Alginate Microbeads Research Articles

Alginate chitosan microbeads and thermos-responsive hyaluronic acid hydrogel for phage delivery

Bacteriophages are potential alternatives to conventional antibiotics in the treatment of multidrug resistant infections. However, optimal treatment administration protocols are not established. The direct application of bacteriophages in local biomaterials-based carriers offers solutions to many of the limitations of current modalities. In this study, thermo-responsive hyaluronic acid-poly(N-isopropylacrylamide) (HA-pNIPAM) hydrogels and alginate-chitosan microbeads have been developed as bioresorbable local bacteriophage delivery systems designed to enable rapid and delayed phage release, respectively. The long-tailed Staphylococcus aureus phage ISP and the short-tailed Pseudomonas aeruginosa phage LUZ19 were selected as structurally diverse phages to load within both biomaterials. HA-pNIPAM hydrogels were synthesized and their thermo-responsive crosslinking was confirmed after rheological assessment. Alginate microbeads where prepared by ionic-gelation and chitosan surface deposition was confirmed by amine sensitive assays. After embedding phages, their antimicrobial potential was retained when stored at 4 °C. Release of both phages from HA-pNIPAM hydrogels was gradual and consistent over 21 days, whereby, continuous release of the short-tailed LUZ19 was observed for 21 days from the microbeads, while release of the long-tailed ISP portrayed burst-like release during early timepoints, with gradually reducing phage release thereafter. These findings suggest that the release kinetics of phage from biomaterials are impacted by both the biomaterial and the phages themselves. These results highlight the potential of HA-pNIPAM hydrogel and alginate-chitosan microbeads for local delivery of ISP and LUZ19 bacteriophages for both rapid burst release and controlled, extended release.

Comparative study of bioethanol production from bagasse pith by Pichia stipitis in free cell system and immobilized electrosprayed micro-beads: SSF and combined hydrolysates fermentation

Biofuels production from biomass are receiving special attention due to the availability of raw materials, suitable conversion yield and renewability. The primary objective of this study was comparison of bioethanol productivity from bagasse pith by free and immobilized Pichia stipitis. Alginate micro-beads at various sizes fabricated by electrospray technique. At the first stage, bioethanol production using free cells in two processes including fermentation of combined acid and enzymatic hydrolysates and simultaneous saccharification and fermentation (SSF) was optimized. In fermentation of combined hydrolysates, maximum production equal to 9.53 and 12.68 g/L was observed by free and immobilized cells, respectively at cell concentration 6 %, portion of acid hydrolysate 40 % and total sugar concentration 50 %. Besides, maximum production in SSF equal to 9.93 and 10.62 g/L was obtained by free and immobilized cells, respectively at 6 % cell, 4 g solid and enzyme content of 50 FPU/g solid. However, in SSF fermentation, the beads size had no significant effect on ethanol production while in combined hydrolysates fermentation, bioethanol production considerably affected by the beads size and 13.9 g/L ethanol was obtained in minimum beads size. Additionally, long-term stability of beads was examined in repeated-batch cultures, showing effective bioethanol production up to five reuse cycles.

Encapsulation of Moringa oleifera leaf extract in chitosan-coated alginate microbeads produced by ionic gelation

Encapsulation of potential bioactive natural substances is a promising preservation method for the natural additives over the synthetic antixodiants. In this study, ionic gelation has been used to encapsulate the Moringa oleifera leaf extract in biopolymers. Automatic solvent extraction was used to extract active material from the plant. Then, the active material was covered by chitosan/alginate via ionic gelation. Ionic gelation system was designed and optimized by Central Composite design of Response Surface Method (RSM). Exposure time to gelling medium (10–30 min), sodium alginate concentration (1%–2%, w/v) and chitosan concentration (0.5%–1%, w/v) were process parameters to be optimized for the maximum encapsulation efficiency concerning total phenolic content (EETPC), rutin (major individual compound) concentration and inhibition effect of the capsules against DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical. Particle shape and morphology of the dried microcapsules show that the capsules obtained are quite spherical, uniform and have a regular structure depending on stereo-microscope and scanning electron microscopy (SEM) analyzes.

Microencapsulation of Bacillus Strains for Improving Wheat (Triticum turgidum Subsp. durum) Growth and Development.

Bio-formulation technologies have a limited impact on agricultural productivity in developing countries, especially those based on plant growth-promoting rhizobacteria. Thus, calcium alginate microbeads were synthesized and used for the protection and delivery of three beneficial Bacillus strains for agricultural applications. The process of encapsulation had a high yield per gram for all bacteria and the microbeads protected the Bacillus strains, allowing their survival, after 12 months of storage at room temperature. Microbead analysis was carried out by observing the rate of swelling and biodegradation of the beads and the released-establishment of bacteria in the soil. These results showed that there is an increase of around 75% in bead swelling on average, which allows for larger pores, and the effective release and subsequent establishment of the bacteria in the soil. Biodegradation of microbeads in the soil was gradual: in the first week, they increased their weight (75%), which consistently results in the swelling ratio. The co-inoculation of the encapsulated strain TRQ8 with the other two encapsulated strains showed plant growth promotion. TRQ8 + TRQ65 and TRQ8 + TE3T bacteria showed increases in different biometric parameters of wheat plants, such as stem height, root length, dry weight, and chlorophyll content. Thus, here we demonstrated that the application of alginate microbeads containing the studied strains showed a positive effect on wheat plants.

3D-printed Mechanically Strong Calcium Phosphate Cement Scaffold with Metformin/Stem Cell-encapsulating Alginate Microbeads for Bone Tissue Engineering

3D-printed Mechanically Strong Calcium Phosphate Cement Scaffold with Metformin/Stem Cell-encapsulating Alginate Microbeads for Bone Tissue Engineering

Removal of lead ions from wastewater using magnesium sulfide nanoparticles caged alginate microbeads

In this study, an adsorbent made of alginate (Alg) caged magnesium sulfide nanoparticles (MgS) microbeads were used to treat lead ions (Pb2+ ions). The MgS nanoparticles were synthesized at low temperatures, and Alg@MgS hydrogel microbeads were made by the ion exchange process of the composite materials. The newly fabricated Alg@MgS was characterized by XRD, SEM, and FT-IR. The adsorption conditions were optimized for the maximum removal of Pb2+ ions by adjusting several physicochemical parameters, including pH, initial concentration of lead ions, Alg/MgS dosage, reaction temperature, equilibration time, and the presence of co-ions. This is accomplished by removing the maximum amount of Pb2+ ions. Moreover, the adsorbent utilized more than six times with a substantial amount (not less than 60%) of Pb2+ ions was eliminated. Considering the ability of sodium alginate (SA) for excellent metal chelation and controlled nanosized pore structure, the adsorption equilibrium of Alg@MgS can be reached in 60 min, and the highest adsorption capacity for Pb2+ was 84.7 mg/g. The sorption mechanism was explored by employing several isotherms. It was found that the Freundlich model fits the adsorption process quite accurately. The pseudo-second-order model adequately described the adsorption kinetics.

Biosorption studies of methylene blue dye using NaOH-treated Aspergillus niger-filled sodium alginate microbeads

The effectiveness of NaOH-functionalized Aspergillus niger-filled sodium alginate microbeads on methylene blue (MB) removal was investigated in this study. First of all, Aspergillus niger biomass was obtained, then functionalized using sodium hydroxide (NaOH). Lastly, the composites were prepared by dispersing NaOH-treated-Aspergillus niger in sodium alginate gel. The cross-linked composite microbeads in a 3% CaCl2 solution were characterized by Fourier-transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy, atomic force microscope (AFM), and scanning electron microscope (SEM). FTIR-ATR results indicated that the hydroxyl, amine, and carboxyl groups on the surface of the composite beads acted as binding sites. It was determined that the biosorption of MB occurred via the electrostatic interactions, Van der Waals interactions, π-π interactions, and H-bonding between functional groups and dye molecules. SEM and AFM analyses proved the presence of adsorbed dye molecules on the surface. Besides, optimum conditions were determined by examining the effects of different physicochemical parameters on biosorption. The maximum biosorption capacity of microbeads was obtained as 322.6 mg/g in pH = 7 at 25 °C. Moreover, various isotherms were used, including Freundlich, Langmuir, Dubinin-Radushkevich, and Harkins-Jura. The Langmuir model was found to be the best feasible when the correlation coefficients were taken into account. Biosorption followed the pseudo-second-order kinetic and occurred exothermically and spontaneously. This study indicates that NaOH-treated Aspergillus niger-filled sodium alginate microbeads are promising material as a biosorbent.

Human periodontal ligament stem cell encapsulation in alginate-fibrin-platelet lysate microbeads for dental and craniofacial regeneration

ObjectiveTissue engineering is promising for dental and craniofacial regeneration. The objectives of this study were to develop a novel xeno-free alginate-fibrin-platelet lysate hydrogel with human periodontal ligament stem cells (hPDLSCs) for dental regeneration, and to investigate the proliferation and osteogenic differentiation of hPDLSCs using hPL as a cell culture nutrient supplement. MethodshPDLSCs were cultured with Dulbecco's modified eagle medium (DMEM), DMEM + 10% fetal bovine serum (FBS), and DMEM + hPL (1%, 2.5%, and 5%). hPDLSCs were encapsulated in alginate-fibrin microbeads (Alg+Fib), alginate-hPL microbeads (Alg+hPL), or alginate-fibrin-hPL microbeads (Alg+Fib+hPL). hPDLSCs encapsulated in alginate microbeads were induced with an osteogenic medium containing hPL or FBS. Quantitative real-time polymerase chain reaction (qRT-PCR), alkaline phosphatase (ALP) activity, ALP staining, and alizarin red (ARS) staining was investigated. ResultshPDLSCs were released faster from Alg+Fib+hPL than from Alg+hPL. At 14 days, ALP activity was 44.1 ± 7.61 mU/mg for Alg+Fib+hPL group, higher than 28.07 ± 5.15 mU/mg of Alg+Fib (p<0.05) and 0.95 ± 0.2 mU/mg of control (p<0.01). At 7 days, osteogenic genes (ALP, RUNX2, COL1, and OPN) in Alg+Fib+hPL and Alg+Fib were 3-10 folds those of control. At 21 days, the hPDLSC-synthesized bone mineral amount in Alg+Fib+hPL and Alg+Fib was 7.5 folds and 4.3 folds that of control group, respectively. ConclusionsThe 2.5% hPL was determined to be optimal for hPDLSCs. Adding hPL into alginate hydrogel improved the viability of the hPDLSCs encapsulated in the microbeads. The hPL-based medium enhanced the osteogenic differentiation of hPDLSCs in Alg+Fib+hPL construct, showing a promising xeno-free approach for delivering hPDLSCs to enhance dental, craniofacial and orthopedic regenerations.

Optimization of co-encapsulation of L. plantarum cells and Silybum marianum seed extract and evaluation of protective effect of extract on cells survival in simulated gastrointestinal fluids

In the present study, Lactobacillus plantarum (MT, ZH593) native cells and Silybum marianum seed extract (SMSE; rich in antioxidant) were co-encapsulated in alginate microbeads by the internal gelatinization emulsion method. The effects of different concentrations of sodium alginate, SMSE and CaCl2 on microbead characteristics were optimized. Under optimum conditions small spherical microbeads (161.82 ± 13.4 μm) with the highest cell encapsulation efficiency (79.84 ± 4.07%) were produced and the highest antioxidant activity (51.92 ± 2.14%) was obtained. The results showed that the co-encapsulation of L. plantarum with SMSE significantly (P < 0.05) increased the viability of cells (approximately 0.71 log CFU/mL) and the release of the extract (approximately 9–10%) in gastric simulation and intestinal simulation fluids, respectively, when compared with microencapsulation without SMSE addition. Also, microbeads coating with whey protein isolate (WPI; 2.76%) resulted in the higher protection in simulated gastrointestinal fluids than coating with chitosan. The results of this study indicated that the WPI-coated alginate microbeads were a good carrier matrix for the controlled delivery of SMSE (as a new non-carbohydrate prebiotic compound) and increased the viability of L. plantarum in the lower part of the gastrointestinal tract.

Design and development of montmorillonite-sodium alginate microbeads for oral sustained delivery of cefaclor

Drug administration via oral route is most suitable for drug delivery to patients. But this route is associated with several drawbacks such as high frequency of administration as well as gastric discomfort. To avoid these problems attempt was made to develop the sustained release system by using nanocomposite clay (MMT) and polymer (Na-Al) based microbeads for oral administration of CFR. The formulations were optimized and validated by using Box–Behnken design with 3 different levels of MMT, Na-Al and CaCl2 wherein the %EE and % drug release at 8 h were considered as dependent responses. The developed formulation was characterized by FT-IR, DSC as well as XRD. The optimized formulation was evaluated for % yield, swelling behavior, particle size, drug release and surface morphology by FESEM. The developed formulation showed physical interaction due to surface adsorption and intercalation of drug with that of MMT. The optimized formulation showed high yield, %EE and swelling behavior due to high amount of MMT and Na-Al. The particle size of the microbeads was below 500 µm and drug release takes place in sustained manner up to 12 h. Polymeric cross-linking and MMT stalking were observed in FESEM study indicated distinctive system. Drug release follow the non-fickian diffusion pattern coupled with matrix release as indicated by Korsemeyer-Peppas (n) value 0.72.

Genipin-crosslinked fibrin seeded with oxidized alginate microbeads as a novel composite biomaterial strategy for intervertebral disc cell therapy

Discectomy procedures alleviate disability caused by intervertebral disc (IVD) herniation, but do not repair herniation-induced annulus fibrosus (AF) defects. Cell therapy shows promise for IVD repair, yet cell delivery biomaterials capable of sealing AF defects and restoring biomechanical function have poor biological performance. To balance the biomechanical and biological demands of IVD cell delivery biomaterials, we engineered an injectable composite biomaterial using cell-laden, degradable oxidized alginate (OxAlg) microbeads (MBs) to deliver AF cells within high-modulus genipin-crosslinked fibrin (FibGen) hydrogels (FibGen + MB composites). Conceptually, the high-modulus FibGen would immediately stabilize injured IVDs, while OxAlg MBs would protect and release cells required for long-term healing. We first showed that AF cells microencapsulated in OxAlg MBs maintained high viability and, upon release, displayed phenotypic AF cell morphology and gene expression. Next, we created cell-laden FibGen + MB composites and demonstrated that OxAlg MBs functionalized with RGD peptides (MB-RGD) minimized AF cell apoptosis and retained phenotypic gene expression. Further, we showed that cell-laden FibGen + MB composites are biomechanically stable and promote extracellular matrix (ECM) synthesis in long-term in vitro culture. Lastly, we evaluated cell-laden FibGen + MB-RGD composites in a long-term bovine caudal IVD organ culture bioreactor and found that composites had low herniation risk, provided superior biomechanical and biological repair to discectomy controls, and retained anabolic cells within the IVD injury space. This novel injectable composite hydrogel strategy shows promise as an IVD cell delivery sealant with potentially broad applications for its capacity to balance biomechanical and biological performance.

Environmentally friendly microbeads to model the dispersal of particulates in aquatic systems

The transport of particulate matter including the gametes, larvae and propagules of reproducing organisms and other organic matter involved in nutrient/contaminant transport are important processes, yet there are few environmentally friendly methods available to examine dispersal empirically. Herein we report on the development and application of a biodegradable and non-toxic physical model, based on alginate microbeads with modifiable size, density (ρ), and colour for use in dispersal studies. Specifically, the microbeads were designed to model the size and ρ of parasitic juvenile freshwater mussels (Unionidae; ρ = 1200 kg m−3), which undergo dispersal upon excystment from fish hosts. We released the juvenile-mussel and neutrally buoyant microbeads (ρ = 1000 kg m−3) in a local river and captured them in drift nets downstream. The concentration of microbeads declined with downstream distance, but neutrally buoyant microbeads were transported farther. Analysis of microbead capture rates could be described using the patterns of several mathematical models (negative exponential, power, and turbulent transport), which were consistent with the reported dispersal of mussel larvae and other benthic macroinvertebrates. These results support the use of alginate microbeads in dispersal studies, because their environmentally friendly and customizable properties offer improvements over non-biodegradable alternatives.

Functional composite microbeads for cell-based meat culture: effect of animal gelatin coating on cell proliferation and differentiation

Hydrogel microbeads are promising matrices for cell-based meat culture because they can be conveniently fabricated from food-grade materials. In this study, food-grade sodium alginate was successfully applied to fabricate edible microbeads with the desired diameters (∼200 µm) using electrospray technology. Pure alginate microbeads have poor cytoaffinity. To enhance the cytoaffinity of alginate microbeads for meat culture, we applied different gelatin coatings, namely porcine gelatin, bovine gelatin, and fish gelatin, onto alginate beads. The modification is non-toxic and environmentally friendly with the use of plant-derived crosslinkers and under mild reaction conditions. As all ingredients are food grade, the modified microbeads are edible and biodegradable, with the mean diameters of 142.96 ± 35.49 μm (porcine gelatin-coated beads), 105.12 ± 80.46 μm (bovine gelatin-coated beads), and 94.81 ± 67.38 μm (bovine gelatin-coated beads). These modified microbeads have self-fluorescence and can be easily distinguished under a fluorescent microscope. The hydrogel core enables the microbeads to have a density of 1.04 g cm−3 for suspension culture. Bovine gelatin-coated microbeads have the best primary porcine myoblast and C2C12 cell adhesion, and can achieve between 16- to 18-fold proliferation of different muscle cells. With the successful differentiation of C2C12 and proliferation of 3T3-L1 adipocytes on bovine gelatin-coated microbeads, cells and microbeads can form clusters, illustrating that our edible microbeads are promising microcarriers for mammalian cell culture for cell-based meat production.

Preparation and Characterization of Sodium Alginate-reduced Graphene Oxide Microbeads for Co-Delivery of Doxorubicin and 5-Fluorouracil

Combination therapy has gained significant interest in recent years due to its potential to produce greater therapeutic success than mono-drug therapy. However, carriers capable of effective and stimuli-responsive codelivery of several drugs are severely lacking at the time. To address this need, the study represents the fabrication of reduced graphene oxide-sodium alginate microbeads for co-delivery and controlled release of doxorubicin (DOX) and 5-fluorouracil (5-FU). The developed beads were characterized by Fourier transform infrared spectroscopy (FTIR), Differential scanning calorimetry (DSC), Thermogravimetric analysis (TGA), X-ray diffraction (XRD) and Scanning electron microscopy (SEM). Swelling and in vitro drug release experiments were performed at pH 7.4, 5.4 and 1.2 at 37 °C. The findings indicated that the pH of the test media could be appropriate for intestinal drug delivery. The release process was studied with the release data being integrated into the Korsmeyer-Peppas equation. The presence of rGO in the polymeric matrix influences the drug release rate. The anticancer activity of the alginate beads was assessed using an MTT assay against MCF-7 cells. The findings indicate that the dual-drug containing microbeads showed good activity compared to 5-FU loaded microbeads. The results showed that the developed microbeads warrant further development for applications that require simultaneous administration of multiple bioactive agents.

Encapsulation of olive leaf antioxidants in microbeads: Application of alginate and chitosan as wall materials

Edible microcapsule technology has been declared as a newly developed technology in 21st century by some certain authorities in order to preserve food products. Encapsulation of the bioactive materials in edible coatings is a blessing that can eliminate many undesirable situations that might arise when it is used as additive. In this study, olive leaf extract has been evaluated as active material to prepare microcapsules by using alginate as coating. Ionic gelation was used to produce microbeads. The experimental design of the encapsulation system, the effects of the process parameters, the modeling of the experimental data and the optimization of the conditions were carried out with Box-Behnken design of response surface method (Box-Behnken-RSM). Box-Behnken-RSM produced 17 experimental runs. Calcium chloride (2–15%, w/v) and sodium alginate concentrations (1–2%, w/v), and hardening time (15–45 min) were selected as independent variables, while encapsulation efficiency (EE) of the capsules in terms of total phenolic content (TPC) and oleuropein concentration were responses. Impact of chitosan as coating layer was also investigated with three different ratios of chitosan (0.4%, 0.7%, 1% w/v). Accelerated oxidation test was employed to measure the stability of the microcapsules against oxidation by means of Rancimat method. Encapsulation of the olive leaf extract in alginate microbeads was satisfying with >70% and >90% efficiencies with respect to TPC and oleuropein under optimum conditions (2.34% calcium chloride concentration and 2% sodium alginate for 26 min of hardening time).

Microencapsulation of Natural Food Antimicrobials: Methods and Applications

The global demand for safe and healthy food with minimal synthetic preservatives is continuously increasing. Some natural food antimicrobials with strong antimicrobial activity and low toxicity have been considered as alternatives for current commercial food preservatives. Nonetheless, these natural food antimicrobials are hardly applied directly to food products due to issues such as food flavor or bioavailability. Recent advances in microencapsulation technology have the potential to provide stable systems for these natural antibacterials, which can then be used directly in food matrices. In this review, we focus on the application of encapsulated natural antimicrobial agents, such as essential oils, plant extracts, bacteriocins, etc., as potential food preservatives to extend the shelf-life of food products. The advantages and drawbacks of the mainly used encapsulation methods, such as molecular inclusion, spray drying, coacervation, emulsification, supercritical antisolvent precipitation and liposome and alginate microbeads, are discussed. Meanwhile, the main current applications of encapsulated antimicrobials in various food products, such as meat, dairy and cereal products for controlling microbial growth, are presented.

Microfibrillated cellulose-reinforced alginate microbeads for delivery of palm-based vitamin E: Characterizations and in vitro evaluation

In this study, palm-based vitamin E-loaded emulsions were immobilized within microfibrillated cellulose-reinforced alginate microbeads by dripping vitamin E-loaded emulsions into the crosslinking solution. FTIR spectrum of crosslinked microbeads demonstrated the characteristic peaks of vitamin E at 1447, 1380, 1255, 1096 and 944 cm−1, implying the entrapment of the therapeutic agent. The microbeads were observed to be spherical under SEM and the external surfaces of the microbeads were rough with visible wrinkles. Results showed that highest compressive strength of 58.8 kPa was obtained for alginate microbeads containing Tween 80:Brij 35-stabilized emulsions with 0.25% w/v hydrophilic oxidized celluloses. This indicated that ammonium persulfate-oxidized celluloses participate in the formation of densely packed and three-dimensional matrix structures of microbeads by interacting with calcium ions via their –COO− groups. Likewise, the highest encapsulation efficiency of 98.7% was also achieved by the 0.25% ammonium persulfate-oxidized cellulose reinforced alginate microbeads containing Tween 80:Brij 35-stabilized emulsion. In addition, the alginate/microfibrillated cellulose microbeads were more resistant against swelling in low pH condition because of the enhanced stability promoted by the biobased microfibrillated celluloses. The microbeads showed slow release of vitamin E in the simulated gastric pH fluid but tend to release in the simulated intestinal pH fluid to allow the absorption of the drug.

Encapsulation of Bacillus pumilus G5 from polyvinyl alcohol‑sodium alginate (PVA-SA) and its implications in improving plant growth and soil fertility under drought and salt soil conditions

Drought and salt stresses adversely affect the growth and yield of plants in agricultural production. Bacillus pumilus, an important plant growth-promoting bacterium, play a significant role in improving plant tolerance to abiotic stresses. In this study, B. pumilus G5 were immobilized in polyvinyl alcohol‑sodium alginate (PVA-SA) microbeads and then applied on the Pharbitis nil under drought and salt stresses by pot experiment. Orthogonal array experiments showed that the optimal immobilization conditions of PVA-SA immobilized G5 microbeads were adsorbent 6.0%, PVA: SA 1:1 (3.0%), CaCl2 4.0%, and bacterium: embedding agent (PVA-SA) 3:4; And the G5 microbeads produced at the optimal condition exhibited better cultivable bacteria count, encapsulation rate, expansion rate and mechanical strength. Pot experiment showed that G5 microbeads significantly increased the length and diameter of root and stem, and dry weight of P. nil during experimental stage under drought and salt stress. G5 microbeads also increased the total cultivable bacteria population, the activities of invertase (INV), urease (URE), phosphatase (PHO) and catalase (CAT), and the contents of available nitrogen (AN) and available phosphorus (AP) in the rhizosphere soil of P. nil. Therefore, our study obtained the optimal process of G5 microbeads, and confirmed its effect on improved plant growth and soil chemical and biological properties of P. nil. Thus it can be used as sustainable tool for eco-friendly bio-inoculants at salinity soil within arid and semi-arid areas.

Microencapsulated Multifunctionalized Graphene Oxide Equipped with Chloroquine for Efficient and Sustained siRNA Delivery.

A multifunctionalized graphene oxide (GO)-based carrier with conjugation of aminated-polyethylene glycol (PEG-diamine), octaarginine (R8), and folic acid (FA), which also contains chloroquine (CQ), a lysosomotropic agent, is introduced. The cellular uptake mechanisms and intracellular targeting of FA-functionalized nanocarriers are examined. The localized releases of CQ and siRNA intracellular delivery are evaluated. Microencapsulation of the nanocarrier complexed with genes in layer-by-layer coating of alginate microbeads is also investigated. The covalently coconjugated FA with PEG and R8 provides a stable formulation with increased cellular uptake compared to FA-free carrier. The CQ-equipped nanocarrier shows a 95% release of CQ at lysosomal pH. The localized release of the drug inside the lysosomes is verified which accelerates the cargo discharge into cytoplasm.

Droplet microfluidics for double lipase immobilisation using TiO2 and alginate microbeads

A double immobilisation technique was developed for C. antarctica lipase (CALB) that improved its enzymatic activity and solved difficulties frequently observed in reactions catalysed by lipases. The first immobilisation consisted of CALB adsorption onto a TiO2 nanoparticle surface (CALB TiO2). The adsorption was carried out by an oriented monolayer formed by CALB’s hydrophilic amino acids and the TiO2 surface, leaving the CALB’s active site accessible for reaction. As a result, an increase in enzymatic activity was achieved. The Relative Enzymatic Activity ((REA) obtained was 289%. The second immobilisation consisted of CALBTiO2 entrapment into calcium alginate microbeads [(CALBTiO2)EDTA-Ca], obtained by an internal crosslinking ion-exchange mechanism and using microfluidic droplet technique. The microbiocatalyst obtained, (CALBTiO2)EDTA-Ca, retained a high enzymatic activity (REAt=0 = 232%) and stability (REAt=30days = 263%) at a size (diameter, Ø=8.9·104 nm) that enabled easier recovery than CALBTiO2 (Ø = 2.3·102 nm) or CALBFree (Ø=5.0 nm), and showed a favourable porosity for diffusion without releasing CALBTiO2. Although the microbeads showed CALBFree leaching, as demonstrated by the loss of REA after a mechanical resistant test, (CALBTiO2)EDTA-Ca maintained an almost constant REA.