Electrostatic Droplet Generation Research Articles

Formulation and Antimicrobial studies of Microencapsulated Musanga cecropioides Stem Bark Extract

Microencapsulation is the process of coating small droplets of liquid or solid materials with a continuous film of polymeric substances. The aim of the research was to formulate microparticles of Musanga cecropioides stem bark extract. The dried, powdered stem bark of Musanga cecropioides was extracted by maceration. The aqueous extract was microencapsulated using polymers such as alginate, pectin and hydroxylpropyl methylcellulose acetate succinate (HPMCAS) by counter-ion coacervation method using an electrostatic droplet generator. The formulated microcapsules were evaluated for flow properties, particle size, drug content, antimicrobial activity and in vitro release kinetics in simulated gastric and intestinal fluids. The microparticles were subjected to stability tests under different temperature (45°C, 25°C and 8°C) and relative humidity (100% RH, 78% RH and 0% RH) conditions for a period of three (3) months. The mean particle size of the microcapsules decreased with increasing applied voltage of the electrostatic generator. Particle size range for the batches of formulated microparticles was 68.20-223.84µm. The percentage entrapment efficiency of the batches of microparticles ranged from 80.26-86.58%. The microparticles lost a significant percent of its drug content after storage at different temperature and relative humidity conditions. The formulated microparticles and crude extract possess significant antibacterial action against both gram positive and negative bacteria as well as antifungal effect against C. albicans. Microparticles of M. cecropioides extract were formulated by counter-ion coacervation method using an electrostatic droplet generator which conferred advantages of sustained drug release, reduced frequency of dosing, masked unpalatable taste, improved stability and enhanced patient acceptance on the crude extract.

Alginate Microsphere Encapsulation of Drug-Loaded Nanoparticles: A Novel Strategy for Intraperitoneal Drug Delivery.

Alginate hydrogels have been broadly investigated for use in medical applications due to their biocompatibility and the possibility to encapsulate cells, proteins, and drugs. In the treatment of peritoneal metastasis, rapid drug clearance from the peritoneal cavity is a major challenge. Aiming to delay drug absorption and reduce toxic side effects, cabazitaxel (CAB)-loaded poly(alkyl cyanoacrylate) (PACA) nanoparticles were encapsulated in alginate microspheres. The PACAlg alginate microspheres were synthesized by electrostatic droplet generation and the physicochemical properties, stability, drug release kinetics, and mesothelial cytotoxicity were analyzed before biodistribution and therapeutic efficacy were studied in mice. The 450 µm microspheres were stable at in vivo conditions for at least 21 days after intraperitoneal implantation in mice, and distributed evenly throughout the peritoneal cavity without aggregation or adhesion. The nanoparticles were stably retained in the alginate microspheres, and nanoparticle toxicity to mesothelial cells was reduced, while the therapeutic efficacy of free CAB was maintained or improved in vivo. Altogether, this work presents the alginate encapsulation of drug-loaded nanoparticles as a promising novel strategy for the treatment of peritoneal metastasis that can improve the therapeutic ratio between toxicity and therapeutic efficacy.

Impact of electrostatic potential on microcapsule-formation and physicochemical analysis of surface structure: Implications for therapeutic cell-microencapsulation.

Cell-encapsulation is used for preventing therapeutic cells from being rejected by the host. The technology to encapsulate cells in immunoprotective biomaterials, such as alginate, commonly involves application of an electrostatic droplet generator for reproducible manufacturing droplets of similar size and with similar surface properties. As many factors influencing droplet formation are still unknown, we investigated the impact of several parameters and fitted them to equations to make procedures more reproducible and allow optimal control of capsule size and properties. We demonstrate that droplet size is dependent on an interplay between the critical electric potential (Uc,), the needle size, and the distance between the needle and the gelation bath, and that it can be predicted with the equations proposed. The droplet formation was meticulously studied and followed by a high-speed camera. The X-ray photoelectron analysis demonstrated optimal gelation and substitution of sodium with calcium on alginate surfaces while the atomic force microscopy analysis demonstrated a low but considerable variation in surface roughness and low surface stiffness. Our study shows the importance of documenting critical parameters to guarantee reproducible manufacturing of beads with constant and adequate size and preventing batch-to-batch variations.

The influence of spatial distribution on add-on therapy of designed Ca-Alg/CS MEMs system

To improve the efficacy and reduce the systemic toxicity of the diabetes mellitus, herewith, we developed a novel microparticles-embedded microcapsules (MEMs) system, synthesized from calcium alginate/chitosan (Ca-Alg/CS), by emulsion gelation using a high voltage electrostatic droplet generator. In our study, we selected two antidiabetic drugs insulin (INS) and metformin (MET) as model drugs to investigate different spatial distribution appropriate of MEMs system. Characterization based on particle size and morphology, encapsulation efficiency and drug loading, as well as drug delivery properties were carried out on the MEMs system. Typical multi-chamber structure was shown by SEM and the optical spectra. The average diameters of microparticles and Ca-Alg/CS MEMs were 2100 nm and 410 μm, respectively. Insulin and MET were embedded into MEMs via electrostatic reaction according to FT-IR spectra. Moreover, drug loading and encapsulation efficiency of INS were higher than that of MET in this system when drugs were loaded alone or together. More importantly, this system has potential for orderly drug release and well sustained release when MET in the inner and INS in the outer space could be applied as a combination therapy for diabetes. The obtained in vivo experimental data on diabetes rats has shown that the designed MEMs system resulted in a higher hypoglycemic effect within add-on therapy.

Establishment of a Model of Microencapsulated SGC7901 Human Gastric Carcinoma Cells Cocultured with Tumor-Associated Macrophages

The important factors of poor survival of gastric cancer (GC) are relapse and metastasis. For further elucidation of the mechanism, a culture system mimicking the microenvironment of the tumor in humans was needed. We established a model of microencapsulated SGC7901 human GC cells and evaluated the effects of coculturing spheres with tumor-associated macrophages (TAMs). SGC7901 cells were encapsulated in alginate-polylysine-sodium alginate (APA) microcapsules using an electrostatic droplet generator. MTT assays showed that the numbers of microencapsulated cells were the highest after culturing for 14 days. Metabolic curves showed consumption of glucose and production of lactic acid by day 20. Immunocytochemistry confirmed that Proliferating Cell Nuclear Antigen (PCNA) and Vascular Endothelial Growth Factor (VEGF) were expressed in microencapsulated SGC7901 cells on days 7 and 14. The expression of PCNA was observed outside spheroids; however, VEGF was found in the entire spheroids. PCNA and VEGF were increased after being cocultured with TAMs. Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expressions were detected in the supernatant of microencapsulated cells cocultured with TAMs but not in microencapsulated cells. Our study confirms the successful establishment of the microencapsulated GC cells. TAMs can promote PCNA, VEGF, MMP-2, and MMP-9 expressions of the GC cells.

Preparation of a MVL-Ca-Alg/CS MEMs system with add-on effect for type 2 diabetes treatment

ABSTRACTAdd-on drugs, often called as synergistic therapy, for diabetes have been comparatively more promising as they can reduce the systemic adverse effects resulted during respective monotherapy [metformin (MET)/insulin (INS)]. Herewith, we formulated a multivesicular liposome microparticles-embedded Ca-Alg/chitosan microcapsules (MVL-Ca-Alg/CS MEMs) system by a double-emulsion method using a high voltage electrostatic droplet generator. Physical characterization of the designed formulation was elucidated based on particle size and distribution, drug loading and encapsulation efficiency, drug delivery properties, and pharmacodynamic evaluation. The multivesicular liposomes microparticles (MVLs MPs) and MVL-Ca-Alg/CS MEMs have shown good sphericity and dispersion, and the average diameters were 37 and 491 µm, respectively. The confocal laser scanning microscopic observations demonstrated that fluorescein isothiocyanate-conjugated INS is uniformly dispersed in MVLs MPs, predominantly within the lumen of the polycystic liposome. This multicomponent system possessing INS in the inner space and MET at the outer space resulted in orderly and sustained drug release patterns. Furthermore, the obtained in vivo experimental data have shown that the designed MEMs system resulted in significantly higher hypoglycemic effect compared to pure INS, demonstrating that our multicomponent design has an enormous potential for treating diabetes.

Novel carboxymethyl chitosan-graphene oxide hybrid particles for drug delivery.

We describe an electrostatic droplet generation method to prepare a novel carboxymethyl chitosan-graphene oxide hybrid particles for delivery purpose. Under an adjustable electrostatic field, graphene oxide and carboxymethyl chitosan mixed solution was sprayed as uniform micro-droplets, which were solidified as particles in CaCl2 solution. Such hybrid particles are wished to have excellent stability in saline solution, and better delivery properties than pristine carboxymethyl chitosan particles. The effects of micro-droplets generation conditions on particles formation were systematically investigated. At conditions of 40 mg/ml of carboxymethyl chitosan, 2 mg/ml of graphene oxide, 3 ml/h of feed speed, electrostatic field parameters was 9 kV and 20 cm, uniformly sized carboxymethyl chitosan-graphene oxide particles in the diameter range of 250-300 μm was successfully prepared. In NaCl saline, these particles could maintain stable for at least a week, while pristine carboxymethyl chitosan particles quickly collapsed within an hour. The results of loading experiments showed that carboxymethyl chitosan-graphene oxide particles could effectively adsorb gatifloxacin, ofloxacin, bovine serum albumin, lysozyme, doxorubicin hydrochloride. And gatifloxacin was chosen as a model drug to study the exact effect of graphene oxide content on the loading and release properties. In 40:2 group, the highest loading capacity of 0.45 ± 0.19 mg/mg was achieved, and also a good sustained release was available. Above all, we believed that carboxymethyl chitosan-graphene oxide particles as a versatile carrier, has great potential in Medicine and Pharmacy.

Heparin-conjugated alginate multilayered microspheres for controlled release of bFGF

In order to effectively immobilize and control release of basic fibroblast growth factor (bFGF) from alginate microspheres, heparin-conjugated alginate (H-Alg) was first synthesized by covalent binding. Then multilayered H-Alg microspheres (multilayered microspheres) were fabricated via an electrostatic droplet generation technique followed by a layer-by-layer (LbL) self-assembly technique. Several techniques such as Fourier transform infrared spectroscopy (FTIR), 1H-NMR, zeta potential analysis and scanning electron microscopy (SEM) were used to characterize the properties of H-Alg (FTIR and 1H-NMR) and multilayered microspheres (FTIR, zeta potential analysis and SEM). bFGF binding efficiency, release profiles of bFGF from multilayered microspheres and the biological activity of released bFGF were well investigated. It was found that the bFGF binding efficiency of H-Alg microspheres was increased up to five times higher than that of the alginate microspheres. Additionally, the release profiles of bFGF from multilayered microspheres were sustained for two weeks with relieved initial burst release, and the release rate to bFGF could be regulated by controlling the number of deposited layers. Importantly, the released bFGF still retained its biological activity as assessed by the in vitro proliferation of NIH-3T3 mouse fibroblasts. In conclusion, this study presented an easy yet effective method for the controlled, sustained release of heparin-binding growth factors, using polyelectrolyte multilayer-coated heparin-conjugated alginate microspheres.

Development of a new approach to investigating the drug transfer from colloidal carrier systems applying lipid nanosuspension-containing alginate microbeads as acceptor

As a new approach to analyzing the release behavior of lipophilic drugs from colloidal carriers, solid trimyristin nanoparticles were incorporated into differently sized (34–1363μm) calcium alginate hydrogel microbeads to serve as acceptor in release studies. The microbeads were prepared by electrostatic droplet generation or by a spraying method. Trimyristin nanoemulsion samples loaded with the fluorescent drug model Nile red were mixed with the nanoparticle-containing microbeads to perform transfer studies. As a result of a rather large diffusion barrier a slow transfer (24–57min) was observed using large acceptor beads (∼330–1360μm). In contrast, Nile red transferred quickly (∼1.4min) into smaller microbeads (<50μm). This new experimental approach applying nanoparticle-containing hydrogel particles with a size below 50μm as acceptor systems is a promising technique to investigate the release of lipophilic substances from lipid nanoparticles under close to realistic conditions. However, there is still room for technical improvement, e.g., with regard to the water loss from microbeads that was observed during sampling by centrifugation and filtration (required to separate the small sized alginate particles) which is expected to have had some effect on the dye content determined during these experiments.

Delivering MC3T3-E1 cells into injectable calcium phosphate cement through alginate-chitosan microcapsules for bone tissue engineering

To deliver cells deep into injectable calcium phosphate cement (CPC) through alginate-chitosan (AC) microcapsules and investigate the biological behavior of the cells released from microcapsules into the CPC. Mouse osteoblastic MC3T3-E1 cells were embedded in alginate and AC microcapsules using an electrostatic droplet generator. The two types of cell-encapsulating microcapsules were then mixed with a CPC paste. MC3T3-E1 cell viability was investigated using a Wst-8 kit, and osteogenic differentiation was demonstrated by an alkaline phosphatase (ALP) activity assay. Cell attachment in CPC was observed by an environment scanning electron microscopy. Both alginate and AC microcapsules were able to release the encapsulated MC3T3-E1 cells when mixed with CPC paste. The released cells attached to the setting CPC scaffolds, survived, differentiated, and formed mineralized nodules. Cells grew in the pores concomitantly created by the AC microcapsules in situ within the CPC. At Day 21, cellular ALP activity in the AC group was approximately four times that at Day 7 and exceeded that of the alginate microcapsule group (P<0.05). Pores formed by the AC microcapsules had a diameter of several hundred microns and were spherical compared with those formed by alginate microcapsules. AC microcapsule is a promising carrier to release seeding cells deep into an injectable CPC scaffold for bone engineering.

Preparation of N,O-carboxymethyl chitosan coated alginate microcapsules and their application to Bifidobacterium longum BIOMA 5920

In order to greatly improve vitality of probiotic bacteria within the application, a novel biocompatible vehicle, N,O-carboxymethyl chitosan (NOCs) with appropriate degrees of substitution coat alginate (ALg) microparticles, was prepared by electrostatic droplet generation. The amount of chitosan (Cs) and N,O-carboxymethyl chitosan (NOCs) coated on the ALg microparticles was determined by differential scanning calorimetry. The surface morphology of ALg microparticles, Cs coated ALg microparticles and NOCs coated ALg microparticles was determined using scanning electron microscopy. The coating thickness of Cs coated ALg microparticles and that of NOCs coated ALg microparticles was directly observed with confocal laser scanning microscopy. In order to assess pH sensitivity of microparticles, the bovine serum albumin release from the microspheres was tested in acid solution (pH2.0) for 2h and subsequently in alkaline solution (pH7.0) for 2h. The survival of Bifidobacterium longum BIOMA 5920 loaded in NOCs coated with ALg microparticle was improved in simulated gastric juice (pH2.0, for 2h) compared to that of B. longum BIOMA 5920 loaded in ALg microparticles and Cs coated ALg microparticles. After incubation in simulated intestinal juices (pH7.0, 2h), the release of microencapsulated B. longum BIOMA 5920 was investigated.

Incorporation of lipid nanoparticles into calcium alginate beads and characterization of the encapsulated particles by differential scanning calorimetry

The aim of this study was to encapsulate lipid nanoparticle dispersions into calcium alginate microbeads and to investigate the feasibility of this incorporation as well as the stability of the enclosed nanoparticles. Two methods were used to prepare the lipid-containing microbeads by external gelation, i.e. an electrostatic droplet generation technique and a spraying method using the two-fluid spray nozzle of a spray dryer. The particle sizes of the hydrogel beads were determined with laser diffraction. The first method produced particles with median sizes between 330 and 1350 μm, depending on the needle applied. Using the second method smaller particles (∼45 μm) could be prepared. Small tyloxapol-stabilized trimyristin nanoparticles (<100 nm) could easily be incorporated into the differently sized alginate beads. The small size of the lipid nanoparticles leads to a typical melting behavior characterized by the occurrence of multiple discrete melting peaks in their differential scanning calorimetry (DSC) thermograms which enabled the characterization of the lipid particles within the microbeads. Thus, any negative influence of the preparation procedure on the lipid dispersions could be detected and it was possible to verify optimization options of the incorporation procedure. Finally, handling and shelf life of the hydrogel beads were improved by a freezing process.

Preparation of N, O-carboxymethyl chitosan-Human-Like Collagen Microspheres for Drug Delivery

In this investigation, the N, O-carboxymethyl chitosan (NOCC) was synthesized. A novel pH-sensitive hydrogel system composed of a water-soluble chitosan derivative (N,O-carboxymethyl chitosan, NOCC)and Human-like collagen (HLC) blended with phosphate was developed for controlling drug delivery and NOCC–HLC microspheres were prepared by the technology of electrostatic droplet generation technique. The release profiles of a model protein drug (bovine serum albumin, BSA) from test hydrogels were studied in simulated gastric and intestinal media.

Molecular Biocompatibility Evaluation of Poly-L-Ornithine-Coated Alginate Microcapsules by Investigating mRNA Expression of Pro-Inflammatory Cytokines

Following a polyelectrolytical complex reaction, the poly-L-ornithine (PLO)-alginate microcapsules were prepared by coating PLO on calcium alginate beads which were produced by a high-voltage electrostatic droplet generator. The biocompatibility of the microcapsules at the molecular level was evaluated through investigating the mRNA expression of pro-inflammatory cytokines; that is, the effect of the PLO coating of alginate beads on the mRNA expression of TNF-α, IL-1β, and IL-6 were measured using the RT-PCR method. The resulting PLO-coated alginate microcapsules have a smooth surface with a mean diameter of 309µm. The molecular biocompatibility studies show that coating microcapsules with PLO has no significant effect on the biocompatibility of alginate microcapsules (p&gt;0.05), and both alginate microcapsules and PLO-coated microcapsules are significantly different from the positive control (p&lt;0.05); however, both are also capable of causing an inflammatory response at a molecular level since both are significantly different from the blank control (p&lt;0.05). Furthermore, with the increase in concentration of microcapsules or co-cultured time, part of the mRNA expression of cytokines is significantly increased. The results also demonstrate that the method used in this study, co-incubating the microcapsules with macrophages and measuring the mRNA expression of cytokines by RT-PCR, may be a useful method for evaluating the biocompatibility of coating materials of microcapsules.

Preparation and characterization of PEM-coated alginate microgels for controlled release of protein

In this study, calcium-alginate microgels coated with a polyelectrolyte multilayer (PEM) were fabricated as a controlled-release system. This system was constructed via an electrostatic droplet generation technique followed by a layer-by-layer (LbL) self-assembly technique. The electrostatic droplet generation technique was reported as an easy method of preparing microgels, due to their mild preparation conditions and ability to preserve the biological activity of the encapsulated drugs. With the LbL self-assembly technique, the PEM could be fabricated on the microgels attributed to the electrostatic attraction between positive-charged chitosan (Chi) and negative-charged dextran sulfate (Dex). The properties of the prepared microgels were investigated using dynamic laser scattering (DLS), scanning electron microscopy (SEM), x-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FTIR) spectrum and zeta potential analyzer. In vitro release study indicated that the initial burst release of the bovine serum albumin (BSA) from PEM-coated microgels was less compared to the uncoated microgels (19% versus 31% in 24 h). In addition, the sustained release of BSA from the PEM-coated microgels was recorded up to 1 month without any damage to BSA integrity. Thus, our results demonstrated that the PEM-coated microgels not only prolonged the release time, but also relieved the initial burst problem to some degree and preserved the biological activity of the encapsulated drugs. Moreover, the release rate of BSA could be regulated by controlling the number of deposited layers. In conclusion, this study presented an easy yet effective method for the controlled, sustained release of biological macromolecules.

Preparation of nanoparticles embedded microcapsules (NEMs) and their application in drug release

The strategy to construct a novel drug delivery system of nanoparticle embedded microcapsules (NEMs) is reported in this work. The Chitosan/Alginate Nanoparticle Embedded Microcapsules (CS/Alg NEMs) have been successfully prepared by emulsion-gelation and high-voltage electrostatic droplet generator. The drug release behavior in vitro of the NEMs was studied, using Capecitabine (CAP) and γ-globulin as model drugs. The results demonstrated that the NEMs could control the drugs' release rate significantly, whether it is a small-molecular drug or peptide and protein. Also, the results indicated that the co-loading drugs exhibited an orderly release property. The NEMs exhibited a great potential for application in drug delivery for multi-functions and sustained release.

Electrostatic Droplet Generator with 3-Coaxial-Nozzle Head for Microencapsulation of Living Cells in Hydrogel Covered by Synthetic Polymer Membranes

The current methods allow for encapsulation of cells inside spherical microcapsules made of a matrix covered by a permselective membrane using an electrostatic droplet generator with 1-nozzle or 2-nozzle heads. However, some potentially useful materials for the outer membranes cannot be put into direct contact with hydrophilic core filled by cells during the manufacturing process. Therefore, we designed a novel 3-coaxial-nozzle head that allows for the third fluid to separate the core material from the membrane material. The equipment was applied for manufacturing spherical microcapsules comprised of cell-friendly alginate core surrounded by semipermeable polyethersulfone membrane. The obtained microcapsules had a diameter between 0.84 mm and 1.79 mm, and the diameter correlated negatively with the applied electric voltage. The thickness of the membrane varied from 171 µm to 450 µm. The SEM images of the interior of microcapsules revealed highly porous membrane structure typical for synthetic membranes obtained by a wet phase inversion method. Bakery yeast cells encapsulated inside the alginate-polyethersulfone microcapsules retained their proliferation ability proving the effectiveness and safety of this encapsulation technique.

Encapsulation of probiotic Bifidobacterium longum BIOMA 5920 with alginate–human-like collagen and evaluation of survival in simulated gastrointestinal conditions

Alginate (ALg)–human-like collagen (HLC) microspheres were prepared by the technology of electrostatic droplet generation in order to develop a biocompatible vehicle for probiotic bacteria. Microparticles were spherical with mean particle size of 400μm. The encapsulation efficiency (EE) of ALg–HLC microspheres could reach 92–99.2%. Water-soluble and fibrous human-like collagen is combined with sodium alginate through intermolecular hydrogen bonding and electrostatic force which were investigated by Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC), thus the matrix of ALg–HLC was very stable. Bifidobacterium longum BIOMA 5920, as a kind of probiotic bacteria, was encapsulated with alginate–human-like collagen to survive and function in simulated gastrointestinal juice. Microparticles were very easy to degradation in simulated intestinal juices. After incubation in simulated gastric (pH 2.0, 2h), the encapsulated B. longum BIOMA 5920 numbers were 4.81±0.38logcfu/g.

Preparation of chitosan/alginate microcapsules by high-voltage electrostatic method

Chitosan and sodium alginate have the opposite charges; they can become a gelatin by the electrostatic attraction, High-voltage electrostatic droplet generator method was used to prepare chitosan-sodium alginate microcapsule. Multi-layer chitosan-sodium alginate microcapsule was prepared through layer-by-layer self-assembly, and the morphology was investigated. In addition, the release property of ofloxacin in microcapsules was studied by UV-Vis microscopy under different conditions such as pH value, layer number, etc. The results showed that the prepared microcapsules have a smooth surface with average particle size about 100 μm. The result of controlled release indicated that the prepared microcapsules are pH-independent, and the rate of release decreased when the layer number increases.

Production of bioethanol from corn meal hydrolyzates by free and immobilized cells of Saccharomyces cerevisiae var. ellipsoideus

The ethanol fermentation of enzymatically obtained corn meal hydrolyzates by free and immobilized Saccharomyces cerevisiae var. ellipsoideus yeast in a batch system was studied. The initial glucose and inoculum concentration and the time required for the efficient ethanol production were optimized taking into account parameters such as ethanol concentration, ethanol yield and volumetric productivity. The yeast cells were immobilized in Ca-alginate by electrostatic droplet generation method. An optimal initial inoculum concentration of 2% (v v−1) and optimal fermentation time of 38 h for both immobilized and free yeasts were determined. Optimal initial glucose concentrations of 150 and 176 g l−1 for free and immobilized system were achieved, respectively. The immobilized cell system was superior to the free cell system since higher ethanol tolerance and productivity and lower substrate inhibition were detected.