorities in Europe. In the light of the foregoing, this study aims to verify that lipoplexes against the murine Ptpn22-R619W, equivalent to the human PTPN22-R620W, could be used for animal experimentation. The murine fibroblast cell line L929 was transfected with the PF62-pLentiPtpn22-R619W plasmid. We designed specific siRNA duplexes for the Ptpn22-R619W allele and formulated them into cationic lipoplexes in order to halt variant expression in the transfected L929 cell line. Transfection of fibroblasts expressing R619W using lipoplexes resulted in efficient silencing at 100 pmol siRNA after 48 h post-transfection, reaching higher significant knockdown after 72 h. Lipoplexes efficiently suppress pathogenic Ptpn22 variant expression in vitro, supporting the feasibility of a pre-clinical platform for testing of in vivo lipoplexes in CRISPR-engineered NOD/ShiLtJ mice carrying the R619W mutation. Full article

Background: Metabolic bone disease (MBD) of prematurity predisposes extremely preterm and extremely low birth weight (ELBW) infants to atraumatic fractures. Evidence on fracture reduction after structured Bone Health Programs (BHPs) remains limited. Methods: We conducted a single-center retrospective cohort of NICU admissions (2014–2024) with gestational age < 28 weeks and/or birth weight < 1000 g, comparing a pre-program era with a standardized BHP that incorporated protocolized biochemical surveillance, a week 4 screening radiograph, optimized mineral targets, pharmacist review of parenteral minerals, and “handle-with-care” practices. The study aimed to evaluate whether implementation of a structured BHP reduced fracture incidence and improved biochemical and clinical outcomes in extremely preterm and ELBW infants. Prespecified effect measures were risk ratio (RR), risk difference (RD) with 95% confidence intervals, Fisher’s exact p values, and number needed to treat (NNT). Among infants with fractures, we compared clinical course and biochemical context across eras. Results: Of 708 eligible infants, 221 were born pre-program and 487 post-program with similar baseline characteristics. Fracture incidence decreased from 9.5% (21/221) to 1.64% (8/487); RR 0.17 (95% CI 0.08–0.38); RD −7.86 percentage points; p < 0.001; NNT ≈ 13. Among infants who fractured, length of stay was lower post-program (104.1 ± 28.3 vs. 172.0 ± 91.5 days). Peak alkaline phosphatase and parathyroid hormone were also lower in the post-program era (ALP 501.3 ± 71.2 vs. 972.5 ± 93.5 IU/L, p = 0.032; PTH 23.1 ± 12.5 vs. 38.4 ± 21.7 pmol/L, p = 0.027), whereas serum phosphate and 25 OH vitamin D did not differ significantly. The fracture burden per infant decreased following the BHP (1.50 ± 0.53 vs. 3.19 ± 3.08, p = 0.024). Age at first fracture was earlier post-program, consistent with scheduled imaging (48.4 ± 34.9 vs. 83.9 ± 37.3 days, p = 0.031). Conclusions: A structured BHP was associated with a large reduction in fracture incidence and more favorable biochemical profiles, together with shorter hospitalization among fracture cases. Program elements that combine scheduled imaging, biochemical triggers, nutritional optimization, parenteral mineral stewardship, and standardized handling may improve skeletal outcomes. Multicenter prospective evaluations should confirm generalizability and define core components. Full article

Background/Objectives: Ochratoxin A (OTA) is a widespread foodborne mycotoxin linked to chronic kidney disease of unknown etiology. Despite evidence from animal models showing OTA accumulation in the kidney, the molecular mechanisms underlying its renal disposition in humans remain only partially understood. Here, we identify human renal transporters responsible for OTA kidney accumulation, elimination, and establish Michaelis–Menten kinetics under matched conditions to directly compare transport mechanisms. We also aim to identify inhibition potential of these transport mechanisms with common dietary polyphenols. Methods: Mammalian cells and membrane vesicles overexpressing human renal transporters were used to screen and profile the uptake and efflux of OTA. Miquelianin, (-)-Epicatechin-3-O-gallate, myricetin, luteolin, and caffeic acid were tested as potential concentration-dependent transporter inhibitors. Results: We demonstrate that OTA is a substrate for human organic anion transporter (hOAT) 1 (K_m: 2.10 ± 0.50 μM, V_max: 396.9 ± 27.0 pmol/mg/min), hOAT3 (K_m: 2.58 ± 0.83 μM, V_max: 141.4 ± 30.3 pmol/mg/min), hOAT4 (K_m: 6.38 ± 1.45 μM, V_max: 96.9 ± 18.8 pmol/mg/min), and human organic anion transporting polypeptide (hOATP) 1A2 (K_m: 37.3 ± 6.2 μM, V_max: 801.0 ± 133.9 pmol/mg/min). Among efflux transporters, OTA was transported only by human breast cancer resistance protein (hBCRP), which has minimal renal expression. While none of the uptake transporters were potently inhibited (>90%) by polyphenols at 10 μM, luteolin inhibited hBCRP-mediated transport of OTA with an IC₅₀ of 22 μM and caffeic acid stimulated hBCRP-mediated efflux with an EC₅₀ of 713.8 μM, both of which are physiologically relevant intestinal lumen concentrations. Conclusions: Our results confirm that exposure to OTA will lead to renal accumulation and increased health risks in affected populations, necessitating increased scrutiny of our food sources. Full article

COVID-19 is a public health issue with a significant mortality rate and potential long-lasting disabling symptoms responsible for the long-COVID syndrome. Mitochondrial dysfunction is a key mechanism but whether peripheral blood mononuclear cell (PBMC) mitochondrial respiration changes might be associated with mortality and/or occurrence and severity of long-COVID syndrome remains to be investigated. We determined mitochondrial respiratory chain oxygen consumption in twenty COVID-19 patients hospitalized in the intensive care unit and analyzed their remaining symptoms at the third year after hospital release. PBMC mitochondrial respiration was decreased in COVID-19 patients compared to the control group (14.13 ± 2.35 vs. 6.21 ± 0.88 pmol/s/10⁶ cell, p = 0.0006 for the OXPHOS state by CII). Considering COVID severity, such a decrease was greater in long-COVID patients and in patients who deceased (4.91 ± 0.75, p = 0.008 and 4.94 ± 1.11 pmol/s/10⁶ cell, p = 0.04, respectively). PBMC markers of inflammation also increased with the severity of COVID (1.0 ± 0.08 vs. 14.45 ± 2.07, p = 0.02 for ISG15 in patients who died) and ISG15 negatively correlated with PBMC mitochondrial respiration (r = −0.67, p = 0.02 for CII). In conclusion, this study shows that the greater the impairment in PBMC mitochondrial respiration in patients hospitalized in the intensive care unit for COVID-19, the greater the mortality rate and the more severe the long-COVID symptoms—three years after hospital discharge. Further, PBMC markers of inflammation also increased with the severity of COVID and ISG15 negatively correlated with PBMC mitochondrial respiration. These results support that PBMC mitochondrial respiration might be a biomarker of COVID severity and further studies investigating whether modulation of PBMC mitochondrial respiration might improve COVID-19 patients’ prognosis. Full article

Background: The objective of this study was to validate reference values for eight selected microRNAs (miRNAs) in a population of healthy individuals. The selected miRNAs (hsa-miR-21-5p, hsa-miR-23a-3p, hsa-miR-142-5p and hsa-miR-126-3p, hsa-miR-499a-5p, hsa-miR-195-5p, hsa-miR-1-3p, hsa-miR-29a-3p) have an important role in heart failure. Methods: Ninety-nine individuals were selected for this study. Specific microRNAs were isolated from whole blood and quantified using the microRNA enzymatic immunoassay (miREIA) method. Reference intervals were evaluated with respect to age and sex. Statistical analyses were performed using MedCalc (v22.021) and R software, Version 4.1.2. Results: Reference values (2.5th and 97.5th percentile values and their 90% confidence intervals) were determined for hsa-miR-21-5p: 1.45 to 96.3 pmol/L, hsa-miR-23a-3p: 13.0 to 432 pmol/L, hsa-miR-126-3p: 5.67 to 66.5 pmol/L, hsa-miR-142-5p: 37.4 to 293 pmol/L, hsa-miR-195-5p: 11.5 to 254 pmol/L, hsa-miR-1-3p: 50.6 to 1800 pmol/L, hsa-miR-499a-5p: 8.90 to 82.5 pmol/L and hsa-miR-29a-3p: 22.9 to 210 pmol/L. The median age of the included individuals was 44 years (range: 23–75 years). No sex-related differences were observed in the reference intervals of the microRNAs (p < 0.05). Except for hsa-miR-21-5p (RS = −0.208; p = 0.043), no significant age-related associations were found for the other microRNAs (p < 0.05). However, due to the limited number of individuals in the stratified subgroups, reference intervals were not calculated for these subgroups. Conclusions: In this study, reference intervals for eight specific miRNAs associated with heart failure were determined. The results are unique for assessment in further clinical research, given that reference intervals in absolute values have not yet been published. Full article

Therapeutics involving small interfering RNA (siRNA) have enormous potential for treating a number of diseases, but their effective delivery to target cells remains a major challenge. We studied the influence of the structure and combination of targeted (folate conjugated, F13) and shield lipoconjugates (P1500, diP1500) on the ability of cationic liposomal formulations based on the 2X3-DOPE system to deliver siRNA into cells in vitro and in vivo. The loop-structured PEG lipoconjugate equipped with two hydrophobic anchor groups (diP1500) demonstrated superior performance across multiple evaluation criteria. The F13/diP1500 composition maintained a compact particle size (126.0 ± 23.0 nm), while F13/P1500 with the same PEG chain equipped with one anchor group maintained an increased particle size of 241.8 ± 65.7 nm. Most critically, F13/diP1500 preserved substantial positive surface charges (21.6–30.5 mV) across all N/P ratios, demonstrating superior ability in avoid the “PEG dilemma”, whereas F13/P1500 suffered substantial charge neutralization (3.9–9.1 mV). Competitive inhibition with free folate confirmed receptor-mediated cellular accumulation of siRNA mediated by F13 containing liposomal compositions. In vivo biodistribution revealed statistically significant circulation advantages: DSPE-PEG2000/diP1500 achieved the highest plasma concentration at 15 min (1.84 ± 0.01 pmol/mL), representing the first direct in vivo comparison of compositions with PEG lipoconjugates of the same length, but formed different structures in the liposomes due to the presence of one or two anchor groups. Our findings provide critical insights for the rational design of targeted liposomal delivery systems, highlighting the importance of balanced optimization between folate targeting functionality and PEG shielding for effective siRNA delivery both in vitro and in vivo. Full article