Preparation and physicochemical characterization of N-succinyl chitosan-coated liposomes for oral delivery of grape seed extract and evaluation of its effect on pulmonary fibrosis induced by bleomycin in rats.

Objectives: This study aimed to develop an oral succinyl chitosan-coated liposomal formulation containing grape seed extract and assess its therapeutic efficacy in rats with bleomycin-induced pulmonary fibrosis. Materials and Methods: N-succinyl chitosan was synthesized, and the liposomal formulations were prepared and characterized regarding phenolic content assay and morphology. Size, zeta potential, in vitro drug release, and stability. Pulmonary fibrosis was induced by intratracheal bleomycin injection, and hydroxyproline measurements, lung weight, animal body weight, as well as histopathological studies were performed. Results: Succinyl chitosan increases the physical stability of the formulation, especially in acidic conditions. Drug release studies revealed that 66.27% of the loaded drug was released from CF2 in an acidic medium in 2 hr, but 92.31% of the drug was released in 8 hr in a pH=7 medium. An in vivo study demonstrated that rats exposed to bleomycin significantly lost weight, while those treated with CF2 (400 mg/kg) partially regained weight. Bleomycin treatment increased the mean lung weight and the amount of hydroxyproline in the lungs; these values were significantly decreased in the group treated with 400 mg/kg CF2 (P<0.05). Histopathological examination confirmed that treatment with 400 mg/kg CF2 improved lung fibrosis. Conclusion: In rats, oral administration of N-succinyl chitosan-coated liposomes containing grape seed extract at the 400 mg/kg dose ameliorates bleomycin-induced pulmonary fibrosis.

Preparation and Characterization of Mucoadhesive Loratadine Nanoliposomes for Intranasal Administration

Objectives: The present study aimed to formulate and characterize mucoadhesive liposomes for intranasal delivery of loratadine. In particular, the formulation was aimed to improve the drug bioavailability and efficacy. Materials and Methods: Liposomes were prepared by thin-film hydration method, with soybean phosphatidylcholine and cholesterol as main components. Liposomes were coated with chitosan solution at a concentration of 0.05% and 0.1%, w/v. The formulations were assessed for particle size, polydispersity index (PDI), encapsulation efficiency (EE), thermodynamic behavior, in vitro drug release, mucoadhesiveness, and stability. Results: Particle size analysis showed that the vesicles of uncoated and coated liposomes with 0.05% and 0.1% chitosan were characterized by size of 193±3.3 nm, 345±4.6, and 438±7.3 nm, respectively. Size distribution for developed formulations was in the acceptable range (PDI <0.7). EE was recorded to be approximately 80%. Chitosan-coated liposomes demonstrated slower release rate as compared to uncoated liposomes. Drug release kinetics profile for all the formulations followed a zero-order model. Chitosan coating improved mucoadhesiveness by more than 3-fold as compared to uncoated liposomes. However, no significant differences were recorded between mucin adsorption behavior of 0.05% and 0.1% chitosan-coated liposomes (p>0.05). For stability studies, liposomes were stored at 4°C for 3 months, and changes in particle diameter, PDI, and EE % were recorded. No significant alternations were reported in particles size, PDI, and drug leakage of coated liposomes. Conclusion: Liposomes coated with 0.05% chitosan were chosen as the optimum formulation, which demonstrated a significant potential for overcoming the nasal drug delivery limits for short residence time and mucociliary clearance.

A double-blind, placebo-controlled randomized trial of skin-lightening cream containing lycopene and wheat bran extract on melasma.

OBJECTIVE: Melasma is an acquired hyperpigmentation disorder, and reactive oxygen species play important role in regulating melanin synthesis. Lycopene is one of the most effective oxygen neutralizers among tomato-derived carotenoids. Also, hydroquinone is a compound that has been used for the treatment of hyperpigmentation by mechanism of tyrosinase inhibition and can be found in wheat. METHODS: An appropriate cream formulation containing 0.05% tomato lycopene and 3.45% wheat bran extract was prepared, and physicochemical characterization was performed. The prepared formulations were applied twice a day for three months in combination with SPF = 30 sunscreen by 22 patients diagnosed with melasma. MASI score in two groups was evaluated at weeks 0, 3, 6, 9, and 12 and 1 month after the treatment. RESULTS: The prepared formulation shows smooth and homogeneous appearance with suitable spreadability and viscosity. The MASI score of intervention group from the sixth week until the end of the treatment was significantly decreased compared to the onset of the treatment (P < .05), and the mean difference of the MASI score and the rate of skin discoloration in intervention group (0.53 ± 0.47 and 3.73 ± 1.90, respectively) were significantly higher than in placebo group (0.14 ± 0.20 and 0.91 ± 0.07, respectively; P < .05). Size of melasma during the study was decreased significantly from 6.59 ± 3.47 to 5.97 ± 3.83 (P < .05). CONCLUSION: The data of mean difference of the MASI score indicated skin improvement in intervention group. Meanwhile, no recurrence was observed one month after the end of the treatment. These data suggest that the prepared formulation containing lycopene and wheat bran extract is safe and could be promising as an efficacious cosmetic treatment.

Effects of Silymarin-Loaded Nanoparticles on HT-29 Human Colon Cancer Cells.

Background and objective: Previous studies have demonstrated the anti-cancer effects of silymarin (SLM). However, the low bioavailability of SLM has restricted its use. This study investigated the toxic effect of nanostructured SLM encapsulated in micelles (Nano-SLM) on the growth of the HT-29 human colon cancer cell line. Materials and methods: HT-29 cells were treated with 25 µM/mL of SLM or Nano-SLM for 48 h. MTT and colony formation assays were used to assess the cytotoxicity and proliferation of HT-29 cells, respectively. The cells were stained with annexin V/PI for assessment of apoptosis. Results: MTT assays revealed that Nano-SLM treatment was able to exert a more pronounced toxic effect on the HT-29 cells as compared to free SLM treatment (p < 0.01). In the Nano-SLM-treated cells, colony numbers were significantly reduced in comparison to the free SLM-treated cells (p < 0.01). Apoptotic and necrotic indexes of Nano-SLM-treated HT-29 cells were also significantly increased in comparison to those of the free SLM-treated cells (p < 0.01). The viability, proliferation and apoptosis of healthy cells (NIH-3T3 cells) were not changed in response to Nano-SLM or SLM. Conclusions: Our results indicate that Nano-SLM enhances the anti-cancer effects of SLM against human colon cancer cells.

Investigation of using pectin and chitosan as natural excipients in pellet formulation.

This study aimed to evaluate the potential of applying pectin and chitosan polysaccharides in pellet formulation. These biopolymers have advantages such as biocompatibility, low toxicity, low price and easy processing which make them interesting candidates for drug delivery purposes. Careful control of pellet porosity is essential to achieve an appropriate drug release profile. Replacing microcrystalline cellulose (MCC) with polysaccharides, especially pectin, leads to increased pellet porosity. Theophylline, dimenhydrinate and ibuprofen were chosen as model drugs. Investigation of possible ionic interactions between drugs and excipients is crucial to optimize the formulation of pellets with acceptable drug release. Differential scanning calorimetry of chitosan showed an endothermic peak; however, this peak was not observed in thermograms of the pectin, implying the lack of interaction between polysaccharides. Fourier transform infrared analysis did not indicate any interaction between drugs and polymers. Incorporation of MCC into the pellet formulation significantly increased the mean dissolution time while substitution of MCC with polysaccharides led to a faster release for each of the three drugs - that were different in their net charges - in both acidic and buffer media. These results highlight the potential value of polysaccharides in improving drug delivery characteristics of pharmaceutical pellets.

Dorzolamide nanoliposome as a long action ophthalmic delivery system in open angle glaucoma and ocular hypertension patients.

IMPORTANCE: To reduce the frequency of dorzolamide eye drop administration and increasing the duration of action. BACKGROUND: This study aims to compare the effect of dorzolamide loaded-nanoliposome with marketed dorzolamide HCl eye drop on intraocular pressure in primary open angle glaucoma and ocular hypertension patients. DESIGN: A randomized study was conducted in a hospital. PARTICIPANTS: Twenty patients with primary open angle glaucoma or ocular hypertension (in both eyes) diagnosis were recruited as participants. METHODS: Dorzolamide loaded-nanoliposome was prepared by thin layer hydration method and characterized. Intra ocular pressure were compared between the two groups who received marketed dorzolamide solution or dorzolamide-loaded nanoliposome. MAIN OUTCOME MEASURES: The main outcome measures include intraocular pressure initially (day 0) and on days 14 and 28 and adverse effect of dorzolamide-loaded nanoliposome eye drop. RESULTS: Based on the results of repeated measure, intra ocular pressure was seen to decrease in both the groups. But these reductions in the intervention group (dorzolamide-loaded eye drop) were significantly higher than those in control group (p < 0.05). CONCLUSION: This study confirmed safety and long-lasting efficacy of dorzolamide loaded-nanoliposome eye drop. The highly enhanced permeation through the cornea can be attributed to similarity of phospholipid bilayer of liposomes to the biological membrane and their small particle size and positive zeta potential.

Ocular Dorzolamide Nanoliposomes for Prolonged IOP Reduction: in-vitroand in-vivo Evaluation in Rabbits.

Dorzolamide ophthalmic drop is one of the most common glaucoma medications but it has a short residence time in the eye. The aim of this study is to develop ocular dorzolamide HCl nanoliposomes (DRZ - nanoliposomes) and to evaluate their potential use for the treatment of ocular hypertension. Nanoliposomes were prepared using Reverse-phase evaporation vesicle (REV) and thin layer hydration (TLH) method with 7:3 and 7:4 molar ratios of phosphatidylcholine:cholesterol. The physicochemical properties of the formulations were investigated. Formulations with 7:4 lipid ratios were evaluated in terms of drug release, physical stability and ex-vivo permeation through the excised albino rabbit cornea. The rabbits in groups of 6 were treated with selected DRZ - nanoliposomes or dorzolamide solution or marketed dorzolamid preparation (Biosopt®) and intraocular pressure (IOP) was monitored. Formulations with 7:4 molar ratio entrapped greater amount of drug compared to those with 7:3 lipid components ratio. DRZ - nanoliposomes with 7:4 lipid ratio showed more transcorneal permeation than Dorzolamide solution (p<0.05); and the formulation prepared by TLH method exhibited higher permeability than that prepared by REV method (p<0.05). The selected DRZ - nanoliposomes showed greater IOP lowering activity and a more prolonged effect compared to dorzolamide solution and Biosopt®. DRZ - nanoliposomes prepared by TLH method with 7:4 ratios showed promising results as a candidate for the treatment of ocular hypertension.

Preparation and physicochemical characterization of topical chitosan-based film containing griseofulvin-loaded liposomes.

Griseofulvin is an antifungal drug and is available as oral dosage forms. Development of topical treatment could be advantageous for superficial fungal infections of the skin. In this study, films prepared from the incorporation of griseofulvin-loaded liposomes in chitosan film for topical drug delivery in superficial fungal infections. The properties of the films were characterized regarding mechanical properties, swelling, ability to transmit vapor, drug release, thermal behavior, and antifungal efficacy against Microsporum gypseum and Epidermophyton floccosum. The presence of liposomes led to decreased mechanical properties but lower swelling ratio. Higher amount of drug permeation and rate of flux were obtained by liposomes incorporated in films compared to liposomal formulations. Antifungal efficacy of formulations was confirmed against two species of dermatophytes in vitro. Therefore, two concepts of using vesicular carrier systems and biopolymeric films have been combined and this topical novel composite film has the potential for griseofulvin delivery to superficial fungal infections.

Statistical optimization of tretinoin-loaded penetration-enhancer vesicles (PEV) for topical delivery.

BACKGROUND: The aim of this study was to develop and optimize deformable liposome for topical delivery of tretinoin. METHODS: Liposomal formulations were designed based on the full factorial design and prepared by fusion method. The influence of different ratio of soy phosphatidylcholine and transcutol (independent variables) on incorporation efficiency and drug release in 15 min and 24 h (responses) from liposomal formulations was evaluated. Liposomes were characterized for their vesicle size and Differential Scanning Calorimetry (DSC) was used to investigate changes in their thermal behavior. The penetration and retention of drug was determined using mouse skin. Also skin histology study was performed. RESULTS: Particle size of all formulations was smaller than 20 nm. Incorporation efficiency of liposomes was 79-93 %. Formulation F7 (25:5) showed maximum drug release. Optimum formulations were selected based on the contour plots resulted by statistical equations of drug release in 15 min and 24 h. Solubility properties of transcutol led to higher skin penetration for optimum formulations compared to tretinoin cream. There was no significant difference between the amount of drug retained in the skin by applying optimum formulations and cream. Histopatological investigation suggested optimum formulations could decrease the adverse effect of tretinoin in liposome compared to conventional cream. CONCLUSION: According to the results of the study, it is concluded that deformable liposome containing transcutol may be successfully used for dermal delivery of tretinoin.

Effect of topical Nanoliposomes of Paromomycin on Rats Liver and Kidney.

BACKGROUND: Hepatotoxicity due to drugs is the most common cause of deaths. Nephrotoxicity of the drugs is usually associated with the drugs accumulation in renal tissue. Paromomycin sulfate (PMS) is an anti-leishmania drug. Although the topical approach for the treatment of leishmania is attractive, its use might cause nephrotoxicity and hepatotoxicity. OBJECTIVES: This study aimed to evaluate probable nephrotoxicity and hepatotoxicity of topically administered PMS liposomes. MATERIALS AND METHODS: Nine groups of male rats were used in this study; each group consisted of 6 rats that were evaluated in 3 time periods of 10, 20, and 30 days. Three groups were placed in each time period; a control group did not receive any medicine; a negative control group received liposome without paromomycin sulfate; and a positive control group received nanoliposomal formulations containing paromomycin sulfate. Pharmaceutical formulation (topical form) was used 2 times a day in a 12-hour interval. At the end of the period, hepatic enzymes (ALT, AST, and ALK), BUN levels, and serum creatinine were measured. RESULTS: The results showed that no significant change was occurred in the amount of the above factors compared with the control group with the negative control group in 3 time periods (P > 0.05). The histopathological results of the liver and kidney showed that there was no difference in the between the negative control and positive control groups and the control group in the 10- and 20-day periods, and they had a normal structure. However, after the 30-day time period a reversible cellular inflammation in the liver and mild kidney necrosis was seen in the positive control group versus the control and negative control groups. CONCLUSIONS: In general, it can be said that the application of nanoliposomal paromomycin sulfate formulations for topical treatment of the cutaneous leishmaniasis does not create serious side effects in the short term, but its long-term use leads to mild renal and liver side effects that requires more attention.

Colloidal, in vitro and in vivo anti-leishmanial properties of transfersomes containing paromomycin sulfate in susceptible BALB/c mice.

The aim of this study was to develop transfersomal formulation with respect to dermal delivery of paromomycin sulfate (PM) for possible topical therapy of cutaneous leishmaniasis (CL). PM transfersomal formulations (PMTFs) with different percent of soy phosphatidylcholine, sodium cholate (Na-Ch) and ethanol were prepared and characterized for the size, zeta potential and encapsulation efficiency. The results showed that the most stable formulations with suitable colloidal properties were obtained by 2% Na-Ch which had average size of around 200 nm. The in vitro permeation study using Franz diffusion cells fitted with mouse skin at 37°C for 24h showed that almost 23% of the PMTFs applied penetrated the mouse skin, and the amount retained in the skin was about 67% for both formulations; however, the percent of penetration and retention for PM conventional cream was 49 and 13, respectively. The 50% effective doses of PMTFs against Leishmania major promastigotes and amastigotes in culture were significantly less than cream and/or solution of PM. Selected PMTFs and empty transfersomes showed no cytotoxicity in J774 A.1 mouse macrophage cell line. Selected PMTFs was used topically twice a day for 4 weeks to treat L. major lesions on BALB/c mice, and the results showed a significantly (P<0.05) smaller lesion size in the mice in the treated groups than in the mice in the control groups, which received either empty transfersomes or phosphate-buffered saline (PBS) and also PM cream. The spleen parasite burden was significantly (P<0.01) lower in mice treated with selected PMTFs than in mice treated with PBS or control transfersomes, and PM cream. The results of this study showed that PMTFs prepared with 2% of Na-Ch with and without 5% ethanol might be useful as a candidate for the topical treatment of CL.

mRNA transfection of cervical carcinoma and mesenchymal stem cells mediated by cationic carriers.

Messenger RNA encoding luciferase (mLUC) was complexed to the cationic lipids Lipofectamine or DOTAP/DOPE, and to the cationic polymer linear poly(ethyleneimine) (linPEI). The complexes were incubated with HeLa cells and luciferase expression was assessed. The type of non-viral carrier used determined the extent and duration of protein expression. Maximal duration of mRNA expression was about 9 days for Lipofectamine complexes, i.e. not very much shorter than with pDNA polyplexes. Interestingly, luciferase activity was already detected 30 min after adding the mRNA complexes to the cells, independent on the type of carrier. We also assessed the proportion of cells that become transfected by means of transfection with an mRNA encoding GFP. For both cationic lipids transfection with mRNA yielded a substantially larger fraction of transfected cells (more than 80%) than transfection with pDNA (40%). In addition we tested the carriers for their ability to mediate delivery of mRNA encoding CXCR4 into mesenchymal stem cells. The fraction of CXCR4-positive cells obtained with the mRNA-cationic lipid complexes was around 80%, as compared to 40% for the linPEI polyplexes. Our results demonstrate that the advantage of the use of mRNA over that of pDNA may under certain conditions outweigh the disadvantage of the somewhat shorter expression period.

Effect of topical liposomes containing paromomycin sulfate in the course of Leishmania major infection in susceptible BALB/c mice.

The aim of this study was to evaluate the antileishmanial effects of topical liposomal paromomycin sulfate (PM) in Leishmania major-infected BALB/c mice. Liposomes containing 10 or 15% PM (Lip-PM-10 and Lip-PM-15, respectively) were prepared by the fusion method and were characterized for their size and encapsulation efficiency. The penetration of PM from the liposomal PM formulations (LPMFs) through and into skin was evaluated in vitro with Franz diffusion cells fitted with mouse skin at 37 degrees C for 8 h. The in vitro permeation data showed that almost 15% of the LPMFs applied penetrated the mouse skin, and the amount retained in the skin was about 60% for both formulations. The 50% effective doses of Lip-PM-10 and Lip-PM-15 against L. major promastigotes in culture were 65.32 and 59.73 microg/ml, respectively, and those against L. major amastigotes in macrophages were 24.64 and 26.44 microg/ml, respectively. Lip-PM-10 or Lip-PM-15 was used topically twice a day for 4 weeks to treat L. major lesions on BALB/c mice, and the results showed a significantly (P < 0.001) smaller lesion size in the mice in the treated groups than in the mice in the control group, which received either empty liposomes or phosphate-buffered saline (PBS). Eight weeks after the beginning of the treatment, every mouse treated with LPMFs was completely cured. The spleen parasite burden was significantly (P < 0.001) lower in mice treated with Lip-PM-10 or Lip-PM-15 than in mice treated with PBS or control liposomes, but no significant difference was seen between the two groups treated with either Lip-PM-10 or Lip-PM-15. The results suggest that topical liposomal PM may be useful for the treatment of cutaneous leishmaniasis.