Assay and drug development technologies最新文献

Antimicrobial resistance (AMR) in Escherichia coli, driven by biofilm formation and quorum sensing (QS), presents a significant challenge in combating infections, particularly urinary tract infections. This study explored the potential of plant bioactive compounds to inhibit LsrR, a key transcriptional regulator of the QS system, in E. coli. The active site of LsrR was identified using the Sitemap module, which demonstrated high druggability, with a D-score of 0.987. Structure-based virtual screening was used to identify plant-derived inhibitors, followed by docking, binding free energy calculations, and induced-fit docking to evaluate ligand interactions and stability. Chebulinic acid, rutin, and vicine have emerged as potent inhibitors with better docking scores and multiple protein-ligand interactions. Molecular dynamics simulations confirmed the stability of these complexes, highlighting their potential to disrupt QS pathways and inhibit bacterial biofilm formation. These findings suggest that plant bioactive compounds are promising novel therapeutic agents for mitigating AMR in E. coli by targeting LsrR.

This study aimed to design and develop flurbiprofen-loaded chitosan nanoparticles for ophthalmic drug delivery, with the objectives of enhancing formulation stability, sustaining drug release, and improving patient comfort. This study introduces an optimized chitosan-based nanoparticle system for ocular delivery of flurbiprofen, achieving high encapsulation efficiency, physiological compatibility, and sustained release for 20 h. The findings demonstrate a practical, patient-friendly approach that enhances drug bioavailability and stability compared with conventional eye drops. Flurbiprofen-loaded nanoparticles were prepared using gelation and optimized through a Box-Behnken design. The influence of formulation variables (0.1%-0.3% w/v chitosan concentration, 0.2-0.6 mL/min dropping rate, and 500-900 rpm mixing speed) was assessed. The optimized nanoparticles were evaluated for particle size, polydispersity index (PDI), zeta potential, pH, osmolarity, and entrapment efficiency (%). The optimized formulation was achieved at 0.2% (w/v) chitosan concentration, a dropping rate of 0.4 mL/min, and a mixing speed of 700 rpm. The nanoparticles exhibited a particle size of 110.0 ± 2.20 nm, PDI of 0.347 ± 0.03, and zeta potential of + 15.4 ± 3.8 mV. The entrapment efficiency was 80.89%. The formulation was adjusted to physiological conditions (pH 6.7, osmolarity 300 mOsm/kg). In vitro studies showed a controlled release of flurbiprofen over 20 h. The optimized chitosan-based nanoparticle system demonstrated favorable physicochemical stability, high encapsulation efficiency, and sustained release of flurbiprofen. Compared with conventional ocular formulations, this structured nanoparticle delivery system offers superior drug retention and extended therapeutic action.

Neurodegenerative disorders such as Alzheimer's and Parkinson's disease remain a significant therapeutic challenge due to the restrictive nature of the blood-brain barrier (BBB) and the limited efficacy of current pharmacological treatments. Intranasal administration has emerged as a promising noninvasive strategy that enables direct drug delivery to the brain by bypassing the BBB. This study aimed to design and optimize a dual-drug nasal hydrogel containing metformin hydrochloride, a hydrophilic AMP-activated protein kinase activator, and curcumin, a lipophilic antioxidant and anti-amyloid agent, and to provide synergistic neuroprotection. The formulation was prepared using carbopol as the gel matrix and characterized in terms of physicochemical stability, drug content uniformity, rheology, in vitro release, and excipient compatibility. A Box-Behnken design was used to systematically evaluate the effects of carbopol, glycerin, and curcumin concentrations on critical quality attributes. The optimized hydrogel exhibited acceptable pH, viscosity suitable for nasal administration, and sustained biphasic drug release with a cumulative 6-h release of approximately 85% for metformin and 39% for curcumin according to the Higuchi drug release model (R² > 0.98). Collectively, these results highlight the feasibility of an integrative intranasal hydrogel platform to overcome the bioavailability challenges of both agents. The proposed system offers a patient-friendly, noninvasive approach for potential nose-to-brain therapy in neurodegenerative disorders and warrants further preclinical and in vivo investigation.

Palbociclib (PLB), an advanced breast cancer drug, shows limited success in clinical scenarios due to toxicity and resistant antitumor activities. The article aimed at preparing folic acid-modified chitosan-based polymeric nanoparticles (PNS) for controlled and targeted release of PLB. PLB-loaded nanoparticles were prepared via solvent evaporation. Central composite design was adopted in optimizing the formulation, with concentration levels of chitosan and polyvinyl alcohol (PVA) making responses of size and zeta potential (ZP) its independent variables. Mean particle size (MPS) ranged from 376 to 412 nm, and ZP ranged from 29.87 to 36.76 mV. Quadratic models were significant for both responses (MPS: F = 9.81, p = 0.0128; ZP: F = 5.24, p = 0.0466), with a nonsignificant lack of fit, confirming model adequacy. Particle size was significantly influenced by chitosan and PVA quadratic terms, while ZP was primarily affected by PVA. Optimization predicted a formulation with 63.35 mg of chitosan, 20 mg of PVA, an MPS of 389.1 nm, and a ZP of 31.2 mV, closely matching experimental observations (MPS of 237.8 ± 1.76 nm and ZP of 32.09 ± 3.38 mV). Entrapment efficiency and drug loading were 81.21 ± 1.80% and 40.79 ± 1.98%, respectively. PLB-PNS exhibited sustained, pH-responsive release, releasing 70.48% at pH 5.4 and 50.77% at pH 7.4 at 12 h, while free drug released 97.20% and 91.52%, respectively, confirming controlled and tumor-microenvironment-responsive drug delivery. Drug release followed Korsmeyer-Peppas kinetics, indicating diffusion-controlled release. Scanning electron microscope analysis revealed smooth, spherical nanoparticles. These developed PLB-loaded nanoparticles were indicated as an attractive tumor microenvironment-responsive drug-delivery carrier system capable of achieving enhanced therapeutic outcomes in breast cancer therapy.

Kinin peptides play a key role in regulating blood vessel tone, renal function, and protection against ischemia-reperfusion injury. Neprilysin is one of the primary enzymes responsible for degrading several vasoactive peptides including bradykinin. Concurrent inhibition of neprilysin and angiotensin II receptors leads to elevated bradykinin levels by reducing its breakdown, potentially enhancing its interaction with bradykinin receptor complexes. It has therefore been hypothesized that increased bradykinin generation may contribute to the neuroprotective effect of neprilysin inhibitors and angiotensin receptor blockers against global cerebral ischemia-reperfusion injury (GCI/R). GCI/R was induced in mice by transient occlusion of bilateral common carotid arteries. A combination of Sacubitril/Valsartan (30/50 mg/kg and 60/100 mg/kg, PO) was administered for 7 days before subjecting to ischemia. Behavioral changes were assessed using the beam walk test for motor coordination and passive avoidance and Y-maze tests for spatial memory, which were further supported by biochemical and histopathological assessment. Our results demonstrated that prophylactic treatment with Sacubitril/Valsartan led to a dose-dependent improvement in neurological deficits, a reduction in oxidative stress and inflammation, and an increased number of intact neurons in the hippocampus and cortex of GCI/R mice. Interestingly, immunohistochemical analysis revealed upregulation of bradykinin B2 receptors accompanied by changes in eNOS and NFκB expression. However, coadministration of the bradykinin B2 receptor antagonist, Icatibant, attenuated the neuroprotective effects of Sacubitril/Valsartan. Our findings suggest that the neuroprotective effects of Sacubitril/Valsartan may be mediated by the endogenous accumulation of vasodilatory bradykinin. Thus, concurrent inhibition of neprilysin and angiotensin II receptors represents a promising therapeutic target for stroke management.

Berberine (BER) is an antioxidant, anti-inflammatory, and antitumor agent, while paclitaxel (PTX) is a widely used synthetic chemotherapeutic agent for breast cancer. Several reports have suggested the use of a PTX and BER combination for the effective treatment of breast cancer, and many pharmaceutical scientists are working to develop a novel drug delivery system incorporating this combination. However, the literature lacks a reliable simultaneous estimation method for this combination. Therefore, in the present study, we aimed to develop a robust reversed-phase high-performance liquid chromatography method for the simultaneous estimation of PTX and BER in free drug form in liposomal formulation. The method employed a C₁₈ column (250 × 4.6 mm, 5 µm) with acetonitrile and water (70:30, v/v) as the mobile phase at a flow rate of 0.8 mL/min and detection at 250 nm. Retention times were 2.84 and 5.62 min for PTX and BER, respectively. Theoretical plates >2000 were demonstrated, and peak tailing of <2 in validation as per ICH Q2(R2) was observed. In the 10-50 ppm range, linearity was found with R² values of 0.9979 (PTX) and 0.9975 (BER). Furthermore, the method achieved within acceptable limits precision (<2% relative standard deviation) and accuracy (90%-110%). Robustness assessments checked method reliability in small variations. In addition, using the method effectively, entrapment efficiencies of 85.27 ± 1.74% and 78.62 ± 2.38% were obtained for PTX and BER in liposomal formulations. Moreover, in vitro release studies revealed 98.83 ± 2.94% (PTX) and 96.56 ± 1.92% (BER) release over 24 h. The validated method was precise, accurate, and reliable, making it suitable for application in drug formulation analysis.

This study presents the first validated High-performance liquid chromatography (HPLC) technique for quantifying naphthol AS-E phosphate (NASEP) in bulk drugs and nanoparticle formulation. A C18 HPLC cartridge (250 × 4.6 mm, 5 µm particle size) served as the stationary phase for quantification. The mobile phase consisted of Milli-Q water with 0.1% trifluoroacetic acid (TFA) in pump A and acetonitrile with 0.1% TFA in pump B, with a flow rate ranging from 0.8 to 1.2 mL/min. A 3² factorial design was employed to evaluate the robustness of the proposed method, using mobile phase composition (X₁), flow rate (X₂), and column temperature (X₃) as independent variables and peak area (R₁), retention time (R₂), and percent recovery (R₃) as response variables. The calibration range curve (10-500 µg/mL) was best fitted by quadratic regression. The linearity was reported in the above-mentioned range. The accuracy was 99.952% ± 0.961% at the 75% level, 99.58% ± 1.483% at the 100% level, and 99.789% ± 1.936% at the 125% level. The coefficient of variation was below 2% for both intraday and interday measurements, and the limits of detection and quantification were 0.038 and 0.115 µg/mL, respectively. The NASEP solution was stable (99.04% ± 0.0251%) for 48 h at 8°C. The forced degradation study also revealed that the NASEP solution remained stable in an acidic environment for 48 h at 40°C but degraded at 80°C (p < 0.046) in a time-dependent manner. In contrast, it was unstable in an alkaline medium, independent of temperature, and degraded in the presence of strong oxidizing agents (p < 0.039). Furthermore, NASEP encapsulated in a Gly-Arg-Gly-Asp-Ser pentapeptide and low-molecular-weight heparin functionalized metal-organic framework exhibited sustained drug release at acidic pH 5.4. The proposed NASEP quantification method was validated and is suitable for routine analysis in pharmaceutical formulations.

Exosomes, nano-sized extracellular vesicles secreted by almost all cell types, have emerged as biologically compatible vehicles for targeted drug delivery, gene therapy, and molecular diagnostics. Their innate ability to traverse biological barriers and deliver diverse cargoes with minimal immunogenicity has catalyzed intense interest in their therapeutic exploitation. However, the intrinsic heterogeneity and limited targeting specificity of native exosomes necessitate advanced engineering strategies to fulfill their clinical potential. This review focuses on the molecular-level nanoengineering of exosomal surfaces to enhance specificity, loading efficiency, and release control of therapeutic payloads. We systematically examine current methodologies, including genetic modification of parental cells, covalent and non-covalent surface conjugation, lipid insertion, click chemistry, and hybrid vesicle fusion. We further detail the quantitative performance of targeting ligands-such as peptides, aptamers, nanobodies, and glycans-in relation to receptor affinity, conjugation efficiency, and biological outcomes. Payload loading techniques, both endogenous and exogenous, are critically analyzed based on loading yield and membrane preservation. Additionally, we highlight disease-specific applications in oncology, neurology, cardiology, and immunotherapy, supported by preclinical and translational case studies. Emerging technologies such as microfluidics, synthetic biology, artificial intelligence-guided modeling, and multi-omics are discussed as integral components of the next generation of precision exosome platforms. Finally, we address key challenges related to scalability, regulatory frameworks, and standardization. This review provides a comprehensive and quantitative framework to guide the design of molecularly engineered exosomes for future translational and clinical success.

Stroke is an intricate oxidative and inflammatory response resulting from cerebral ischemia followed by reperfusion injury. The complex pathophysiology of stroke poses challenges for treatment. Peroxisome proliferated-activated receptor (PPAR) expression in the rat hippocampus is markedly elevated post cerebral ischemia/reperfusion injury (I/R injury). Hence, Saroglitazar, a dual PPAR-α/γ agonist, was investigated against cerebral I/R injury in rats. Male Sprague Dawley rats were subjected to bilateral common carotid artery occlusion for 30 min and reperfusion for 3 days. During the reperfusion, animals were treated with vehicle or Saroglitazar once a day for 3 days. The behavioral parameters were assessed, and animals were sacrificed to measure oxidative markers (Malondialdehyde, Superoxide dismutase, catalase, and reduced glutathione), inflammatory markers (interleukin-6, tumor necrosis factor-α, nuclear factor kappa-light chain enhancer of activated B cells [NF-κB], and high mobility group box 1 protein [HMGB-1]), infarction, and histopathology changes. Following I/R injury, antioxidant enzymes were reduced, while nitric oxide and inflammatory markers were increased in the disease group. In the rat hippocampus, these changes led to neurobehavioral impairment and cerebral infarction. Saroglitazar improved the levels of antioxidants and reduced inflammation. 2,3,5-triphenyltetrazolium chloride staining and histopathological analysis revealed the neuroprotective effect of Saroglitazar in the hippocampus region. The neuroprotective effects of Saroglitazar were attributed to its activation of both PPAR-α and PPAR-γ. It improved antioxidant levels and inhibited pro-inflammatory cytokines by suppressing the HMGB-1/NF-κB signaling pathway. These findings underscore the potential of Saroglitazar in mitigating cerebral I/R injury.

The inward-rectifier potassium channel (Kir) 2.x family is an important family of ion channels in the context of human health. These potassium channels are involved in processes such as cardiac action potential, formation of skeletal muscle, bone development, vasodilation, and neuronal activity and are expressed centrally and peripherally. Given their importance, they are an attractive target for the development of tool compounds. The high homology between the members of the Kir family has made isoform selectivity challenging. In an effort to discover novel chemical matter related to this intriguing target, we performed a high-throughput screen utilizing compounds from the Vanderbilt Institute of Chemical Biology Discovery Collection. This screen of over 20,000 compounds resulted in 48 verified hits consisting of six novel chemical scaffolds. Of these hits, VU0523203 and VU0606851 were selected as promising starting points for initial medicinal chemistry optimization to improve potency and distribution, metabolism, and pharmacokinetic (DMPK) properties. These efforts resulted in the discovery of VU6073995, a compound with modest potency at Kir2.1 and improved DMPK properties compared with ML133.