Formulation development and in vitro evaluation of transferrin-conjugated liposomes as a carrier of ganciclovir targeting the retina.

Ganciclovir (GCV) is an antiviral drug approved for treatment of cytomegalovirus (CMV) retinitis. It can be delivered to the eye via systemic administrations. However, local delivery of GCV that targets the retina is considered as an alternative to increase efficacy of the treatment and lessen side effects. Thus, this study aimed to develop formulations of transferrin (Tf)-conjugated liposomes containing GCV (Tf-GCV-LPs) for intravitreal injection and topical instillation. Tf-GCV-LPs were prepared by the reverse-phase evaporation technique and then conjugated to Tf. Their physicochemical properties were evaluated. The optimized formulation was selected and subjected to the cytotoxicity test, cellular uptake study in the human retinal pigment epithelial cells (the ARPE-19 cells) and antiviral activity evaluation. The results showed that physicochemical properties of Tf-GCV-LPs were affected by formulation compositions. The optimized Tf-GCV-LPs had a particle size lower than 100 nm with a negative value of zeta potential. They were safe for the ARPE-19 cells. These Tf-GCV-LPs were taken up by these cells via Tf receptors-mediated endocytosis and showed inhibitory activity on CMV in the infected cells. Therefore, the optimized Tf-GCV-LPs could be accepted as a promising drug delivery system for targeted GCV delivery to the retina in the treatment of CMV retinitis.

Clinical efficacy of 0.5% topical mangosteen extract in nanoparticle loaded gel in treatment of mild-to-moderate acne vulgaris: A 12-week, split-face, double-blinded, randomized, controlled trial.

BACKGROUND: Acne vulgaris is the most common inflammatory sebaceous gland disorder in young adults. The resistant strains of Propionibacterium acnes (P. acnes) are of increasing concern in the treatment of acne. OBJECTIVES: To evaluate the efficacy of 0.5% topical mangosteen extract in nanoparticle loaded gel (containing alpha-mangostin) compared with 1% clindamycin gel for treatment of mild-to-moderate acne vulgaris. METHODS: Patients aged 18-40 years were enrolled in this double-blinded, split-face, randomized, control study. The 2.5% benzoyl peroxide cream was applied to both sides of the faces once daily for 5 minutes and washed off. Each patient was randomly treated with the mangosteen fruit rind extract on one side and 1% clindamycin on another side of the face twice daily for 12 weeks. Treatment efficacies and side effects were evaluated on every follow-up. RESULTS: Twenty-eight patients, 24 female (85.7%), mean ± SD age of 25.14 ± 5.8, with Global Acne Grading system (GAGs) score of 15.43 ± 5.96 were included. Mangosteen fruit rind extract significantly showed significant 66.86% and 67.05% reduction of comedone and inflammatory lesions (P < 0.001) after 12-week treatment. The improvement on both treated sides significantly showed since 2 weeks after treatment, without statistical difference between two groups. Nonetheless, the mangosteen fruit rind extract revealed significantly better improvement of clinical severity, with no severe side effects. CONCLUSIONS: The mangosteen fruit rind extract formation could be a phytopharmaceutical medication for effective treatment of mild and moderate acne vulgaris treatment comparable to 1% clindamycin gel, with no severe side effects.

Ethanolic extract of Thevetia peruviana flowers enhances TNF-α and TRAIL-induced apoptosis of human cervical cancer cells via intrinsic and extrinsic pathways.

Tumor necrosis factor-α (TNF-α) and TNF-related apoptosis-inducing ligand (TRAIL) are promising candidates for cancer treatment due to their ability to induce apoptosis through death receptor stimulation. However, their usage may be limited due to the resistance of cancer cells to TNF-α- and TRAIL-induced apoptosis. Currently, there is interest in screening for natural products that can sensitize cancer cells to TNF-α- and TRAIL-induced apoptosis for their use in combination with TNF-α or TRAIL. It was previously reported that the bark extract of Thevetia peruviana showed a reversal effect on TRAIL-resistance in human gastric adenocarcinoma cell lines. In the present study, the effects of the ethanolic extract of T. peruviana flowers on TNF-α- and TRAIL-induced apoptosis of human cervical cancer HeLa cells were investigated in vitro by determining cell viability and apoptosis using a WST-1 cell proliferation assay and immunoblot analysis, respectively. The ethanolic extract of T. peruviana flowers promoted TNF-α and TRAIL-mediated cell death through the activation of the caspase cascade, poly(ADP-ribose) polymerase and BH3-interacting domain death agonist cleavage. Combined treatment using the extract plus TNF-α resulted in downregulation of anti-apoptotic protein, including myeloid cell leukemia sequence-1, B-cell lymphoma-extra large (Bcl-XL), X-linked inhibitor of apoptosis protein and survivin, while the combined treatment with TRAIL downregulated Bcl-XL. Thus, the ethanolic extract of T. peruviana flowers has potential in sensitizing the TNF-α- and TRAIL-induced apoptosis of HeLa cells via the intrinsic and extrinsic pathways.

Thermal, mechanical and drug release characteristics of an acrylic film using active pharmaceutical ingredient as non-traditional plasticizer.

The objective of this study was to investigate thermal and mechanical properties as well as in vitro drug release of Eudragit® RL (ERL) film using chlorpheniramine maleate (CPM) as either active pharmaceutical ingredient or non-traditional plasticizer. Differential scanning calorimeter was used to measure the glass transition temperature (Tg) of 0-100% w/w CPM in ERL physical mixture. Instron testing machine was used to investigate Young's modulus, tensile stress and tensile strain (%) of ERL film containing 20-60% w/w CPM. Finally, a Franz diffusion cell was used to study drug release from ERL films obtained from four formulations, i.e. CRHP0/0, CRHP0/5, CRHP2/0 and CRHP2/5. The Tg of ERL was decreased when the weight percentage of CPM increased. The reduction of the Tg could be described by Kwei equation, indicating the interaction between CPM and ERL. Modulus and tensile stress decreased whereas tensile strain (%) increased when weight percentage of CPM increased. The change of mechanical properties was associated with the reduction of the Tg when weight percentage of CPM increased. ERL films obtained from four formulations could release the drug in no less than 10 h. Cumulative amount of drug release per unit area of ERL film containing only CPM (CRHP0/0) was lower than those obtained from the formulations containing traditional plasticizer (CRHP0/5), surfactant (CRHP2/0) or both of them (CRHP2/5). The increase of drug release was a result of the increase of drug permeability through ERL film and drug solubility based on traditional plasticizer and surfactant, respectively.

Effect of galactose density on asialoglycoprotein receptor-mediated uptake of galactosylated liposomes.

Galactosylated (Gal) liposomes containing various molar ratios of cholesten-5-yloxy-N-(4-((1-imino-2-D-thiogalactosylethyl)formamide (Gal-C4-Chol) as a ligand for asialoglycoprotein receptors were prepared to study the effect of the galactose content of Gal-liposomes labeled with [3H]cholesteryl hexadecyl ether on their targeted delivery to hepatocytes. The uptake characteristics of Gal-liposomes having Gal-C4-Chol of 1.0%, 2.5%, 3.5%, 5.0%, and 7.5% were evaluated. The uptake and internalization by HepG2 cells was enhanced by the addition of Gal-C4-Chol to the Gal-liposomes. In the presence of excess galactose, the uptake of Gal-liposomes having Gal-C4-Chol of 3.5%, 5.0%, and 7.5% was inhibited suggesting asialoglycoprotein receptor mediated uptake. After intravenous injection, Gal-liposomes having Gal-C4-Chol of 3.5%, 5.0%, and 7.5%, rapidly disappeared from the blood and exhibited rapid liver accumulation with up to about 80% of the dose within 10 min whereas Gal-liposomes having low Gal-C4-Chol (1.0% and 2.5%) showed a slight improvement in liver accumulation compared with bare-liposomes. Gal-liposomes with high Gal-C4-Chol are preferentially taken up by hepatocytes and the highest uptake ratio by parenchymal cells (PC) and nonparenchymal cells (NPC) (PC/NPC ratio) was observed with Gal-liposomes having of 5.0% Gal-C4-Chol. We report here that the galactose density of Gal-liposomes prepared by Gal-C4-Chol is important for both effective recognition by asialoglycoprotein receptors and cell internalization.

Uptake characteristics of galactosylated emulsion by HepG2 hepatoma cells.

Galactosylated (Gal) emulsions containing various molar ratios of cholesten-5-yloxy-N-(4-((1-imino-2-D-thiogalactosylethyl)amino)butyl)formamide (Gal-C4-Chol) as a ligand for asialoglycoprotein receptors were prepared to study the effect of the galactose content of Gal-emulsions labeled with [3H]cholesteryl hexadecyl ether on their targeted delivery to hepatocytes. The uptake characteristics of Gal-emulsions having Gal-C4-Chol of 1, 3, 4, 6, and 9 mol% were evaluated in HepG2 cells which possess asialoglycoprotein receptors and NIH3T3 cells which are lack of asialoglycoprotein receptors. The uptake and internalization by HepG2 cells was enhanced by the addition of Gal-C4-Chol to the Gal-emulsions whereas the uptake of Gal-emulsions by NIH3T3 cells was not much and was comparable with that of bare-emulsions. In the presence of excess Gal-BSA, the uptake of Gal-emulsions having Gal-C4-Chol of 4, 6, and 9% was inhibited suggesting asialoglycoprotein receptor mediated uptake. Moreover, Gal-emulsions having Gal-C4-Chol of 4, 6, and 9% showed a slight increase in surface binding and exhibited extensive uptake and internalization into HepG2 cells. The present study strongly suggested that the Gal-emulsions are taken up by the asialoglycoprotein receptor-mediated endocytosis and galactose density of Gal-emulsions is important for effective recognition and cell internalization.

Biodistribution characteristics of galactosylated emulsions and incorporated probucol for hepatocyte-selective targeting of lipophilic drugs in mice.

PURPOSE: Galactosylated emulsions containing cholesten-5-yloxy-N-(4-((1-imino-2-D-thiogalactosylethyl)amino)butyl)formamide (Gal-C4-Chol) as a "homing device" were developed for hepatocyte-selective drug targeting. The targeting efficiency of galactosylated emulsions was evaluated by a distribution study in mice. METHODS: Soybean oil/EggPC/cholesterol (Chol) (weight ratio, 70:25: 5) (bare) emulsions and soybean oil/EggPC/Gal-C4-Chol (weight ratio, 70:25:5) (Gal) emulsions were prepared and labeled with [3H]cholesteryl hexadecyl ether (CHE). [14C]probucol as a model lipophilic drug was incorporated in the emulsions or EggPC/Chol/Gal-C4-Chol (Gal) liposomes. Their tissue and intrahepatic distribution were evaluated following intravenous injection in mice. RESULTS: After intravenous injection, Gal-emulsions were rapidly eliminated from the blood and accumulated in the liver, in contrast to the bare-emulsions. The liver uptake clearance of Gal-emulsions was 3.2- and 1.2-times greater than that of bare-emulsions and Gal-liposomes, respectively. The uptake ratio in liver parenchymal cells (PC) and nonparenchymal cells (NPC) of Gal-emulsions was higher than that of Gal-liposomes, being 7.4 and 3.0, suggesting that Gal-emulsions are an effective PC-selective carrier. The hepatic uptake of Gal-emulsions, but not that of bare-emulsions, was significantly inhibited by the pre-dosing of not only lactoferrin but also Gal-liposomes, suggesting asialoglycoprotein receptor-mediated endocytosis. Furthermore, [14C]probucol incorporated in Gal-emulsions was efficiently delivered to the liver compared with Gal-liposomes. CONCLUSION: Gal-emulsions have been proven to be an alternative carrier for hepatocyte-selective drug targeting.

Targeted and sustained drug delivery using PEGylated galactosylated liposomes.

To achieve a sustained and targeted delivery of liposomes to liver parenchymal cells (PC), we modified distearoyl-L-phosphatidylcholine (DSPC)/cholesterol (Chol) (60:40) (DSPC/Chol) liposomes with a galactosylated cholesterol derivative (Gal-C4-Chol), and polysorbate (Tween) 20 or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol (PEG(x)-DSPE). After intravenous injection, DSPC/Chol/Gal-C4-Chol (60:35:5) (Gal) liposomes were rapidly eliminated from the blood circulation and mostly recovered in the liver. The blood elimination of DSPC/Chol/Gal-C4-Chol/Tween 20 (55:35:5:5) (Tween 20-Gal) liposomes was slightly reduced as compared to Gal-liposomes. In contrast, a significant reduction in the blood elimination was observed with DSPC/Chol/Gal-C4-Chol/PEG(2000)-DSPE (59:35:5:1) (PEG(2000)-Gal) liposomes. Hepatic uptake of DSPC/Chol/Gal-C4-Chol/PEG(350)-DSPE (59:35:5:1) (PEG(350)-Gal) liposomes was intermediate between PEG(2000)-Gal-liposomes and Tween 20-Gal-liposomes. The uptake of PEG(350)-Gal-liposomes by liver PC was 7.7-fold higher than that by non-parenchymal cells (NPC). These results suggest that PEG(350)-DSPE can control the delivery rate of Gal-liposomes to liver PC without losing its targeting capability.

Targeting efficiency of galactosylated liposomes to hepatocytes in vivo: effect of lipid composition.

PURPOSE: To investigate the effects of the lipid composition of galactosylated liposomes on their targeted delivery to hepatocytes. METHODS: Several types of liposomes with a particle size of about 90 nm were prepared using distearoyl-L-phosphatidylcholine (DSPC), cholesterol (Chol) and cholesten-5-yloxy-N-(4-((1-imino-2-D- thiogalactosylethyl)amino)butyl)formamide (Gal-C4-Chol), and labeled with [3H]cholesterol hexadecyl ether. Their tissue disposition was investigated in mice following intravenous injection. The binding and internalization characteristics were also studied in HepG2 cells. RESULTS: Compared with [H]DSPC/Chol (60:40) liposomes, [3H]DSPC/Chol/Gal-C4-Chol (60:35:5) liposomes exhibit extensive hepatic uptake. Separation of the liver cells showed that galactosylated liposomes are preferentially taken up by hepatocytes, whereas those lacking Gal-C4-Chol distribute equally to hepatocytes and nonparenchymal cells (NPC). Increasing the molar ratio of DSPC to 90% resulted in enhanced NPC uptake of both liposomes, suggesting their uptake via a mechanism other than asialoglycoprotein receptors. DSPC Chol/Gal-C4-Chol (60:35:5) and DSPC/Chol/Gal-C4-Chol (90:5:5) liposomes exhibited similar binding to the surface of HepG2 cells, but the former were taken up faster by the cells. CONCLUSIONS: The recognition of galactosylated liposomes by the asialoglycoprotein receptors is dependent on the lipid composition. Cholesterol-rich galactosylated liposomes, exhibiting less non-specific interaction and greater receptor-mediated uptake, are better for targeting drugs to hepatocytes in vivo.