Mozafari Method Research Articles

Insight into heating method and Mozafari method as green processing techniques for the synthesis of micro- and nano-drug carriers

Abstract Drug delivery systems, also known as bioactive carriers, are currently an important contribution to the pharmaceutical and biomedical industries. A leading category of these drug carriers is lipid- and phospholipid-based systems including liposomes, nanoliposomes, solid lipid nanoparticles, nanostructured lipid vesicles, archaeosomes, and tocosomes. At present, there are several methods available for the preparation of the lipidic drug carriers at the micro- and nanoscales. There are some misunderstandings and confusion in the literature regarding two of the scalable and environment-friendly (green) techniques developed in our laboratory, namely the heating method and the Mozafari method. These methods are superior to conventional procedures used in the synthesis of drug carriers due to the fact that they do not involve utilization of potentially toxic solvents, detergents, or high-shear homogenizations. This entry is aimed to clarify differences between these methods to the peers and colleagues in academia as well as relevant industries. Some details of the industrially applied patented instrument used in the manufacturing of lipidic carriers are also provided.

The effect of antioxidant properties of free and encapsulated rosemary extract in liposome on the oxidation process of canola oil

SummaryThe antioxidant property of rosemary extract as a natural antioxidant on the oxidation process of canola oil has been reported and compared with the synthetic antioxidant butylated hydroxytoluene (BHT) (at 200 mg Lˉ1). The effect of free and liposomal encapsulated rosemary extract at concentrations of 200, 600 and 1000 mg Lˉ1 was evaluated on improving the oxidative stability of canola oil at 120°C using the Rancimat method. The changes in peroxide value, anisidine, TOTOX and thiobarbituric acid (TBA) have been determined by the Schaal oven test at 60°C. Liposomal rosemary extracts were prepared by the Mozafari method. Then physicochemical properties (Z‐potential, particle size and encapsulation efficiency (EE)) were determined. EE and the Z‐potential of liposomes were obtained at 54.59% and −65.1 mV, respectively. The particle size of empty and extract containing liposomes was determined 583.5 nm and 265.4 nm, respectively. The total polyphenol content of the rosemary extract by reagent of the Folin–Ciocalteu's phenol was determined as 164.2 mg gallic acid gˉ1 extract. Also, the activity of free radical scavenging of both liposomal encapsulated and free extracts, by the 2‐2‐diphenyl‐1‐picrylhydrazyl (DPPH) method, were 84.57% and 92.5%, respectively. The induction periods of oil containing liposomal rosemary extract at 600, 1000 mg Lˉ1 and free extract in level 1000 mg Lˉ1 was higher in comparison with samples containing BHT and blank (P ≤ 0.05). The incorporation of rosemary extract especially in liposome form into oil delayed the oxidation process. Based on the results of this study, to inhibit oil oxidation, rosemary extract, especially in the liposomal form, can be applied instead of synthetic antioxidants such as BHT.

A Review of Patents on “Mozafari Method” as a Green Technology for Manufacturing Bioactive Carriers

Encapsulation and controlled release of bioactive compounds is now an established protocol to enhance the bioavailability and long-lasting efficacy of therapeutic and disease-preventive agents while minimizing or eliminating side effects. Currently, there are a number of products approved by the regulatory authorities for Human or animal use, which contain encapsulated drugs, nutraceuticals, diagnostic agents, cosmetics, or even vaccines. Manufacturing the encapsulation/carrier systems requires special considerations with respect to scalability, environmental issues, and cost-effectiveness. Among the many available procedures for large-scale preparation of micro- and nanocarriers, the "Mozafari method" has proven to be simple to implement and reproducible technique that does not require sophisticated equipment or use of potentially toxic solvents. The mentioned proprietary method and patents in which this method is utilized or incorporated is the focus of the present review article.

Investigation of Temperature-Responsive Tocosomal Nanocarriers as the Efficient and Robust Drug Delivery System for Sunitinib Malate Anti-Cancer Drug: Effects of MW and Chain Length of PNIPAAm on LCST and Dissolution Rate

In this study, for the first time, the coated tocosome by blend of chitosan, CS, and poly(N-isopropylacrylamide), PNIPAAm, was developed as the efficient and robust drug delivery system with improved drug encapsulation efficiency, extended stability, proper particle size and industrial upscaling for Sunitinib malate anti-cancer drug. Tocosome was synthesized by using Mozafari method as a scalable and robust method and without the need for organic solvents. The effects of tocosome composition and drug concentration on the stability, particle size of tocosome, zeta potential, encapsulation efficacy and loading of drug into it were investigated by Taguchi method, and optimum composition was selected for combining with the polymeric blend. Homopolymer of PNIPAAm was synthesized by two different polymerization methods, including free radical and reversible addition–fragmentation chain transfer (RAFT). Effects of molecular weight (MW) and chain length of the polymers on lower critical solution temperature (LCST) were examined. The developed nanocarrier in this research, CS-Raft-PNIPAAm-tocosome, indicated LCST value beyond 37°C (about 45°C) and this is suitable for hyperthermia and spatio-temporal release of drug particles.

Entrapment of rosemary extract by liposomes formulated by Mozafari method: physicochemical characterization and optimization

A major obstacle in the utilization of phenolic antioxidant compounds is their sensitivity and as a result stability issue. The current study aimed to encapsulate polyphenolic compounds, extracted from Rosemary, in liposomes prepared by the Mozafari method without the utilization of toxic solvents or detergents. The extract was prepared and converted into a powder by freeze-drying. The process conditions were optimized using response surface analysis, and the optimal parameters were as follows: phosphatidylcholine (PC), 2.5% (25 mg/mL); extract, 0.7% (7 mg/mL); process temperature, 70 °C and process time, 60 min. The entrapment efficiency in optimal sample was 54.59%. Also, optimal glycerosomes formulation were finally physicochemical characterized (permeability, zeta potential, and size distribution). The mean size of empty and containing rosemary extract glycerosome were 265.4 nm and 583.5 nm, respectively, and the Z-potential of optimal glycerosome was -65.1 mV. Total phenolic content was obtained 151.38 mg gallic acid/g extract, in optimal liposomal formulation, which was measured by Folin-Ciocalteu's phenol reagent. Also, the antioxidant activity of rosemary extract by DPPH for the free and optimal liposomal formulation was determined to be 84.57% and 92.5% respectively. It can be concluded that the liposomal rosemary extract formulation prepared in this study, employing a safe, scalable, and green technology, has great promise in food and pharmaceutical applications.

Synthesis of Curcumin Loaded Smart pH-Responsive Stealth Liposome as a Novel Nanocarrier for Cancer Treatment

The innovation of drug delivery vehicles with controlled properties for cancer therapy is the aim of most pharmaceutical research. This study aims to fabricate a new type of smart biocompatible stealth-nanoliposome to deliver curcumin for cancer treatment. Herein, four different types of liposomes (with/without pH-responsive polymeric coating) were synthesized via the Mozafari method and then characterized with several tests, including dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FTIR), Zeta potential, and field emission scanning electron microscopes (FE-S EM). The loading and release profile of curcumin were evaluated in two pH of 7.4 and 6.6. Finally, the MTT assay was used to assess the cytotoxicity of the samples. FE-SEM results revealed a mean size of about 40 and 50 nm for smart stealth-liposome and liposome, respectively. The results of drug entrapment revealed that non-coated liposome had about 74% entrapment efficiency, while it was about 84% for PEGylated liposomes. Furthermore, the drug released pattern of the nanocarriers showed more controllable release in stealth-liposome in comparison to non-coated one. The results of the cytotoxicity test demonstrated the toxicity of drug-loaded carriers on cancer cells. Based on the results of this study, the as-prepared smart stealth pH-responsive nanoliposome could be considered as a potential candidate for cancer therapy.

Liposomal Green Tea Extract: Optimization and Physicochemical Characterization

Introduction: Although Green tea is a rich source of antioxidant, use of this herbal in food industries is limited due to its oxygen and light instabilities. Materials and Methods: Green tea polyphenols were encapsulated into liposomes using Mozafari method (with no solvents and detergents) to improve bioavailability of tea polyphenols. Screening design was used to find the major variables within all possible process variables. Then, optimal conditions were studied using response surface. Results: The most appropriate condition was achieved using phosphatidylcholine of 4.5% (w/w), extract concentration of 0.7%, mixing time of 30 min and temperature of 50 °C. Encapsulation efficiency (EE) depended on concentrations of the green tea extract and phosphatidylcholine. The EE in optimal formulation was reached as 51.34%. The particle size and Z-potential of liposomal green tea extract were assessed at 419 nm and -59.7 mV, respectively. The total polyphenol content (TPC) of green tea included 164.2 mg gallic acid/g extract. Free radical scavenging activities of free and liposomal extracts were calculated as 90.6 and 93.37%, respectively, using 2-2-diphenyl-1-pycrylhydrazyl (DPPH) method. Conclusions: Results revealed that liposomal green tea extract can be used extensively in food industries due to its high antioxidant activity. No size decreasing methods (e.g. sonication and homogenization) were needed to produce nanosize liposomal extracts to avoid structure instability.

Entrapment of Rosemary Extract by Liposomes Formulated by Mozafari Method Without Employing Toxic Solvents or Detergents: Physicochemical Characterization and Optimization

A major obstacle in the utilization of phenolic antioxidant compounds is their sensitivity and as a result stability issue. The current study aimed to encapsulate polyphenolic compounds, extracted from Rosemary, in liposomes prepared by the Mozafari method without the utilization of toxic solvents or detergents. The extract was prepared and converted into a powder by freeze-drying. The encapsulation protocol was optimized by changing the main process variables (i.e. liposomal ingredients, process temperature, and time). Results demonstrated that to achieve the highest rate of encapsulation, the best concentration of phosphatidylcholine (PC) and the extract were ca. 2.5% and 0.7% (w/w) respectively. The optimum process temperature and time were 70°C and 60 minutes respectively. It can be concluded that the liposomal rosemary extract formulation prepared in this study, employing a safe, scalable, and green technology, has great promise in food and pharmaceutical applications.

Physical stability of microliposomes in bene (Pistacia atlantica) oil with different formulations

Bene oil (pistacia atlantica), as a plant source, is rich in phenolic and tocopherol compounds and has significant antioxidant, therapeutic and antimicrobial effects. Encapsulation of hydrophobic compounds in liposome system is an ideal solution for protecting them against destruction during storage. An important advantage of liposomes is the encapsulation of hydrophilic, hydrophobic and amphiphilic compounds by using natural phospholipids, such as lecithin, with beneficial effects. The aim of this study is to encapsulate the bene kernel oil in the form of microliposomes. For this purpose, the effect of composition of liposomes based on lecithin and cholesterol was studied using the Mozafari method. Liposomes are prepared using lecithin and cholesterol in the ratios of 60:0, 50:10, 40:20 and 30:30. Particle size, size distribution, zeta potential and encapsulation efficiency were charac-terised. According to the result, the size of liposomes was dependent on their composition, but the wasaffected significantly affected by adding cholesterol (P < 0.05). Average diameter of the particles was between 4 and 9 µm. Liposome with a ratio of 40:20 had the smallest size. By applying cholesterol, zeta potential increased from 16.4 mV to 32.39 mV, which indicates electrostatic stability of liposomes. In general, with encapsulation efficiency of 84.33%, the ratio of 40:20 is considered as an ideal concentration in the formulation of microliposomes. Based on the results, bene oil-loaded liposomes with a lecithin:cholesterol formulation ratio of :as 40:20 was chosen as an optimal formulation because of its smaller particle size, higher zeta potential and suitable stability, which can be used in trapping, delivering and releasing hydrophilic, adipose-friendly and amphiphilic compounds (dual-friendly).

Physical stability of microliposomes in bene (Pistacia atlantica) oil with different formulations

Bene oil (pistacia atlantica), as a plant source, is rich in phenolic and tocopherol compounds and has significant antioxidant, therapeutic and antimicrobial effects. Encapsulation of hydrophobic compounds in liposome system is an ideal solution for protecting them against destruction during storage. An important advantage of liposomes is the encapsulation of hydrophilic, hydrophobic and amphiphilic compounds by using natural phospholipids, such as lecithin, with beneficial effects. The aim of this study is to encapsulate the bene kernel oil in the form of microliposomes. For this purpose, the effect of composition of liposomes based on lecithin and cholesterol was studied using the Mozafari method. Liposomes are prepared using lecithin and cholesterol in the ratios of 60:0, 50:10, 40:20 and 30:30. Particle size, size distribution, zeta potential and encapsulation efficiency were characterised. According to the result, the size of liposomes was dependent on their composition, but the wasaffected significantly affected by adding cholesterol (P < 0.05). Average diameter of the particles was between 4 and 9 µm. Liposome with a ratio of 40:20 had the smallest size. By applying cholesterol, zeta potential increased from 16.4 mV to 32.39 mV, which indicates electrostatic stability of liposomes. In general, with encapsulation efficiency of 84.33%, the ratio of 40:20 is considered as an ideal concentration in the formulation of microliposomes. Based on the results, bene oil-loaded liposomes with a lecithin: cholesterol formulation ratio of :as 40:20 was chosen as an optimal formulation because of its smaller particle size, higher zeta potential and suitable stability, which can be used in trapping, delivering and releasing hydrophilic, adipose-friendly and amphiphilic compounds (dual-friendly).

Development and Characterization of Liposomal Formulations Containing Phytosterols Extracted from Canola Oil Deodorizer Distillate along with Tocopherols as Food Additives.

Phytosterols are plant sterols recommended as adjuvant therapy for hypercholesterolemia and tocopherols are well-established anti-oxidants. However, thermo-sensitivity, lipophilicity and formulation-dependent efficacy bring challenges in the development of functional foods, enriched with phytosterols and tocopherols. To address this, we developed liposomes containing brassicasterol, campesterol and β-sitosterol obtained from canola oil deodorizer distillate, along with alpha, gamma and delta tocopherol. Three approaches; thin film hydration-homogenization, thin film hydration-ultrasonication and Mozafari method were used for formulation. Validated liquid chromatographic tandem mass spectrometry (LC-MS/MS) was utilized to determine the entrapment efficiency of bioactives. Stability studies of liposomal formulations were conducted before and after pasteurization using high temperature short time (HTST) technique for a month. Vesicle size after homogenization and ultrasonication (<200 nm) was significantly lower than by Mozafari method (>200 nm). However, zeta potential (−9 to −14 mV) was comparable which was adequate for colloidal stability. Entrapment efficiencies were greater than 89% for all the phytosterols and tocopherols formulated by all three methods. Liposomes with optimum particle size and zeta potential were incorporated in model orange juice, showing adequate stability after pasteurization (72 °C for 15 s) for a month. Liposomes containing phytosterols obtained from canola waste along with tocopherols were developed and successfully applied as a food additive using model orange juice.

OPTIMIZATION OF MIXING TEMPERATURE AND SONICATION DURATION IN LIPOSOME PREPARATION

Liposomes are a delivery system used in pharmaceutical products and cosmetics. Liposomes have many advantages such as increase stability and efficacy, can be targeted to reduce toxicity and increase accumulation at the target site and are biocompatible. Preparation of liposomes can be done by conventional or new methods which are still being developed. Conventional methods often require a long time and organic solvents which may be toxic. Heating (Mozafari method) is one of the new methods developed in the manufacture of liposomes without organic solvents. Mixing temperature can affect the physical properties of liposomes. The particle size has become one of the important physical properties because it affects the absorption of the drug. Sonication is an easy method of choice in reducing the size of liposomes. Optimization of mixing temperature and duration of sonication in liposomes’ preparation using new heating methods and sonication were performed by factorial design with 2 factors and 3-levels to obtain optimal liposome size. Data were analyzed with two-way ANOVA. The results showed that both mixing temperature and sonication duration significantly affect liposome size, but the interaction was not statistically significant. Data analysis also showed that mixing temperature, sonication, and their interaction do not affect the polydispersity index of liposome. Results showed the optimum mixing temperature and sonication duration that can produce liposomes with size below 100 nm is at 60°C for 30 minutes.

Tocosome: Novel drug delivery system containing phospholipids and tocopheryl phosphates

The potential of two derivatives of vitamin E, i.e. α-tocopheryl phosphate (TP) and di-α-tocopheryl phosphate (T2P), for the protection and delivery of therapeutics is being investigated in our laboratory and some promising results have been obtained so far. Novel nanocarrier systems containing TP, T2P and different lipids/phospholipids (phosphatidylcholine, stearyl amine, Phospholipon 90H, Phospholipon 100H) with and without cholesterol were prepared using Mozafari method. The preparation method did not require utilization of potentially toxic solvents, detergents or employment of harsh treatments such as sonication or high-shear force homogenization. Tocosomes, containing 5-fluorouracil (as a model drug), were shown to possess narrow size distribution, acceptable encapsulation efficiency and long-term stability (more than two years).

Novel nanoliposomal encapsulated omega-3 fatty acids and their applications in food.

The aim of the present research was to evaluate the application, stability and suitability of ω3 polyunsaturated fatty acids (PUFAs) incorporated nanoliposomes in food enrichment. Nanoliposomal ω3 PUFAs was prepared by Mozafari method, and their application in bread and milk was compared with unencapsulated (fish oil) and microencapsulated ω3 PUFAs. Sensory evaluation was conducted to determine the perceptible sensory difference/similarity between control, unencapsulated, microencapsulated, and nanoliposomal ω3 PUFAs enriched foods. Results showed no significant (p=0.11) detectable difference between control and nanoliposomal ω3 PUFAs enriched samples while, samples enriched with unencapsulated or microencapsulated ω3 PUFAs showed significant (p=0.02) fishy flavor. Moreover, significantly (p<0.01) higher ω3 PUFAs % recovery and lower peroxide and anisidine values were observed in nanoliposomal ω3 PUFAs enriched samples in comparison with other samples. In conclusion, an effective and reproducible method for application of ω3 PUFAs in the food system was developed.

Optimization on preparation condition of polyunsaturated fatty acids nanoliposome prepared by Mozafari method

This study presents the application of the response surface methodology (design) to develop an optimal preparation condition (independent variables) namely shear rate (600–1000 rpm), mixing time (30–60 min), and sonication time (10–20 min) for polyunsaturated fatty acids (docosahexaenoic acid and eicosapentaenoic acid) nanoliposomes. Fifteen lipid mixtures were generated by the Box–Behnken design and nanoliposomes were prepared by the Mozafari (direct hydration and without using organic solvents) method. Nanoliposomes were characterized with respect to entrapment efficiency (EE) and vesicle size as Y1 and Y2 dependent variables, respectively. The results were then applied to estimate the coefficients of response surface model and to find the optimal preparation conditions with maximum EE and minimum vesicle size. The response surface analysis exhibited that the significant (p < 0.05) second-order polynomial regression equations were successfully fitted for all dependent variables with no significant (p > 0.05) lack of fit for the reduced models. The response optimization of experiments was the shear rate: 795 rpm; mixing time: 60 min; and sonication time: 10 min. The optimal nanoliposome had an average diameter of 81.4 nm and EE of 100%. The experimental results of optimal nanoliposomes characterization confirmed an accurate fitness of the predicted values by reduced response surface models.

Stability and catalytic kinetics of protease loaded liposomes

The purpose of this study was to prepare liposomes through Mozafari method to encapsulate Flavourzyme as the flavor-enhancing enzyme at cheese ripening. Some kinetic parameters of encapsulated protease were determined and compared with corresponding free enzyme. Kinetic studies indicated an increase in both Michaelis–Menten constant and maximum velocity in encapsulated Flavourzyme. Optimum incubation temperature and pH of encapsulated enzyme did not show any significant difference with the free one. Evaluation of the thermal stability (at 35, 45 and 55°C) shows that encapsulated enzyme was stable at 35°C during in 6h. Also decreased stability of encapsulated enzyme has been observed at 65 and 75°C in 1h. The results show increase of stability of encapsulated enzymes in pHs 7 and 8 (7h), while it was decreased in acidic condition. Results indicate the gradually fulfilling proteolysis of casein by liposomal loaded Flavourzyme in comparison with the free enzyme.

Comparative study of the oxidative and physical stability of liposomal and nanoliposomal polyunsaturated fatty acids prepared with conventional and Mozafari methods

The relative oxidative stability of freshly prepared and stored liposomal and nanoliposomal systems of docosahexaenoic acid (DHA, 22:6 n−3) and eicosapentaenoic acid (EPA, 20:5 n−3) were investigated. The effects of organic solvents on the oxidative stability of liposomal polyunsaturated fatty acids (PUFAs) produced by two methods, the Bangham thin-film hydration (conventional rotary evaporation method and using organic solvents) and Mozafari (direct hydration and without using organic solvents) methods, were compared. The highest physicochemical stability was observed in PUFA liposomes prepared by the Mozafari method, followed by conventional liposomes and bulk PUFAs. There was no significant change in physicochemical stability during 10months of cold storage (4°C) in the dark. Moreover, the comparison between liposomes (>200nm) and nanoliposomes (50–200nm) revealed that the surface charge, physical stability and oxidative stability of liposomal PUFAs increased as the size of the liposomes decreased. The differences in the oxidative stability of PUFAs may be due to the protective effects of aqueous systems, which indicate the advantage of using non-organic solvent (water and CO2) techniques in liposome manufacturing.

Microscopical investigations of nisin-loaded nanoliposomes prepared by Mozafari method and their bacterial targeting

Nanoencapsulation may improve activity of protein or polypeptide antimicrobials against a variety of microorganisms. In this study, nanoliposomes prepared from different lipids (Phospholipon 90H, Phospholipon 100H, dipalmitoylphosphatidylcholine (DPPC), stearylamine (SA), dicetyl phosphate (DCP) and cholesterol) by a new, non-toxic and scalable method, were tested for their capacity to encapsulate nisin Z and target bacteria ( Bacillus subtilis and Pseudomonas aeruginosa). Factors affecting the entrapment efficiency (charge and cholesterol concentration in the vesicles) and stability of nanoliposomes were assessed. The nanoliposomes and their bacterial targeting were visualised, using different microscopes under air and liquid environments. Nisin was entrapped in different nanoliposomes with encapsulation efficiencies (EE) ranging from 12% to 54%. Anionic vesicles possessed the highest EE for nisin while increase in cholesterol content in lipid membranes up to 20% molar ratio resulted in a reduction in EE. Stability of nanoliposome-encapsulated nisin was demonstrated for at least 14 months at 4 °C (DPPC:DCP:CHOL vesicles) and for 12 months at 25 °C (DPPC:SA:CHOL vesicles).