Complex Coacervation Technique Research Articles

Coacervação complexa: uma técnica para a encapsulação de probióticos

Microencapsulation is the protection of a compound and modulation of its release and now, several techniques are available and among them there is the complex coacervation. The complex coacervation technique involves the association of two polymers and presents some advantages over other techniques such as the possibility of working with biopolymers, the absence of organic solvent and temperature conditions in mild processing. Functional foods containing probiotic bacteria are becoming increasingly popular in the market due to the beneficial health effects attributed to probiotics. However, these microorganisms are sensitive to various factors found in the environment that are exposed primarily to tratogastrointestinal and storage conditions. Thus, the microencapsulation is an alternative protection and controlled release for probiotics. However, factors such as temperature, pH and concentration of the hydrocolloid, particularly, can influence the formation of microcapsules. For the encapsulation of probiotics, although there are many related studies, however, according to these results, this technique can be regarded as promising for these microorganisms. Thus, this review article aims to address the key technological aspects related to microencapsulation of probiotics by the complex coacervation technique as well as the parameters related to the technique and its application potential.

Encapsulação de compostos bioativos por coacervação complexa

The bioactive compounds are chemicals found in plants and are widely known for bringing health benefits, so it can be designated as functional foods. However, they are sensitive to various factors found in the environment that are exposed primarily to tratogastrointestinal and, moreover, are lipophilic, therefore, have low solubility in aqueous media. In this sense, the encapsulation of these compounds is an alternative protection, and the provision of such compounds in suitable amounts. The complex coacervation microencapsulation is a technique commonly used for such compounds, besides having front advantages to other techniques such as the use of biopolymers, no use of toxic agents, among others. Currently, there are a lot of research in this direction, which will be presented in this paper. Thus, the present review aims to present the main bioactive compounds studied and encapsulated as well as the parameters for complex coacervation technique.

Microencapsulation by Complex Coacervation Using Whey Protein Isolates and Gum Acacia: An Approach to Preserve the Functionality and Controlled Release of β-Carotene

β-Carotene is a red–orange pigment, a known source of vitamin A and has exceptional antioxidant and free radical scavenging potential. However, uses of β-carotene in food industry are inadequate mostly because of their poor water solubility and low stability. Using the complex coacervation technique, the work is meant to fabricate the microcapsules of β-carotene, to examine the physicochemical properties of microcapsules and finally to evaluate the extent of stability improvement. The configuration of electrostatic complexes between whey protein isolate (WPI) and gum acacia (Acacia arabica, GA) was optimized as a function of pH, ionic strength, WPI/GA ratio, core material load and size of final micromolecules. The optimum process conditions were balanced by the ratio of wall materials WPI/GA 2.0/1.0 % and pH value 4.2. Morphological observations showed that microcapsules presented spherical shape, and smooth and continuous surface. The effective amount of encapsulated core was greater than 70 % for all formulations evaluated. In vitro release data indicated an initial burst release followed by sustained release behavior. The microstructure and viscoelastic properties of WPI and GA complex were studied using dynamic rheometer. The encapsulation method and the wall materials used in this work gave effective protection during storage and eventually resulted sustained release of bioactive while used in food matrix, at suitable conditions.

Whey Protein-Carboxymethylcellulose Obtained by Complex Coacervation as an Ingredient in Probiotic Fermented Milk

Discharge of whey proteins is still a current practice by small cheese producers. The development of low-cost alternatives for recovery of these proteins is fundamental for small producers who cannot apply expensive techniques. The present study investigated the complex coacervation technique as a cheap technology to recover proteins from sweet whey using carboxymethylcellulose, and the coacervate used as an ingredient in the formulation of probiotic fermented milk. The nutritional properties of whey-carboxymethylcellulose coacervates (WP-CMC) were evaluated in trials with animals (rats) using casein as a reference. All these parameters—the coefficient of feed efficiency (CEA), protein digestibility-corrected amino acid score (PDCAAS), and net protein ratio (NPR), as well as weight gain—were determined to evaluate protein quality. A sensory acceptance test was applied to evaluate the sensory characteristics of the product. The complex coacervation technique recovered 86% of the protein from sweet whey. No significant (p > 0.05) differences were observed in the biological tests for both groups (WP-CMC and Casein groups) when NPR (4.98 to 5.04), digestibility (92.35 to 90.64), and CEA (0.40 to 0.42) were evaluated. Probiotic fermented milk beverage containing WP-CMC (0.78%) and guar gum (0.68%) presented good acceptability as determined by sensory evaluation. WP-CMC can be considered an ingredient with high nutritional and biological value that could be applied in probiotic fermented milk as an alternative to small producers to allocate the residual whey from cheese manufacture.

СОВРЕМЕННОЕ СОСТОЯНИЕ И ПЕРСПЕКТИВЫ РАЗВИТИЯ АРОМАТНОЙ ОТДЕЛКИ ТЕКСТИЛЬНЫХ МАТЕРИАЛОВ

The review of scientific literature characterising basic directions of modern researches on the creation of textile materials with function of prolonged aromatization is presented. The mechanism of human smells recognition and influence of essential oils and aromatic on a human body is described.Various methods of formation of the microcapsules including flavouring substances are considered including a method of interfacial monomer polymerisation when on border of the water dispersive medium and disperse phase – oils is created by a solid polymer shell forming a spherical microcapsule with a fragrant oils as a core; a complex coacervation technique based on the abilities of cationic and anionic water-soluble polymers to interact with water, forming a polymer-rich phase; self-organising of supramolecular host-guest assemblies comprising guest molecule without strong chemical bonds formation; microemulsion or dispersion of perfume compositions based on organic compounds. Classification of supramolecular assemblies and the structure of host-guest microcapsules where flavoring agent acts as a guest is shown. Due to the absence of strong chemical bonds in this case the effect of mild and prolonged release of aroma are achieved. The modern perfume compositions having a therapeutic effect are described. The ways of fragrant substances immobilization on the textile material surface by means of electrostatic interaction of oppositely charged synthetic polyelectrolytes are reported. Effective ways of developing textile materials with ability to neutralize unpleasant odors and to prevent the life of bacteria based on the fixation on the surface of preliminary activated by deodorizing preparation fibrous materials are given.The publications devoted to themethods of unpleasant smells elimination using sorbents and bacteria with the subsequent action on the human olfactory receptors of such substances as menthol, camphor, methyl lactate and other esters are analysed.

Effect of microencapsulation of Lactobacillus acidophilus LA-5 on physicochemical, sensory and microbiological characteristics of stirred probiotic yoghurt

The aim of this study was to evaluate the characteristics of probiotic stirred yoghurt and the survival of free and microencapsulated cells of Lactobacillus acidophilus LA-5 during refrigerated storage. L. acidophilus LA-5 was microencapsulated by ionic gelation and complex coacervation techniques using pectin and whey protein as wall and coating materials, respectively. The survival of probiotics subjected to conditions simulating the passage through the gastrointestinal tract and the sensory acceptance of yoghurts were evaluated after 35days of refrigerated storage. The yoghurt containing encapsulated L. acidophilus LA-5 showed lower post-acidification and improved probiotics survival (62%) when compared to the yoghurt containing free cells of L. acidophilus LA-5 (10%) after 35days of refrigerated storage. The encapsulated L. acidophilus LA-5 showed higher survival than the free microorganism during simulated gastrointestinal conditions. After 35days of storage, no significant difference was observed for the attributes appearance, aroma, flavor, and overall impression for both samples. However, with respect to the attribute texture, the yoghurt containing encapsulated L. acidophilus LA-5 was less accepted than yoghurt containing free cells of L. acidophilus LA-5. Microencapsulation of L. acidophilus LA-5 by ionic gelation and complex coacervation provided lower post-acidification in probiotic yoghurts, and protection to the microorganism, during 35days of refrigerated storage and during the passage through the simulated gastrointestinal conditions.

Enhanced efficiency fertilisers: a review of formulation and nutrient release patterns.

Fertilisers are one of the most important elements of modern agriculture. The application of fertilisers in agricultural practices has markedly increased the production of food, feed, fuel, fibre and other plant products. However, a significant portion of nutrients applied in the field is not taken up by plants and is lost through leaching, volatilisation, nitrification, or other means. Such a loss increases the cost of fertiliser and severely pollutes the environment. To alleviate these problems, enhanced efficiency fertilisers (EEFs) are produced and used in the form of controlled release fertilisers and nitrification/urease inhibitors. The application of biopolymers for coating in EEFs, tailoring the release pattern of nutrients to closely match the growth requirement of plants and development of realistic models to predict the release pattern of common nutrients have been the foci of fertiliser research. In this context, this paper intends to review relevant aspects of new developments in fertiliser production and use, agronomic, economic and environmental drives for enhanced efficiency fertilisers and their formulation process and the nutrient release behaviour. Application of biopolymers and complex coacervation technique for nutrient encapsulation is also explored as a promising technology to produce EEFs.

Nanoparticles With Surface Antibody Against CD98 and Carrying CD98 Small Interfering RNA Reduce Colitis in Mice

Nanoparticles have been explored as carriers of small interfering RNAs (siRNAs) and might be developed to treat patients with inflammatory bowel disease (IBD). Overexpression of CD98 on the surface of colonic epithelial cells and macrophages promotes the development and progression of IBD. We developed an orally delivered hydrogel that releases nanoparticles with single-chain CD98 antibodies on their surface (scCD98 functionalized) and loaded with CD98 siRNA (siCD98). We tested the ability of the nanoparticles to reduce levels of CD98 in the colons of mice with colitis. scCD98-functionalized siCD98-loaded nanoparticles were fabricated using a complex coacervation technique. We investigated the cellular uptake and lysosome escape profiles of the nanoparticles in Colon-26 cells and RAW 264.7 macrophages using fluorescence microscopy. Colitis was induced by transfer of CD4(+)CD45RB(high) T cells to Rag(-/-) mice or administration of dextran sodium sulfate to C57BL/6 mice. Mice were then given hydrogel (chitosan and alginate) containing scCD98-functionalized nanoparticles loaded with siCD98 or scrambled siRNA (control) via gavage. The scCD98-functionalized nanoparticles were approximately 200 nm in size and had high affinity for CD98-overexpressing cells. The scCD98-functionalized siCD98-loaded nanoparticles significantly reduced levels of CD98 in Colon-26 cells and RAW 264.7 macrophages, along with production of inflammatory cytokines (tumor necrosis factor α, interleukin-6, and interleukin-12). In mice with colitis, administration of the scCD98-functionalized siCD98-loaded nanoparticles reduced colon expression of CD98. Importantly, the severity of colitis was also reduced compared with controls (based on loss of body weight, myeloperoxidase activity, inflammatory cytokine production, and histological analysis). Approximately 24.1% of colonic macrophages (CD11b(+)CD11c(-)F4/80(+)) in the mice had taken up fluorescently labeled siRNA-loaded nanoparticles within 12 hours of administration. Nanoparticles containing surface CD98 antibody and loaded with siCD98 reduce expression of this protein by colonic epithelial cells and macrophages, and oral administration decreases the severity of colitis in mice. This nanoparticle in hydrogel (chitosan/alginate) formulation might be developed to treat patients with IBD.

Development of ultrasmall chitosan/succinyl β‐cyclodextrin nanoparticles as a sustained protein‐delivery system

ABSTRACTIn this article, we introduce a new method for preparing ultrasmall chitosan (CS)/succinyl β‐cyclodextrin (SCD) nanoparticles (NPs) intended for loading bovine serum albumin (BSA) as a model protein. The proposed method is based on the complex coacervation technique followed by ionotropic gelation with tripolyphosphate. SCD, an anionic derivative of cyclodextrin, was synthesized and used in CS‐based NPs to enhance the entrapment efficiency of BSA. The results show that with this approach, ultrasmall, compact, and neutralized NPs with a mean particle size near 30 nm were obtained. A high degree of protein entrapment in the NPs led to a significant improvement in the BSA release profile with a low initial burst release (ca. 3% w/v of the initially loaded BSA) and a sustained release over time. This enabled a suitable nanocarrier for long‐term protein delivery (30% release over 120 h). © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 39648.

Improvement in antibacterial activity of moxa oil containing gelatin-arabic gum microcapsules

In the present study, moxa oil was encapsulated into the gelatin-arabic gum microcapsules using a complex coacervation technique in order to improve the antibacterial property of moxa oil. The physical properties including surface morphology, particle size, drug loading as well as release profile and anti-Staphylococcus aureus activity of moxa oil containing microcapsules were investigated. The results showed that the moxa oil containing microcapsules had relatively spherical appearance with the average particle size (6.42 µm) and the average oil loading (0.20 ± 0.01 mg/ml). The moxa oil was demonstrated to be gradually released from the microcapsules. The antibacterial study revealed that the moxa oil microcapsule-treated cotton fabrics showed a significantly stronger growth inhibition towards Staphylococcus aureus (S. aureus).

Formulation, characterization, and expression of a recombinant MOMP Chlamydia trachomatis DNA vaccine encapsulated in chitosan nanoparticles

Chlamydia trachomatis is a bacterial sexually transmitted infection affecting millions of people worldwide. Previous vaccination attempts have employed the recombinant major outer membrane protein (MOMP) of C. trachomatis nonetheless, with limited success, perhaps, due to stability, degradation, and delivery issues. In this study we cloned C. trachomatis recombinant MOMP DNA (DMOMP) and encapsulated it in chitosan nanoparticles (DMCNP) using the complex coacervation technique. Physiochemical characterizations of DMCNP included transmission and scanning electron microcopy, Fourier transform infrared and ultraviolet-visible spectroscopy, and zeta potential. Encapsulated DMOMP was 167–250 nm, with a uniform spherical shape and homogenous morphology, and an encapsulation efficiency > 90%. A slow release pattern of encapsulated DMOMP, especially in acidic solution, was observed over 7 days. The zeta potential of DMCNP was ~8.80 mV, which indicated that it was highly stable. Toxicity studies of DMCNP (25–400 μg/mL) to Cos-7 cells using the MTT assay revealed minimal toxicity over 24–72 hours with >90% viable cells. Ultra-violet visible (UV-vis) spectra indicated encapsulated DMOMP protection by chitosan, whereas agarose gel electrophoresis verified its protection from enzymatic degradation. Expression of MOMP protein in DMCNP-transfected Cos-7 cells was demonstrated via Western blotting and immunofluorescence microscopy. Significantly, intramuscular injection of BALB/c mice with DMCNP confirmed the delivery of encapsulated DMOMP, and expression of the MOMP gene transcript in thigh muscles and spleens. Our data show that encapsulation of DMOMP in biodegradable chitosan nanoparticles imparts stability and protection from enzymatic digestion, and enhances delivery and expression of DMOMP in vitro and in mice. Further investigations of the nanoencapsulated DMCNP vaccine formulation against C. trachomatis in mice are warranted.

Physicochemical and sensory properties of yogurt enriched with microencapsulated fish oil

Encapsulation of marine omega-3 oil by complex coacervation technique has been introduced as most effective approach to delay its oxidation and extend shelf life of ω(3)-enriched food products. Therefore, to produce enriched yogurt, fish oil containing long-chain omega-3 polyunsaturated fatty acids was microencapsulated in complex coacervates of gelatin/acacia gum. Then, the microcapsules were dried and their surface oil was extracted. Set yogurt was prepared by enriched milk with microcapsules powder. Physicochemical and sensory properties of enriched yogurt were measured during 21 days storage. Acidity, apparent viscosity and water holding capacity of enriched samples were higher and gel strength and amount of whey separation were lower compared to the control. The enriched yogurt samples were more yellowish compared to control. The peroxide value of free and encapsulated fish oil in enriched yogurt samples, after 22 days storage, were increased to 72% and 260%, respectively. Fish oil release of microcapsules was not detected by gas chromatography in extracted oil from enriched yogurt. Sensory results showed that untrained panelists evaluated overall acceptance of enriched yogurt with treated-fish oil microcapsules by lime juice as 'neither liked nor disliked to slightly liked'.

Microencapsulation of oil droplets using freezing-induced gelatin–acacia complex coacervation

A freezing process was applied to control the kinetics of a gelatin–acacia complex coacervation technique for encapsulating emulsified oil droplets. An oil-in-water emulsion was stabilized with a gelatin–acacia solution, and the pH of the solution was adjusted to a selected value with acetic acid. When the pH of the emulsion was adjusted to 4.7, the system was visibly stable at ambient temperature for up to 12h. Freezing the emulsion caused polymer phase separation (complex coacervation) in the cryoconcentrated phase and resulted in encapsulated oil droplets and the accumulation of a cream layer in the freeze-thawed solution. Observation by transmission electron microscopy clarified the formation of 50–4000nm core–shell nano-microparticles, the surfaces of which were surrounded by polymeric membranes. The membrane properties of the particulate systems were dependent on the cooling rate that was used during freezing. For example, when an emulsion with a pH of 4.7 was frozen, a cooling rate of −1.0°C/min maximized the encapsulation yield, whereas a rate of −2.0°C/min was effective in limiting the release rate of the ingredient (β-carotene) from the oil phase through the shell membrane. The results of this study suggest that the formation of nano-microparticles could be highly associated with the kinetics of freezing, so their resultant properties could be fine-tuned using a freezing operation. This is a potential concept providing a novel strategy for engineering core–shell nanoparticles.

Whey protein coating bead improves the survival of the probiotic Lactobacillus rhamnosus CRL 1505 to low pH

To evaluate the efficacy of a novel microencapsulation procedure using whey protein and pectin to improve the survival rate of Lactobacillus rhamnosus CRL 1505 to low pH and bile. Lactobacillus rhamnosus CRL 1505 was encapsulated by ionotropic gelation using pectin (PE) and pectin-whey protein (PE-WP). Both types of beads (MC(PE/WP) and MC(PE-WP/WP)) were covered with a layer of whey protein by complex coacervation. The noncapsulated lactobacilli were not sensitive to bile salts but to acid. Both microparticles protected Lact. rhamnosus CRL 1505 at pH 2.0, but only MC(PE/WP) was effective at pH 1.2. The combination of ionotropic gelation and complex coacervation techniques is efficient to obtain microcapsules of pectin covered with whey proteins. The MC(PE/WP) beads were more stable than the MC(PE-WP/WP) beads in simulated gastric conditions, thus offering better protection to Lact. rhamnosus CRL 1505 at low pH. Pectin beads with a whey protein layer (MC(PE/WP)) could be used as probiotic carrier in functional foods of low pH (e.g. apple juice), thus protecting Lact. rhamnosus CRL 1505 against the stressful conditions of the gastric tract.

Enhanced Immune Response Against Pertussis Toxoid by IgA-Loaded Chitosan–Dextran Sulfate Nanoparticles

The objective of the present study was to evaluate immunological activities of chitosan-dextran sulfate (CS-DS) nanoparticle formulation of pertussis toxoid (PTXd) and its combination with a potential immunological adjuvant, immunoglobulin A (IgA). CS-DS nanoparticles were prepared using a complex coacervation (polyelectrolyte complexation) technique. CS-DS nanoparticle formulations with size and zeta potential in a range of 300-350 nm and +40-+55 mV, respectively, were obtained. An entrapment efficiency of more than 90% was obtained for pertussis toxin and IgA in CS-DS nanoparticles. All loaded nanoparticle formulations showed less than 20% of release within 24 h in in vitro release studies. The immunological evaluation of developed formulations in female Balb/c mice groups showed that the CS-DS nanoparticles formulations induced significantly higher serum IgG and IgG1 titers (p < 0.05) as compared with conventional alum-adjuvanted PTXd formulation administered by subcutaneous route. This study indicated the potential of CS-DS nanoparticles to be a simple and effective particulate delivery system with in-built immunological adjuvant property for acellular protein antigens. The study also revealed the potential important role of IgA-loaded CS-DS nanoparticles as a novel immunological adjuvant for vaccine delivery.

PREPARATION AND PROPERTIES OF NANOCAPSULATED CAPSAICIN BY COMPLEX COACERVATION METHOD

The application of gelatin and acacia protective colloid for encapsulating capsaicin was demonstrated using a complex coacervation technique. The effects of shearing force, the gelatin and acacia viscosity, and cross-linking time on the particle size of the nanocapsulated capsaicin (NC) agents were investigated. Structure and morphology of the nanocapsules prepared by the most suitable conditions was demonstrated by Fourier transform-infrared (FT-IR) spectroscopy, transmission electron microscopy (TEM), and atomic force microscopy (AFM). Thermal properties of the NC agents were measured by differential scanning calorimeter (DSC) and thermogravimetric analysis (TGA). Results showed that the NC agents had a mean diameter of about 100 nm, and the melting point and degradation properties were relatively improved.

Genipin crosslinked microcapsules of gelatin A and κ-carrageenan polyelectrolyte complex for encapsulation of Neem (Azadirachta Indica A.Juss.) seed oil

Microcapsules containing neem seed oil (NSO) were prepared using complex coacervation technique and employing gelatin A and κ-carrageenan polyelectrolyte complex. The yield of the coacervate was dependent on the ratio of the two polymers and on the pH of the medium. Viscosity and turbidity measurements were carried out in order to support the ratio of the two polymers that produced the highest yield. The encapsulation efficiency and the release rates of NSO were dependent on the amount of crosslinker, oil loading and polymer concentration. Scanning electron micrographs showed the formation of free flowing spherical microcapsules. The size of microcapsules increased with the increase of the concentration of the polymer. Fourier Transform Infrared Spectroscopy and Differential Scanning Calorimetry study showed that there was no significant interaction between NSO and carrageenan–gelatin complex.

Chitosan/pshRNA plasmid nanoparticles targeting MDR1 gene reverse paclitaxel resistance in ovarian cancer cells

In order to investigate the effect of chitosan/pshRNA plasmid nanoparticles targeting MDR1 genes on the resistance of A2780/TS cells to paclitaxel, chitosan/pshRNA plasmid nanoparticles were synthesized by means of a complex coacervation technique and transfected into A2780/TS cells. The cells transfected with MDR1-targeted chitosan/pshRNA plasmid nanoparticles were experimental cells and the cells transfected with chitosan/pGPU6/GFP/Neo no-load plasmid nanoparticles served as negative control cells. Morphological features of the nanoparticles were observed under transmission electron microscope (TEM). MDR1 mRNA expression was assessed by RT-PCR. Half-inhibitory concentration (IC50) of paclitaxel for A2780/TS cells was determined by MTT method. TEM showed that the nanoparticles were round-shaped, smooth in surface and the diameters varied from 80 to 120 nm. The MDR1 mRNA in the transfected cells was significantly decreased by 17.6%, 27.8% and 52.6% on the post-transfection day 2, 4 and 7 when compared with that in A2780/TS cells control (P<0.05). MTT assay revealed that the relative reversal efficiency was increased over time and was 29.6%, 51.2% and 61.3% respectively in the transfected cells 2, 4, 7 days after transfection and IC50 (0.197+/-0.003, 0.144+/-0.001, 0.120+/-0.004) were decreased with difference being significant when compared with that in A2780/TS (0.269+/-0.003) cells control (P<0.05). It was concluded that chitosan/pshRNA plasmid nanoparticles targeting MDR1 can effectively reverse the paclitaxel resistance in A2780/TS cells in a time-dependent manner.

Enhanced gene transfer into brain capillary endothelial cells using Antp-modified DNA-loaded nanoparticles

Brain capillary endothelial cells (BCECs) have been considered as one of the primary targets for cerebral gene therapy. However, the cells, well-known for their poor function of endocytosis, are difficult to be transfected by general non-viral vectors. The aim of this study was to enhance the efficiency of transfection and expression in BCECs of DNA/polymer nanoparticles with the modification of membrane-penetrating peptide, Antennapedia peptide (Antp) polyethylenimine (PEI) and polyamidoamine (PAMAM) were chosen to prepare Antp-modified DNA-loaded nanoparticles with a complex coacervation technique. After a 20-min transfection, the efficiency, in terms of transfection and expression, of DNA/PEI NP or DNA/PAMAM NP was enhanced significantly with the modification of Antp. After a 3-h transfection of DNA/Antp/PEI NP, there was no difference in cellular uptake but an enhancement in gene expression, compared to DNA/PEI NP alone. However, both the transfection and expression efficiency of DNA/PAMAM NP were enhanced using Antp. These observations suggest that Antp can increase the membrane-penetrating ability of DNA-loaded nanoparticles, which can be employed as novel non-viral gene vectors.

Chitosan nanoparticle as gene therapy vector via gastrointestinal mucosa administration: Results of an in vitro and in vivo study

This study was designed to investigate the in vitro and in vivo transfection efficiency of chitosan nanoparticles used as vectors for gene therapy. Three types of chitosan nanoparticles [quaternized chitosan − 60% trimethylated chitosan oligomer (TMCO-60%), C(43–45 KDa, 87%), and C(230 KDa, 90%)] were used to encapsulate plasmid DNA (pDNA) encoding green fluorescent protein (GFP) using the complex coacervation technique. The morphology, optimal chitosan-pDNA binding ratio and conditions for maximal in vitro transfection were studied. The in vivo transfection was conducted by feeding the chitosan/pDNA nanoparticles to 12 BALB/C-nu/nu nude mice. Both conventional and TMCO-60% could form stable nanoparticles with pDNA. The in vitro study showed the transfection efficiency to be in the following descending order: TMCO-60% > C(43–45 KDa, 87%) > C(230 KDa, 90%). TMCO-60% proved to be the most efficient and the optimal chitosan/pDNA ratio being 3.2:1. In vivo study showed most prominent GPF expression in the gastric and upper intestinal mucosa. GFP expression in the mucosa of the stomach and duodenum, jejunum, ileum, and large intestine were found, respectively, in 100%, 88.9%, 77.8% and 66.7% of the nude mice examined. TMCO-60%/pDNA nanoparticles had better in vitro and in vivo transfection activity than the other two, and with minimal toxicity, which made it a desirable non-viral vector for gene therapy via oral administration.