Efficient encapsulation of proteins in submicro liposomes using a supercritical fluid assisted continuous process

Objective To prepare PLGA/PEG microsphcres for sustained release of bovine serum albumin (BSA), and evaluate the effect of the micrnsphere composition, proportion of the components and integration of PEG on the size, BSA encapsulation rate and in vitro BSA release properties of the microspheres. Methods BSA-loaded PLGA/PEG microspheres were prepared by water-in-oil-in-water double emulsions-solvent evaporation method (W/O/W). The morphology of the microspheres was observed with scanning elect'on microscope, the diameter was determined by Mastersizer 2000, and the encapsulation efficiency and in vitro BSA release were evaluated by UV spectrophotometry. Results Increased GA to LA ratio was associated with increased size of the microspheres and accelerated BSA release. The incorporation of PEG obviously increased the BSA encapsulation rate to as much as 88.22% with also increased BSA release rate. The BSA-PLGA/PEG biodegradable microspheres prepared was characterized by high encapsulation rate, low burst release, and slow BSA release for over 3 weeks at high drug concentrations. Conclusion BSA-PLGA/PEG microspheres with relatively high encapsulation efficiency and proper drug loading can be prepared by adjusting the parameters during the preparation process. Key words: PLGA/PEG microspheres; Bovine serum albumin; Double emulsious-solvent evaporation technique; Controlled release

Liposomes are spherical vesicles formed by a inner aqueous core and a double lipidic layer around it. Conventional techniques for the production of liposomes are characterized by several drawbacks, like the production of micrometric vesicles, a difficult control of the Particle Size Distribution (PSD) and low encapsulation efficiencies (EE) of hydrophilic compounds. Many supercritical semi-continuous techniques were proposed in literature. They are successful in the intent of producing liposomes of smaller diameter, but the EE of hydrophilic compounds and the reproducibility are still a challenge. For this reason, it was recently proposed a new supercritical process whose aim is to invert the steps of production of liposomes, by first creating water droplets and then to fast surround them by phospholipids. We discovered that the high diffusion coefficient of phospholipids in supercritical carbon dioxide allows a fast coverage of water droplets preserving the drug content into the liposome core. In this work, hydrophilic compounds were encapsulated in the vesicles produced using SuperLip, such as Fluorescein, Bovine Serum Albumin (BSA) and Ampicillin, obtaining monodispersed spherical vesicles with a mean size from 100 to 300 nm. Operative parameters like water flow rate and lipid to water mass ratio were optimized. The EEs were evaluated with UV-Vis spectroscopy according to methods reported in literature, and obtaining high values up to 99 % for the three investigated compounds.

The affinities of bovine serum albumin (BSA) with six kinds of steroid hormones (alpha-estradiol, ethynylestradiol, progesterone, androsterone, dehydroisoandrosterone and testosterone) were studied, and BSA was employed as a carrier to transport these hormones through the liquid membrane system, which consists of a hexane sauce phase (I), a BSA solution phase (1/15 phosphate buffer) and a hexane receiving phase (II). These steroid hormones dissolved in hexane were extensively transferred into a water phase containing BSA compared to the case of control (without added BSA). This suggests that these hormones are bound to BSA in a water phase. The ratio of hormone, BSA-binding form/free-form, is in the following order: ethynylestradiol greater than androsterone greater than testosterone greater than progesterone greater than dehydroisoandrosterone greater than alpha-estradiol in the water phase at the equilibrium conditions. The initial rate of hormone transfer from the hexane phase (I) to the water phase is followed by the first-order kinetics, and BSA induces an increase in the transfer rate except for the case of alpha-estradiol and ethynylestradiol. On the other hand, as for the increase rate of hormone concentration in the hexane phase (II), BSA makes the rate of transfer of progesterone faster than that of control, but it makes the rate of transfer of dehydroisoandrosterone more slowly. The results show the transport behaviors of steroid hormones across a water membrane are governed primarily by the partition coefficient between water and hexane phase and the addition of BSA accelerates the transport of hormones in the case of lower partition coefficients.

The effects of fabrication parameters on the morphology, drug loading, and initial burst release of poly(lactide‐co‐glycolide) microspheres loaded with bovine serum albumin were investigated to establish an optimal process and system for the in vivo delivery of therapeutic proteins. Through the addition of salts or sugars to induce an osmotic pressure in the external water phase, large microspheres were seen to have their morphology, drug loading, and initial burst release significantly affected. However, the effect was not observed for compact microspheres less than 10 μm in diameter. The presence of poly(vinyl alcohol), Pluronic F127, and Tween 80 in the internal water phase had detrimental effects on the drug loading because of the depressed stability of the primary emulsion and competitive interactions of surface‐active substances with the polymer. However, the simultaneous addition of salts to the external water phase resulted in enhanced drug loading and decreased initial burst. The polymer concentration and volume of the internal water phase were important factors influencing the characteristics of the microspheres. These parameters were optimized for achieving the maximal drug loading and a low initial burst. The solvent extraction method yielded microspheres with a higher drug loading and a lower initial burst in comparison with the solvent evaporation method. Different ranges of protein encapsulation efficiencies were obtained with blends of poly(lactide‐co‐glycolide) and poly(ethylene glycol), depending on the molecular weight and content of poly(ethylene glycol). © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

Biodegradable copolymeric polymersomes have been used for controlled drug delivery of proteins. These polymersomes important areas to overcome formulation associated problems of the proteins. The aim of this study was to develop polymersomes using biodegradable copolymers for delivery of bovine serum albumin (BSA) as a model protein. Encapsulated BSA by mPEG-PCL polymersomes led to formation of BSA-loaded mPEG-PCL polymersomes. The polymersomes synthesized with the protein-polymer ratio of 1:4 at 15 000 rpm gave maximum loading, minimum polydispersion with maximally sustained protein release pattern, among the prepared polymersomes. Investigation on FTIR and DSC results revealed that such a high encapsulation efficiency is due to strong interaction between BSA and the copolymer.The particles size and their morphology of polymersomes were determined by DLS and AFM.The encapsulation efficiency of BSA was 91.02%. The results of AFM showed that the polymersomes had spherical shapes with size of 49 nm.The sizes of BSA-loaded polymersomes ranged from 66.06 nm to 84.97 nm. The results showed that polymersomes exhibited a triphasic release, for BSA. Overall, the results indicated that mPEG-PCL polymersomes can be considered as a promising carrier for proteins.

Preparation and characterization of liposomes-in-alginate (LIA) for protein delivery system

Biodegradable polymer nano‐ and microparticles are of interest as drug carriers due to their capability to modulate drug release. Two different methods, including solvent displacement and spray drying, are used, resulting in the fabrication of Artemisone (ART)‐loaded polymeric nano‐ and microparticles, respectively. Scanning electron microscopy, transmission electron microscopy, dynamic light scattering, and asymmetric flow field flow fractionation (AF4) are employed to evaluate the morphology and size of the particles. The encapsulation and loading efficiency of the drug as well as cumulative release are characterized using high‐performance liquid chromatography (HPLC). HPLC results confirm that the spray drying method provides higher encapsulation efficiencies of about 97%, whereas, in nanoparticles prepared by solvent displacement, the highest encapsulation efficiency is 71%. Using these production methods, round‐shaped particles with homogeneous size distribution are fabricated. Moreover, the preparation procedure is optimized to obtain particles with the highest encapsulation and drug loading efficiency. Using the solvent displacement method, stable dispersions in water are obtained without adding any surfactant, whereas, for spray‐dried microparticles, tween 80 is needed to redisperse particles. The micro‐ and nanoparticles show different release properties, which are of interest for drug delivery application of hydrophobic drugs like ART.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the formation of intracellular cholesterol esters in various tissues. We recently reported the cloning and expression of human macrophage ACAT cDNA. In the current study, we report the production of specific polyclonal antibodies against ACAT by immunizing rabbits with the recombinant fusion protein composed of glutathione S-transferase and the first 131 amino acids of ACAT protein. Immunoblot analysis showed that the antibodies cross-reacted with a 50-kDa protein band from a variety of human cell lines. These antibodies immunodepleted more than 90% of detergent-solubilized ACAT activities from six different human cell types, demonstrating that the 50-kDa protein is the major ACAT catalytic component in these cells. In multiple human tissues examined, the antibodies recognized protein bands with various molecular weights. These antibodies also cross-reacted with the ACAT protein in Chinese hamster ovary cells. Immunoblot analysis showed that the ACAT protein contents in human fibroblast cells, HepG2 cells, or Chinese hamster ovary cells were not affected by sterol in the medium, demonstrating that the main mechanism for sterol-dependent regulation of ACAT activity in these cells is not change in ACAT protein content. As revealed by indirect immunofluorescent microscopy, the ACAT protein in tissue culture cells was located in the endoplasmic reticulum. This finding, along with earlier studies, suggests that cholesterol concentration in the endoplasmic reticulum may be the major determinant for regulating ACAT activity in the intact cells.

In this study, gemcitabine-loaded methoxy poly(ethylene glycol)-b-poly(L-lactide) (MPEG-PLLA) microparticles with different PEG block lengths were prepared by a W/O/W double emulsion technique. The present study focuses on the investigation of the influence of various preparative parameters such as the ratio of internal water phase and oil phase, polymer concentration, solvent composition of organic phase and salt concentration of external water phase on the morphology and encapsulation efficiency of the microparticles. The microparticles fabricated at high volume ratios of internal water phase to oil phase and at high polymer concentrations showed a relatively high encapsulation efficiency and low porosity. When a dichloromethane/ethyl acetate mixture was used as solvent, both the encapsulation efficiency and drug loading of the microparticles decreased as the level of ethyl acetate increased. The addition of a salt (NaCl) to the external water phase significantly improved the encapsulation efficiency up to 40%, and the microparticles became more spherical with their size and porosity decreased.

Recently, metformin (Met) has shown to have antineoplastic properties in cancer treatment by improving hypoxic tumor conditions, and causing reduction in the synthesis of biomolecules, which are vital for cancer growth. However, as an orally administered drug, Met has low bioavailability and rapid renal clearance. Thus, the goal of this study was to vectorize Met inside liposomes in the context of triple negative breast cancer (TNBC), which currently lacks treatment options when compared to other types of breast cancer. Vectorization of Met inside liposomes was done using Bangham method by implementing double design of experiment methodology to increase Met drug loading (minimum-run resolution V characterization design and Box-Behnken design), as it is generally extremely low for hydrophilic molecules. Optimization of Met-loaded liposome synthesis was successfully achieved with drug loading of 190 mg/g (19% w/w). The optimal Met-liposomes were 170 nm in diameter with low PdI (< 0.1) and negative surface charge (-20 mV), exhibiting sustained Met release at pH 7.4. The liposomal Met delivery system was stable over several months, and successfully reduced TNBC cell proliferation due to the encapsulated drug. This study is one the first reports addressing liposome formulation through thin-film hydration using two design of experiment methods aiming to increase drug loading of Met.

Liposomes are one of the most well-known and promising nanoscale drug delivery systems. Liposomal medicinal products are successfully used in clinical practice for cardiovascular, oncological, dermatological, and other indications. The development of liposomes and their widespread implementation in clinical practice are relevant tasks.The aim of the study was to summarise and analyse scientific data regarding the structure, composition, and classification of liposomes, as well as specific aspects of liposome production methods.This review covers up-to-date information on marketed liposomal medicinal products. The authors illustrate how production methods affect the composition and structure of liposomes. The size and lamellarity are important characteristics of liposomes that determine the encapsulation efficiency and biodistribution of active pharmaceutical ingredients (APIs). The choice of excipients depends on the intended use of liposomal medicinal products. The article summarises the main liposome production methods, highlighting specific usage aspects, advantages and disadvantages. Conventional liposome production methods are easy to apply and do not require complex equipment, and their principal disadvantages include the low efficiency of API encapsulation within liposomes and the high complexity of scaling up technological processes. The authors pay special attention to microfluidic techniques for liposome preparation, which are characterised by a highly controlled technological process (in terms of size distribution and lamellarity), high reproducibility, and scalability to the level of industrial production and are applicable to encapsulating different APIs.

Nanoparticles have been promptly studied and developed for oral protein delivery. Selection of excipients and formulation method depends on the physicochemical characteristics of the protein carried. Therefore, this study aims to design a formulation and characterize the alginate coated chitosan nanoparticles which carried different protein. Alginate coated chitosan nanoparticles were formed using an ionic gelation method. Optimization was done by varying the parameter such as crosslinker concentration, agitation method, rate, and time. The results show that chitosan nanoparticles formed by sonication using sodium tripolyphosphate (STPP): chitosan (1:0.8) was the best method to form nanoparticles. The particle size and polydispersity index (PDI) of bovine serum albumin (BSA) and lysozyme nanoparticles were 812.2 nm and 0.412 for BSA while 793.3 nm and 0.438 for lysozyme. Encapsulation efficiency (EE) of BSA is 52 % and lysozyme is 68 %. In vitro tests on acidic/gastric conditions showed that lysozyme (±32 %) was released faster than BSA (±10 %) during the 24 h incubation. Under neutral/terminal intestine conditions, percentage of BSA (±17 %) release is slightly higher than lysozyme (±13 %) for 8 h incubation period. It was concluded that the formulation of alginate coated chitosan nanoparticles in this study appears to be more effective for BSA delivery than lysozyme.&#x0D; HIGHLIGHTS&#x0D; &#x0D; Nanoparticle vesicular system is an effective approach for protein drug delivery system&#x0D; Factors affecting the formation of chitosan nanoparticle are crosslinker concentration, agitation method, rate, and time&#x0D; Alginate coated chitosan nanoparticle was more effective for oral delivery of BSA compared to lysozyme&#x0D; &#x0D; GRAPHICAL ABSTRACT

![Figure][1] J. Patrick Kampf Torrey Pines Institute for Molecular Studies, San Diego, California ![Figure][1] Alan M. Kleinfeld Torrey Pines Institute for Molecular Studies, San Diego, California akleinfeld{at}tpims.org The mechanism of free fatty acid (FFA)

Dactolisib (NVP-BEZ235, also referred to as: ′BEZ235′ or ′BEZ′) is a dual mTOR/PI3K inhibitor that is of potential interest in the treatment of inflammatory disorders. This work focuses on formulation of BEZ-loaded polymeric nanoparticles composed of a blend of poly(D,L-lactide-co-glycolide) (PLGA) and poly(D,L-lactide-co-glycolide)-poly(ethylene glycol)-2000 (PLGA-PEG). The nanoparticles were prepared by an oil/water emulsion solvent evaporation method, and were subsequently characterized for yield, encapsulation efficiency, morphology, particle size, drug-polymer interaction and in vitro drug release profiles. A targeted formulation was developed by conjugation of a S-acetyl-thioacetyl (SATA)-modified mouse-anti human E-selectin antibody to the distal end of PLGA-PEG-SPDP containing nanoparticles. Our results show the successful preparation of spherical PLGA/PLGA-PEG nanoparticles loaded with BEZ. The particle size distribution showed a range from 250 to 360nm with a high (>75%) BEZ encapsulation efficiency. Approximately 35% of the loaded BEZ was released within 10days at 37°C in a medium containing 5% bovine serum albumin (BSA). Evaluation of efficacy of anti-E-selectin decorated BEZ-loaded nanoparticles was carried out in tumor necrosis factor-α (TNF-α) activated endothelial cells. Confocal microscopy analysis showed that cellular uptake of the targeted nanoparticles and subsequent internalization. Cell functional assays, including migration assay and phosphowestern blot analysis of the mTOR and pI3K signaling pathways, revealed that the E-selectin targeted nanoparticles loaded with BEZ had a pronounced effect on inflammation-activated endothelial cells as compared to the non-targeted BEZ-loaded nanoparticles. In conclusion, E-selectin targeted nanoparticles have a high potential in delivering the potent mTOR/pI3K inhibitor dactolisib to inflamed endothelial cells and are an interesting nanomedicine for anti-inflammatory therapy.

Preparation and characterization of galactosylated chitosan coated BSA microspheres containing 5-fluorouracil