Double Emulsion Evaporation Method Research Articles

Advance in transdermal delivery of calcitonin using nanostructured lipid carrier-based emulgel

Context: Peptide-protein drugs have a very important role as therapeutic agents for various diseases. However, their therapeutic use has many barriers to delivery, such as large molecular weight, reduced stability during the manufacturing process and storage, and poor absorption when administered orally. One of peptide-protein drugs is calcitonin, a polypeptide of 32 amino acids used to overcome high levels of calcium in the blood, such as hyperparathyroidism. Nevertheless, drug delivery is still challenging to develop. Aims: To evaluate a calcitonin nanostructured lipid carrier-based emulgel, which could penetrate through the stratum corneum, and meet the stability requirements. Methods: Four formulas of calcitonin nanostructured lipid carrier (NLC) were prepared by the double emulsion-evaporation method, then all formulas were characterized in terms of particle size, polydispersity index, zeta potential, entrapment efficiency, and particle morphology. Calcitonin NLCs were then formulated into NLC-based emulgel. Further, in vitro penetration and stability of NLC calcitonin emulgel studies were conducted. Results: The 75:1 ratio of total lipid to the drug (F2) was optimal for calcitonin-loaded NLC with a particle size of 135.6 nm, an index polydispersity of 0.1, the zeta potential of 34.7 mV, and an entrapment efficiency of 99.6%. According to the percutaneous penetration study, the calcitonin NLC-based-emulgel resulted in a fivefold enhancement compared to the non-NLC calcitonin emulgel. Moreover, the stability study illustrated calcitonin levels after six months were 46.09-60.95% and 43.45-68.59% at storage conditions of 5 ± 3ºC and 25 ± 2ºC/RH 60 ±5 %, respectively. Conclusions: The calcitonin NLC-based-emulgel produced a fivefold enhancement permeation through the stratum corneum.

Orally deliverable sequence-targeted astaxanthin nanoparticles for colitis alleviation

Orally targeted strategy of anti-inflammatory agents has attracted tremendous attention for reducing highly health-care costs and enhancing the intervention efficiency of ulcerative colitis (UC). Herein, we developed a new kind of sequence-targeted astaxanthin nanoparticles for UC treatment. Astaxanthin nanoparticles were firstly designed by self-assembly method using (3-carboxypentyl) (triphenyl) phosphonium bromide (TPP)-modified whey protein isolate (WPI)-dextran (DX) conjugates. Subsequently, lipoic acid (LA) modified hyaluronic acid (HA) was coated on the surface of the nanoparticles by double emulsion evaporation method. Exhilaratingly, the constructed sequence-targeted astaxanthin nanoparticle exhibited excellent macrophages and mitochondria targeting ability, with a Pearson's correlation coefficient of 0.84 adstnd 0.92, respectively. In vivo imaging elucidated an obvious accumulation of the sequence-targeted nanoparticles in colon tissues in UC mice. Meanwhile, the reduction stimulus release features of astaxanthin were observed in the presence of 10 mM of glutathione (GSH) at pH 7.4. Most importantly, in vivo experiments indicated that sequence-targeted astaxanthin nanoparticles could markedly alleviate inflammation by moderating the TLR4/MyD88/NF-κB signaling pathway. What's more, the composition of gut microbiota and the production of short chain fatty acid were also improved upon the uptake of sequence-targeted astaxanthin nanoparticles. Our results suggested this novel astaxanthin nanoparticles, which showed sequence-targeted ability and reduction response feature, could be exploited as a promising strategy for effective UC treatment.

The synthesis of polycaprolactone (PCL) microspheres containing cerium (III) nitrate (Ce(NO3)3) self-healing agent via double emulsion evaporation method

In this study, the synthesis of polycaprolactone (PCL) microspheres containing cerium (III) nitrate (Ce(NO3)3) as a self-healing agent was performed by water-in-oil-in-water (W1/O/W2) double emulsion evaporation (DEE) method. The parameters including stirring speeds (100, 200, 400, 600, 800, and 1000 rpm) and mixing temperatures (room temperature, 50 °C, 60 °C, and 70 °C) were investigated during the DEE formation. Based on the results, an increase in stirring speeds resulted in a decrease in an average particle size of the PCL microspheres containing Ce(NO3)3; yet it did not affect the average particle size of the PCL microspheres without Ce(NO3)3 inside. The suitable temperature for synthesizing PCL microspheres containing Ce(NO3)3 as well as PCL microspheres without Ce(NO3)3 inside should not exceed 50 °C during DEE formation because the PCL compound started to melt at temperature above 50 °C. Based on the results, the conditions of DEE formation at the stirring speed of 1000 rpm and mixing temperature of 50 °C yielded the PCL microspheres containing Ce(NO3)3 with an average size of 5.48 μm.

Implication of nano-antioxidant therapy for treatment of hepatocellular carcinoma using PLGA nanoparticles of rutin.

The present work describes the development of poly(lactic co-glycolic acid) (PLGA) nanoparticles (NPs) of rutin (RT) for the treatment of hepatocellular carcinoma in rats. RT-loaded PLGA NPs (RT-PLGA-NPs) were prepared by double emulsion evaporation method. Further these are optimized by Box-Behnken design. PLGA NPs were evaluated for size, polydispersity index, drug-loading capacity, entrapment, gastric stability, invitro drug release, invivo preclinical studies and biochemical studies. Preclinical evaluation of RT-PLGA-NPs for anticancer activity through oral route exhibited significant improvement in hepatic, hematologic and renal biochemical parameters. Highly superior activity was observed in regulating oxidative stress and inflammatory markers, antioxidant enzymes, cytokines and inflammatory mediators and their role on plasma membrane ATPases responsible for destruction in liver tissues. Histopathological evaluation indicated reduced incidence of hepatic nodules, necrosis formation, infiltration of inflammatory cells, blood vessel inflammation and cell swelling with RT-PLGA-NP treatment along with considerable downregulation in the levels of proinflammatory cytokines.

PH-responsive and porous vancomycin-loaded PLGA microspheres: evidence of controlled and sustained release for localized inflammation inhibition in vitro.

Adequate delivery of antibiotics to infected sites is crucial for the effective treatment of bacterial infections. A controlled and sustained release system based on porous and pH-responsive poly(lactic-co-glycolic acid) (PLGA)–vancomycin (Van) microspheres was developed. In this system, drug release is triggered by the weakly acidic environment, like local inflamed tissues. The microspheres, developed through the W1/O/W2 double-emulsion evaporation method, comprised a PLGA-based shell and a core containing Van and the bubble-generating agent of NaHCO3. The optimized preparation conditions for PLGA–NaHCO3–Van microspheres were investigated and characterized. The PLGA–NaHCO3–Van microspheres exhibited porous microstructures with regular shape and uniform size and the characteristic of controlled drug release, which could be attributed to the incorporation of NaHCO3. The results of the Kirby–Bauer assay confirmed that released Van retained effective antibacterial activity towards standard Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) infected clinical samples, suggesting their further promising application in local anti-infection.

The influence of hydrophilic mPEG segment on formation, morphology, and properties of PCL‐mPEG microspheres

AbstractThis research displayed the differences of formation process, morphology, degradation, and release behaviors between PCL and PCL‐mPEG microspheres. Lysozyme loaded microspheres were prepared by double emulsion evaporation method, and the double droplets transformation process was observed and compared under microscope. Microspheres morphology, in vitro degradation and release behaviors were evaluated by scanning electron microscope, gel permeation chromatography, and bicinchoninic acid protein assay kit. PCL‐mPEG microspheres got a rough surface and the outer water phase penetrated into PCL‐mPEG double droplet easier than PCL during the transformation, accompanied with a high broken percentage and size swelling. PCL‐mPEG microspheres get a faster degradation/erosion rate than PCL microspheres, and the rate was in direct proportion to mPEG chain length. In vitro release analysis showed that PCL‐mPEG microspheres get a faster release rate than PCL microspheres, and the rate was also in direct proportion to mPEG chain length.

Imipenem/cilastatin encapsulated polymeric nanoparticles for destroying carbapenem-resistant bacterial isolates

BackgroundCarbapenem-resistance is an extremely growing medical threat in antibacterial therapy as the incurable resistant strains easily develop a multi-resistance action to other potent antimicrobial agents. Nonetheless, the protective delivery of current antibiotics using nano-carriers opens a tremendous approach in the antimicrobial therapy, allowing the nano-formulated antibiotics to beat these health threat pathogens. Herein, we encapsulated imipenem into biodegradable polymeric nanoparticles to destroy the imipenem-resistant bacteria and overcome the microbial adhesion and dissemination. Imipenem loaded poly Ɛ-caprolactone (PCL) and polylactide-co-glycolide (PLGA) nanocapsules were formulated using double emulsion evaporation method. The obtained nanocapsules were characterized for mean particle diameter, morphology, loading efficiency, and in vitro release. The in vitro antimicrobial and anti adhesion activities were evaluated against selected imipenem-resistant Klebsiella pneumoniae and Pseudomonas aeruginosa clinical isolates.ResultsThe obtained results reveal that imipenem loaded PCL nano-formulation enhances the microbial susceptibility and antimicrobial activity of imipenem. The imipenem loaded PCL nanoparticles caused faster microbial killing within 2–3 h compared to the imipenem loaded PLGA and free drug. Successfully, PCL nanocapsules were able to protect imipenem from enzymatic degradation by resistant isolates and prevent the emergence of the resistant colonies, as it lowered the mutation prevention concentration of free imipenem by twofolds. Moreover, the imipenem loaded PCL eliminated bacterial attachment and the biofilm assembly of P. aeruginosa and K. pneumoniae planktonic bacteria by 74 and 78.4%, respectively.ConclusionsThese promising results indicate that polymeric nanoparticles recover the efficacy of imipenem and can be considered as a new paradigm shift against multidrug-resistant isolates in treating severe bacterial infections.

Encapsulation of TOPO stabilized NaYF4:Er3+,Yb3+ nanoparticles in biocompatible nanocarriers: Synthesis, optical properties and colloidal stability

The emulsification process leading to up-converting NaYF4:Er3+,Yb3+ NPs encapsulation, was performed using a modified water/oil/water double emulsion evaporation method, where poly(lactic-co-glycolic acid) was used as biocompatible polymer. Span 80 and Cremophor A25 were applied as non-ionic surfactants and dichloromethane as oily phase. The use of trioctylphosphine oxide ligands for the synthesis of up-converting NaYF4:Er3+,Yb3+ NPs allowed to obtain spherical particles with sizes below 10nm, what further facilitated the efficient encapsulation process. Those newly designed nanosystems were subjected to analysis of their morphology, colloidal stability and optical properties by: dynamic light scattering, ζ-potential, atomic force microscopy, transmission electron microscopy and measuring the up-conversion emission spectra of free and loaded NaYF4:Er3+,Yb3+ NPs. The encapsulated NaYF4:Er3+,Yb3+ NPs showed increased colloidal stability for a long period of 60 days of storage in different conditions. Simultaneously, the encapsulation process did not significantly influenced their optical properties and strong visible emission could be observed upon nearinfrared excitation.

Laser-Activatible Gold Particles for Phase Transformation Imaging and Therapy in Metastatic Lymph Nodes

The gold nanoparticles were incorporated into PLGA/PFH (Au-PLGA/PFH) particles to observe their enhanced ultrasound imaging and treatment effect in rabbit metastatic lymph nodes. Double emulsion evaporation method was used to prepare the Au-PLGA/PFH particles. The size distribution, the morphological and structural characterization, the optical obsorbing property were estimated. The rabbit VX2 tumor was inoculated in rabbits subcutaneous thigh muscles to build popliteal lymph node metastasis of tumor models. After the Au-PLGA/PFH particles were introduced into lymph nodes by rabbit footpad, US were used to image the popliteal lymph nodes in real-time, also the Color Doppler Flow Image (CDFI) were acquired. With a wavelength of 532nm laser irradiation in vitro, the image of lymph nodes were observed under 2D and contrast enhance mode. Improving the laser energy, the irradiation was done every 48 h for a total of three times, this process was observed by US and CDFI. 48 h after the last irradiation, the rabbits were sacrificed, and the lymph node tissue were removed to measure the size, also the HE, immunohistochemistry and transmission electron microscopy (TEM) measurement were carried. The Au-PLGA/PFH particle is about 1 μm in diameter, the gold nanoparticles are located around the shell, these are clearly visible as small black dots in the PLGA shell. A UV-Vis shows there is a strong optical obsorbing peak of the particles in 525nm. Thus, with a 532 nm laser irradiation in vitro, the particles in lymph nodes absorb light energy to enhance the local temperature, when the temperature is over the phase transition threshold of PFH, the particles transfer into bubbles and enhance the US imaging. In addition, as the laser fluence increased, the creating bubble that expanded within the tumor cells, eventually destroyed the cells. Therefore, the lymph nodes enlarge slowly or smaller than before. HE, immunohistochemisty and TEM further conform this effect. This work demonstrates PLGA/PFH particles containing gold nanoparticles can use as US contrast agents and theragnostic agents.

HematoPorphyrin Monomethyl Ether polymer contrast agent for ultrasound/photoacoustic dual-modality imaging-guided synergistic high intensity focused ultrasound (HIFU) therapy.

This study is to prepare a hematoporphyrin monomethyl ether (HMME)-loaded poly(lactic-co-glycolic acid) (PLGA) microcapsules (HMME/PLGA), which could not only function as efficient contrast agent for ultrasound (US)/photoacoustic (PA) imaging, but also as a synergistic agent for high intensity focused ultrasound (HIFU) ablation. Sonosensitizer HMME nanoparticles were integrated into PLGA microcapsules with the double emulsion evaporation method. After characterization, the cell-killing and cell proliferation-inhibiting effects of HMME/PLGA microcapsules on ovarian cancer SKOV3 cells were assessed. The US/PA imaging-enhancing effects and synergistic effects on HIFU were evaluated both in vitro and in vivo. HMME/PLGA microcapsules were highly dispersed with well-defined spherical morphology (357 ± 0.72 nm in diameter, PDI = 0.932). Encapsulation efficiency and drug-loading efficiency were 58.33 ± 0.95% and 4.73 ± 0.15%, respectively. The HMME/PLGA microcapsules remarkably killed the SKOV3 cells and inhibited the cell proliferation, significantly enhanced the US/PA imaging results and greatly enhanced the HIFU ablation effects on ovarian cancer in nude mice by the HMME-mediated sono-dynamic chemistry therapy (SDT). HMME/PLGA microcapsules represent a potential multifunctional contrast agent for HIFU diagnosis and treatment, which might provide a novel strategy for the highly efficient imaging-guided non-invasive HIFU synergistic therapy for cancers by SDT in clinic.

A novel study on the mechanisms of drug release in PLGA-mPEG microspheres with fluorescent drug

The purpose of this research was to proof the microspheres release mechanism by a novel method-detecting and comparing the drugs fluorescent changes on the microspheres surface. Fluorescein sodium (FS, 0.4 kDa) and fluorescein isothiocyanate-bovine serum albumin (FITC-BSA, 66.8 kDa) were employed as model drugs. FS and FITC-BSA were encapsulated into PLGA-mPEG microspheres through double emulsion evaporation method, and the drug-loaded microspheres in vitro degradation and release behaviors were evaluated by scanning electron microscope, gel permeation chromatography, confocal laser scanning microscopy (CLSM), BCA assay kit, and UV–vis spectrophotometry. FS-loaded microspheres revealed a severe initial burst release, followed by a sustained release, and we could observe a bright fluorescent on the microspheres surface during the early release period under the CLSM. The bright fluorescent gradually faded out in the later period as only 1~2% FS was remained after 14 days release. FITC-BSA-loaded microspheres revealed a typical tri-phase release profile, and we observed a weak fluorescent on the microspheres surface after the initial burst release, and the fluorescent came bright again after an obvious erosion appeared on the microspheres surface. In the later release stage, the fluorescent gradually faded out as the fast release of FITC-BSA.

Preparation of biodegradable PCL particles via double emulsion evaporation method using ultrasound technique

Polymeric nanoparticles have attracted growing attention because of their unique properties and extensive application. In this study, polycaprolactone (PCL) nanoparticles were prepared via double emulsion solvent evaporation-like process using power ultrasound, and the effects of various process parameters on particle size, zeta potential, and morphology were investigated and optimized. Nanoparticles (NPs) were prepared by two-step emulsification process. In the first step, the inner aqueous phase (W1) was homogenized with organic phase (PCL in dichloromethane) to obtain primary emulsion. In the second step, the primary emulsion was emulsified with outer aqueous phase (W2) containing polyvinyl alcohol (PVA) as stabilizer using power ultrasound, followed by evaporation of solvent which resulted in a particulate suspension at the end. Effects of various parameters like ultrasound exposure time and amplitude, outer aqueous phase volume, PVA concentration, and PCL content were investigated. It has been shown that, by increasing ultrasound exposure time, amplitude, and outer aqueous phase volume, the particle size decreases. Additionally, particle size was also related to amount of PCL and PVA concentration. Spherical NPs with smooth surfaces were observed by scanning electron microscopy (SEM).

Evaluation of superparamagnetic iron oxide-polymer composite microcapsules for magnetic resonance-guided high-intensity focused ultrasound cancer surgery

BackgroundSuperparamagnetic poly (lactic-co-glycolic acid) (PLGA)-coated Fe3O4 microcapsules are receiving increased attention as potential diagnostic and therapeutic modalities in the field of oncology. In this study, PLGA-coated Fe3O4 microcapsules were combined with a magnetic resonance imaging-guided high-intensity focused ultrasound (MR-guided HIFU) platform, with the objective of investigating the effects of these composite microcapsules regarding MR-guided HIFU liver cancer surgery in vivo.MethodsPLGA-coated Fe3O4 microcapsules consisting of a liquid core and a PLGA-Fe3O4 shell were fabricated using a modified double emulsion evaporation method. Their acute biosafety was confirmed in vitro using MDA cells and in vivo using rabbits. To perform MR-guided HIFU surgery, the microcapsules were intravenously injected into a rabbit liver tumor model before MR-guided HIFU. T2-weighted images and MR signal intensity in normal liver parenchyma and tumor tissue were acquired before and after injection, to assess the MR imaging ability of the microcapsules. After MR-guided HIFU ablation tissue temperature mapping, the coagulative volume and histopathology of the tumor tissue were analyzed to investigate the ablation effects of MR-guided HIFUs.ResultsScanning and transmission electron microscopy showed that the microcapsules displayed a spherical morphology and a shell-core structure (mean diameter, 587 nm). The hysteresis curve displayed the typical superparamagnetic properties of the microcapsules, which are critical to their application in MR-guided HIFU surgery. In MR-guided HIFU surgery, these microcapsules functioned as an MRI contrast agent, induced significant hyperthermal enhancement (P < 0.05) and significantly enhanced the volume of coagulative necrosis (P < 0.05).ConclusionsThe administration of PLGA-coated Fe3O4 microcapsules is a potentially synergistic technique regarding the enhancement of MR-guided HIFU cancer surgery.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-800) contains supplementary material, which is available to authorized users.

Methotrexate-loaded PLGA nanobubbles for ultrasound imaging and Synergistic Targeted therapy of residual tumor during HIFU ablation

High intensity focused ultrasound (HIFU) has attracted the great attention in tumor ablation due to its non-invasive, efficient and economic features. However, HIFU ablation has its intrinsic limitations for removing the residual tumor cells, thus the tumor recurrence and metastasis cannot be avoided in this case. Herein, we developed a multifunctional targeted poly(lactic-co-glycolic acid) (PLGA) nanobubbles (NBs), which not only function as an efficient ultrasound contrast agent for tumor imaging, but also a targeted anticancer drug carrier and excellent synergistic agent for enhancing the therapeutic efficiency of HIFU ablation. Methotrexate (MTX)-loaded NBs were synthesized and filled with perfluorocarbon gas subsequently using a facile but general double emulsion evaporation method. The active tumor-targeting monoclonal anti-HLA-G antibodies (mAbHLA-G) were further conjugated onto the surface of nanobubbles. The mAbHLA-G/MTX/PLGA NBs could enhance the ultrasound imaging both in vitro and in vivo, and the targeting efficiency to HLA-G overexpressing JEG-3 cells has been demonstrated. The elaborately designed mAbHLA-G/MTX/PLGA NBs can specifically target to the tumor cells both in vitro and in vivo, and their blood circulation time in vivo was much longer than non-targeted MTX/PLGA NBs. Further therapeutic evaluations showed that the targeted NBs as a synergistic agent can significantly improve the efficiency of HIFU ablation by changing the acoustic environment, and the focused ultrasound can promote the on-demand MTX release both in vitro and in vivo. The in vivo histopathology test and immunohistochemical analysis showed that the mAbHLA-G/MTX/PLGA NBs plus HIFU group presented most serious coagulative necrosis, the lowest proliferation index and the highest apoptotic index. Therefore, the successful introduction of targeted mAbHLA-G/MTX/PLGA NBs provides an excellent platform for the highly efficient, imaging-guided and non-invasive HIFU synergistic therapy of cancer with the supplementary functions of killing residual tumor cells and preventing tumor recurrence/metastasis.

Superparamagnetic maghemite loaded poly (ε- caprolactone) nanocapsules : characterization and synthesis optimization

Iron oxide nanoparticles (ION) have been studied for essential applications, like detection of biological constituents (virus, bacterials, cell, nucleic acids, protein, enzyme, etc.), magnetic bioseparation and clinic therapy and diagnosis (such as MRI magnetic fluid and hyperthermia). In this work, γ-Fe2O3 has been synthetized by a adapted sol-gel method and entraped in poly e-caprolactone (PCL) nanocapsules. The superparamagnetic nanocapsules have been formulated by double emulsion evaporation method. Some variables affecting the polydispersity index, zeta potential surface and size of nanocapsules were studied aiming optimize the formulation process of maghemite-loaded PCL nanocapsules. The following parameters were selected: sonication time, PCL concentration in organic phase, PVA concentration in external aqueous phase and maghemite/PCL weight ratio. Under these experimental conditions, the resulting nanocapsules displayed a mean size of about 346 nm and a maghemite content of about 7.5 µg/mg of nanocapsules and superparamagnetic behaviour at room temperature.

Modified double emulsion process as a new route to prepare submicron biodegradable magnetic/polycaprolactone particles for in vivo theranostics

A modified double emulsion evaporation method was devised to encapsulate active ingredients to be used for theranostic purposes. In comparison with classical double emulsion methods, the solvents used for this modified method are miscible. Aqueous and organic ferrofluids having different solid contents of iron oxide nanoparticles were prepared by the co-precipitation method. Different materials were screened for the preparation of biodegradable and biocompatible nanoparticles via this new modified double emulsion evaporation method. Factors affecting the final particle size were investigated by encapsulating a hydrophilic model drug that permitted us to prepare a recipe using polycaprolactone (PCL) as polymer, dichloromethane (DCM) as organic solvent and polyvinyl alcohol (PVA) as stabilizing agent. The characterization of aqueous and organic ferrofluids alone, Stilbene and iron oxide loaded nanoparticles was done by studying particle size, size distribution, zeta potential and morphology, the magnetic properties and chemical composition. All the characterization results showed that the process is praiseworthy for encapsulation of iron oxide nanoparticles (in both aqueous and organic medium) along with an active ingredient for theranostic purposes with a final size of about 300 nm.

Novel Approach for Delivery of Insulin Loaded Poly(lactide-co-glycolide) Nanoparticles Using a Combination of Stabilizers

Insulin stability during microencapsulation and subsequent release is essential for retaining its biological activity. The successful delivery of insulin relies on the proper selection of stabilizers in addition to other parameters. Attempts were made to address the problem with a few combination of stabilizers for maintaining the integrity of insulin during formulation and delivery. Insulin loaded nanoparticles with different stabilizers such as pluronic F68, trehalose, and sodium bicarbonate were prepared by the double emulsion evaporation method using two different copolymer ratios of poly(DL-lactide-co-glycolide) (50:50 and 85:15). The presence of stabilizers in the nanoparticles resulted in an increase in the particle size but a reduction of encapsulation efficiency. Insulin release rate was comparatively higher for the batches containing stabilizers when compared with controls for both the copolymer ratios. Also the presence of stabilizers resulted in sustained release of insulin resulting in prolonged reduction of blood glucose levels in streptozotocin induced diabetic rats. From the in vitro and in vivo studies, we concluded that a combination of stabilizers results in beneficial effects without compromising the advantages of delivery systems.

Preparation of insulin-loaded PLA/PLGA microcapsules by a novel membrane emulsification method and its release in vitro

Uniform-sized biodegradable PLA/PLGA microcapsules loading recombinant human insulin (rhI) were successfully prepared by combining a Shirasu Porous Glass (SPG) membrane emulsification technique and a double emulsion-evaporation method. An aqueous phase containing rhI was used as the inner water phase (w 1), and PLA/PLGA and Arlacel 83 were dissolved in a mixture solvent of dichloromethane (DCM) and toluene, which was used as the oil phase (o). These two solutions were emulsified by a homogenizer to form a w 1/o primary emulsion. The primary emulsion was permeated through the uniform pores of a SPG membrane into an outer water phase by the pressure of nitrogen gas to form the uniform w 1/o/w 2 droplets. The solid polymer microcapsules were obtained by simply evaporating solvent from droplets. Various factors of the preparation process influencing the drug encapsulation efficiency and the drug cumulative release were investigated systemically. The results indicated that the drug encapsulation efficiency and the cumulative release were affected by the PLA/PLGA ratio, NaCl concentration in outer water phase, the inner water phase volume, rhI-loading amount, pH-value in outer water phase and the size of microcapsules. By optimizing the preparation process, the drug encapsulation efficiency was high up to 91.82%. The unique advantage of preparing drug-loaded microcapsules by membrane emulsification technique is that the size of microcapsules can be controlled accurately, and thus the drug cumulative release profile can be adjusted just by changing the size of microcapsules. Moreover, much higher encapsulation efficiency can be obtained when compared with the conventional mechanical stirring method.

PELA microspheres loaded H. pylori lysates and their mucosal immune response.

To prepare poly (D,L-lactide)-polyethylene glycol copolymer (PELA) microspheres loaded H.pylori lysates or Cystografin and observe their targeting in gastrointestinal mucous membrane or analyze the mucosal immune responses by oral administration. PELA microspheres loaded H.pylori lysates or Cystografin were prepared by double emulsion evaporation method. Their distribution in gastrointestinal mucous membrane was observed by CT. Balb/c mice orally immunized in mucosal immune responses, whose antibody production in salivary and gut washing and antibody secreting cells in Peyer's patches (PP) were estimated by ELISA and ELISPOT, respectively. The microspheres physical properties, such as particle size, protein level and morphology were investigated. All prepared microspheres were found to have a smooth surface morphology from 3.20-4.05 microm in diameter and high encapsulation efficiency from 74.9-82.2 %. No significant correlation in their physical properties was shown, depending on their molecular weight at the similar composition ratio. Immunization with all types of PELA-Hp microspheres elevated the saliva sIgA level at week 3 by approximately 3-4 times that with soluble antigen, which was greatly enhanced after boosting. At one week after last immunization with all types of PELA-Hp microspheres (week 8), the specific sIgA-ASCs, IgG-ASCs and sIgA in salivary rose obviously. In intestinal Peyer's patches, the specific sIgA-ASCs were 5.92-6.98X10(4)/ml cell and IgG-ASCs were 3.47-4.02X10(4)/ml cell, about 5-9 times higher than those with soluble antigen (P<0.01). ASCs in intestine were more than those in stomach and the majority of the ASCs were sIgA-ASCs. The sIgA in gut washing fluid was 1.62-1.85 OD, about 3-6 times tthat of those with soluble antigen. There were significant differences of the ASCs and sIgA in gut washing fluid as compared with those of PBS and MS-0 (P<0.05). There appeared to be good correlation between sIgA level in gut washing fluid and sIgA-ASCs in intestinal Peyer's patches. PELA microspheres may be used as vehicle to delivery antigen and adjuvant in designing oral vaccination.

Polylactide Microparticles Prepared by Double Emulsion-Evaporation: II. Effect of the Poly(Lactide-co-Glycolide) Composition on the Stability of the Primary and Secondary Emulsions

Polylactide and copolymers with glycolide have been used as coating polymers in the microencapsulation technique based on the w 1/o/w 2 double emulsion-evaporation method. Stability of the primary emulsion is of critical importance and clearly predetermines the internal morphology of the microspheres, in agreement with the very fast hardening of the oil phase (CH 2Cl 2) as soon as the primary emulsion is dispersed in water. When the peptide or protein to be encapsulated is able to interact with the coating polymer and possibly with the surfactant, the stabilization mechanism of the primary emulsion is very complex. It has been shown that the interaction of BSA (bovine serum albumin) with the polyester is as strong as the glycolide content is high, which results in the formation of a solid film at the w 1/o interface and in a high emulsion stability. Addition of a surfactant, such as poloxamer 188, in the oil phase has a deleterious effect on both the emulsion stability and the internal structure of the microspheres. Use of a dye tracer in the internal aqueous phase was not useful in probing the double emulsion stability and the encapsulation efficiency, because of specific interactions between the dye (indigocarmine) and the coating polyester.