Hepatocellular carcinoma (HCC) is a fatal disease with limited therapeutic choices. The stroma-rich tumor microenvironment hinders the in vivo delivery of most nanomedicines. Ultra-small lipid nanoparticles (usLNPs) were designed for the selective co-delivery of the cytotoxic drug, sorafenib (SOR), and siRNA against the Midkine gene (MK-siRNA) to HCC in mice. The usLNPs composed of a novel pH-sensitive lipid, a diversity of phospholipids and a highly-selective targeting peptide. A microfluidic device, iLiNP, was used and a variety of factors were controlled to tune particle size aiming at maximizing tumor penetration efficiency. Optimizing the composition and physico-chemical properties of the usLNPs resulted in an enhanced tumor accumulation, selectivity and in vivo gene silencing. The optimized usLNPs exerted potent gene silencing in the tumor (median effective dose, ED50~0.1 mg/Kg) with limited effect on the healthy liver. The novel combination synergistically-eradicated HCC in mice (~85%) at a surprisingly-low dose of SOR (2.5 mg/Kg) which could not be achieved via individual monotherapy. Toxicity studies revealed the biosafety of the usLNPs upon either acute or chronic treatment. Furthermore, the SOR-resistant HCC established in mice was eradicated by 70% using this approach. We conclude that our strategy is promising for potential clinical applications in HCC treatment.

Adipose tissue in the body is classified as white adipose tissue (WAT); a fat-accumulating tissue, or brown adipose tissue (BAT); an energy-dissipating tissue. Transforming WAT-to-BAT (browning) is a promising strategy for the treatment of obesity, since it would lead to an increase in energy expenditure. Rosiglitazone (Rosi), an agonist of the peroxisome proliferator-activated receptor γ (PPARγ), is known to be a potent browning inducer in subcutaneous WAT. However, the effectiveness of Rosi has been quite limited because of several off-target effects. The objective of this study was to develop locally administered Rosi-loaded nanoparticles (Rs-NPs) with the ability to target adipocytes to achieve the adipose tissue-specific activation of PPARγ, thus causing the browning of WAT. We prepared dual targeted Rs-NPs that were modified with a specific peptide that targets prohibitin that are expressed in adipocytes, and a cell penetrating peptide for enhancing cellular uptake and controlling intracellular trafficking. The Rs-NPs modified with a single ligand were internalized into mature adipocytes and induced browning activity in vitro but they failed to significantly affect the body weight of the diet-induced obese mice model. The dual-targeted Rs-NPs induced a strong browning activity, both in vitro and in vivo, and successfully inhibited the progression of obesity, as evidenced by the shrinkage of hypertrophied adipocytes without any detectable systemic adverse effects. Meanwhile, free Rosi aggravated hepatic steatosis and did not cause adipose tissue browning nor the inhibition of body weight gain. We conclude that the increased energy expenditure via adipose tissue browning using dual-targeted Rs-NP is a promising strategy for the treatment of obesity and its related metabolic syndrome.

This study describes the development of lipid nanoparticles (LNPs) for the efficient and selective delivery of plasmid DNA (pDNA) to the lungs. The GALA peptide was used as a ligand to target the lung endothelium and as an endosomal escape device. Transfection activity in the lungs was significantly improved when pDNA was encapsulated in double-coated LNPs. The inner coat was composed of dioleoylphsophoethanolamine and a stearylated octaarginine (STR-R8) peptide, while the outer coat was largely a cationic lipid, di-octadecenyl-trimethylammonium propane, mixed with YSK05, a pH-sensitive lipid, and cholesterol. Optimized amounts of YSK05 and GALA were used to achieve an efficient and lung-selective system. The optimized system produced a high gene expression level in the lungs (>107 RLU/mg protein) with high lung/liver and lung/spleen ratios. GALA/R8 modification and the double-coating design were indispensable for efficient gene expression in the lungs. Despite the fact that NPs prepared with 1-step or 2-step coating have the same lipid amount and composition and the same pDNA dose, the transfection activity was dramatically higher in the lungs in the case of 2-step coating. Surprisingly, 1-step or 2-step coatings had no effect on the amount of nanoparticles that were delivered to the lungs, suggesting that the double-coating strategy substantially improved the efficiency of gene expression at the intracellular level.

This study reports on the development of a novel lipid combination that permits the efficient and highly selective delivery of plasmid DNA (pDNA) to immune cells in the spleen. Using DODAP, an ionizable lipid that was previously thought to be inefficient for gene delivery, we show for the first time, that this ignored lipid can be successfully used for efficient and targeted gene delivery in vivo, but only when combined with DOPE, a specific helper lipid. Using certain DODAP and DOPE ratios resulted in the formation of lipid nanoparticles (LNPs) with a ~ 1000-fold higher gene expression, and this expression was specific for the spleen, making it the most spleen-selective system for transfection using pDNA. The developed DODAP/DOPE-LNPs target immune cells in the spleen via receptors for complement C3 and this pathway is critical for efficient gene expression. We hypothesize that the high spleen transfection activity of DODAP/DOPE-LNPs is caused by the promotion of gene expression associated with B cell activation via complement receptors. LNPs encapsulating tumor-antigen encoding pDNA showed both prophylactic and therapeutic anti-tumor effects. The optimized LNPs resulted in the production of different cytokines and antigen-specific antibodies as well as exerting antigen-specific cytotoxic effects. This study revives the use of DODAP in gene delivery and highlights the importance of using appropriate lipid combinations for delivering genes to specific cells.

The emergence of strains resistant to certain antibiotics is turning into an important issue worldwide that threatens global health with the increasing incidence of carbapenemase-producing Klebsiella pneumoniae (KPC). Thus, successful doripenem–ertapenem (DOR–ERTA) combination is highly recommended in treatment of bacteremic ventilator-associated pneumonia due to Klebsiella pneumoniae. Hence, a fast and highly-sensitive HPLC-UV method was developed for the estimation of the cited drugs simultaneously in their pure form, pharmaceutical dosage forms and in simulated synthetic mixtures. The DOR–ERTA mixture was successfully separated within 6 min on a reversed-phase ODS column using an isocratic elution; a mobile phase mixture consists of 0.05 M phosphate buffer (pH 3.0 adjusted by 85% ortho-phosphoric acid) : acetonitrile : methanol (86 : 12 : 2; % v/v/v). The proposed method was optimized and validated according to ICH guidelines, where the calibration graph was constructed from 0.04 to 50 μg mL−1 and from 0.05 to 50 μg mL−1 with low detection limits reached 1.7 and 1.4 ng mL−1 for DOR and ERTA respectively. The proposed method showed higher sensitivity than several previous methods, which allowed an effective estimation of the DOR and ERTA in human plasma after a simple extraction method with high recovery results ranged from 96.30% ± 1.55 to 97.90% ± 1.45 and without any interference from plasma components.

The emergence of strains resistant to certain antibiotics is turning into an important issue worldwide that threatens global health with the increasing incidence of carbapenemase-producing Klebsiella pneumoniae (KPC). Thus, successful doripenem–ertapenem (DOR–ERTA) combination is highly recommended in treatment of bacteremic ventilator-associated pneumonia due to Klebsiella pneumoniae. Hence, a fast and highly-sensitive HPLC-UV method was developed for the estimation of the cited drugs simultaneously in their pure form, pharmaceutical dosage forms and in simulated synthetic mixtures. The DOR–ERTA mixture was successfully separated within 6 min on a reversed-phase ODS column using an isocratic elution; a mobile phase mixture consists of 0.05 M phosphate buffer (pH 3.0 adjusted by 85% ortho-phosphoric acid) : acetonitrile : methanol (86 : 12 : 2; % v/v/v). The proposed method was optimized and validated according to ICH guidelines, where the calibration graph was constructed from 0.04 to 50 μg mL−1 and from 0.05 to 50 μg mL−1 with low detection limits reached 1.7 and 1.4 ng mL−1 for DOR and ERTA respectively. The proposed method showed higher sensitivity than several previous methods, which allowed an effective estimation of the DOR and ERTA in human plasma after a simple extraction method with high recovery results ranged from 96.30% ± 1.55 to 97.90% ± 1.45 and without any interference from plasma components.

Thioglycolic acid-capped cadmium sulphide quantum dots (TGA-CdS QDs) have been synthesized and utilized as a fluorescent probe for the estimation of doripenem (DOR). Monitoring of DOR in different biological fluids is required to estimate the efficient dose to avoid bacterial infections and resistance. The investigated method is based on the measurement of fluorescence quenching of TGA-CdS QDs after the addition of DOR. The synthesized TGA-CdS QDs were characterized using transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, X-ray powder diffraction (XRD) and ZETA sizer. The TGA-CdS QDs showed unique photophysical properties with high quantum yield (0.32) using a comparison method with rhodamine B. Different experimental parameters affecting the synthesis process of the TGA-CdS QDs and their behavior with the studied drug DOR were examined and optimized. The values of the fluorescence quenching were linearly correlated to DOR concentration over the range of 10–500 ng mL−1 with a good correlation coefficient of 0.9991. The proposed method showed higher sensitivity over several reported methods, with LOD reaching 2.0 ng mL−1. The method was effectively applied for the estimation of DOR in human plasma and urine with good recovery results ranged from 95.16% to 99.51%. Furthermore, the stability of DOR in the human plasma was studied and a pharmacokinetic study of DOR in real human plasma was conducted.

Thioglycolic acid-capped cadmium sulphide quantum dots (TGA-CdS QDs) have been synthesized and utilized as a fluorescent probe for the estimation of doripenem (DOR). Monitoring of DOR in different biological fluids is required to estimate the efficient dose to avoid bacterial infections and resistance. The investigated method is based on the measurement of fluorescence quenching of TGA-CdS QDs after the addition of DOR. The synthesized TGA-CdS QDs were characterized using transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, X-ray powder diffraction (XRD) and ZETA sizer. The TGA-CdS QDs showed unique photophysical properties with high quantum yield (0.32) using a comparison method with rhodamine B. Different experimental parameters affecting the synthesis process of the TGA-CdS QDs and their behavior with the studied drug DOR were examined and optimized. The values of the fluorescence quenching were linearly correlated to DOR concentration over the range of 10–500 ng mL−1 with a good correlation coefficient of 0.9991. The proposed method showed higher sensitivity over several reported methods, with LOD reaching 2.0 ng mL−1. The method was effectively applied for the estimation of DOR in human plasma and urine with good recovery results ranged from 95.16% to 99.51%. Furthermore, the stability of DOR in the human plasma was studied and a pharmacokinetic study of DOR in real human plasma was conducted.

Glyoxylic acid (GA) is the intermediate metabolite in various mammalian metabolic pathways. GA showed high reactivity towards formation of advanced glycation end-products (AGEs); the main cause of pathogenesis and complications of many diseases. The presented study aimed to detect GA in healthy and cardiovascular patients' (CV) sera; however analysis of GA in biological fluid is a challenge and requires chemical derivatization. Hence, a new, highly sensitive, time saving and reproducible pre-column fluorescence derivatization procedure coupled with high performance liquid chromatography (HPLC) method was developed. The derivatization method was based on reaction of 2-aminobenzenthiol (2-ABT), a fluorogenic reagent, with GA in acidic medium to form highly fluorescent thiazine derivative (290 and 390 nm for excitation and emission wavelengths respectively). The fluorescent derivative was separated within 6 min on a reversed-phase ODS column using an isocratic elution with a mixture of methanol-water (70:30, v/v%). The proposed method parameters were optimized and the method was validated. A good linearity in the concentration range (0.05-5.0 µM) was obtained with detection limit (LOD) of 10 nM (200 fmol/injection), which is more sensitive than several previous methods. Moreover, the recovery results were within the range of 85.0-95.5 % and the intra- and inter-day precision results were ≤3.5%. It should be emphasized that this method is the first one for monitoring of GA in CV patients; to investigate its role for diagnosis and monitoring the severity and complications of this disease in clinical laboratory.

In continuation of our efforts to develop new antiproliferative agents that could be effective and selective in treatment of cancer, we designed and synthesized new hybrid structures containing an arylsulfonamide scaffold linked to a steroidal skeleton through a 1,2,3-triazole ring. Both in vitro cytotoxicity on breast MCF-7 cancer cells and human placental aromatase enzyme (pAROM) inhibition assays were performed on new hybrids. All new hybrids showed marked cytotoxic activity against breast MCF-7 cancer cells (IC50 = 3.56–43.76 μM) in comparison to staurosporine (IC50 = 4.06 μM). Tumor selectivity index was higher for some of the new hybrids on normal fibroblast (F-180) cells (TS = 1.5–25) in comparison to staurosporine (TS = 2.5). The p-nitro derivative exhibited the best inhibitory activity on the pAROM with an IC50 of 64.6 ng/cm3, compared to hybrids unsubstituted derivative, p-bromo derivative, and letrozole (IC50 = 375.14, 269.86, and 132.86 ng/cm3, respectively). Furthermore, the p-nitro hybrid arrested the cell cycle selectively at the G2/M phase, in addition to inducing both early and late apoptotic processes of breast MCF-7 cancer cells. Molecular docking studies were performed within pAROM to explore the binding modes of the new hybrids. Collectively, the antiproliferative profile of new hybrids indicates how good they are as promising leads for developing tumor-specific cytotoxins, and deserve further studies to optimize their structure and in vivo activity.