Oral Delivery Applications Research Articles

Perspectives on important considerations in designing nanoparticles for oral delivery applications in food

With rapid technological advancements in nanotechnology, it is important to revisit the knowledge of nanoparticles design and fundamental principles that have played major roles in the development of encapsulation and delivery technologies for food applications. In this perspective paper, important considerations for designing edible, safe, and effective nanoparticles for oral delivery of bioactive compounds in food products are highlighted. Physicochemical characterizations, biochemical evaluations, and biological assessments of biomaterials-based colloidal nanoparticles intended for food applications are particularly discussed to guide future research in this area.

Preparation and Evaluation of Resveratrol-Loaded Composite Nanoparticles Using a Supercritical Fluid Technology for Enhanced Oral and Skin Delivery.

We created composite nanoparticles containing hydrophilic additives using a supercritical antisolvent (SAS) process to increase the solubility and dissolution properties of trans-resveratrol for application in oral and skin delivery. Physicochemical properties of trans-resveratrol-loaded composite nanoparticles were characterized. In addition, an in vitro dissolution–permeation study, an in vivo pharmacokinetic study in rats, and an ex vivo skin permeation study in rats were performed. The mean particle size of all the composite nanoparticles produced was less than 300 nm. Compared to micronized trans-resveratrol, the trans-resveratrol/hydroxylpropylmethyl cellulose (HPMC)/poloxamer 407 (1:4:1) nanoparticles with the highest flux (0.792 μg/min/cm2) exhibited rapid absorption and showed significantly higher exposure 4 h after oral administration. Good correlations were observed between in vitro flux and in vivo pharmacokinetic data. The increased solubility and flux of trans-resveratrol generated by the HPMC/surfactant nanoparticles increased the driving force on the gastrointestinal epithelial membrane and rat skin, resulting in enhanced oral and skin delivery of trans-resveratrol. HPMC/surfactant nanoparticles produced by an SAS process are, thus, a promising formulation method for trans-resveratrol for healthcare products (owing to their enhanced absorption via oral administration) and for skin application with cosmetic products.

Mucoadhesive Particles: A Novel, Prolonged-Release Nanocarrier of Sitagliptin for the Treatment of Diabetics.

Sitagliptin (MK–0431) is a widely and commonly used oral hypoglycemic drug in the treatment of type 2 diabetes mellitus; patients typically take higher doses of this drug (50 mg, twice daily). One drawback is that only 38% of the drug is bound reversibly to plasma proteins and 79% is excreted in urine without being metabolized. To overcome this issue, there is a need for a better drug-delivery method to improve its efficacy in patients. It has been found that in existing formulations, the drug content is 72.5% ± 5% and the percentage yield is 84.9% ± 3%. In this study, sitagliptin nanoparticles (sizes ranging from 210 to 618 nm) were developed. The bioadhesion properties of the nanoparticles, as well as the swelling of the nanoparticles on the mucus membrane aided in sustained drug release. The pattern of drug release was in accordance with the Peppas model. Fourier-transform infrared (FTIR) spectroscopy demonstrated that there were no significant interactions between sitagliptin and chitosan. Differential scanning calorimetry (DSC) results showed an absence of drug peaks due to the fact that the drug was present in an amorphous state. Mucoadhesive nanoparticles were formulated using sitagliptin and were effective for about 12 hours in the gastrointestinal tract. When compared to conventional sitagliptin administration, use of a nanoparticle delivery system demonstrated greater benefits for use in oral delivery applications. This is the first time that a drug-delivery method based on the mucoadhesive properties of nanoparticles could prolong the drug-release time of sitagliptin.

Lipid reducing potential of liposomes loaded with ethanolic extract of purple pitanga (Eugenia uniflora) administered to Caenorhabditis elegans

The ethanolic extract obtained from purple pitanga fruit (Eugenia uniflora – PPE) has been previously described by its potential to reduce lipid accumulation in vitro. In this study, we aimed to study this potential in vivo using Caenorhabditis elegans as animal model. Considering the low pH of the extract, its hydrophilic characteristic, its absorption by the medium where the worms are cultivated and the need of a chronic exposure in the worms solid medium, we have loaded liposomes with PPE and investigated its potential for oral administration. Following 48 h exposure to the PPE-loaded liposomes on worms nematode growth medium, we did not observe any toxic effects of the formulation. Under high cholesterol diet, which increased worms total lipid and also triacylglycerides levels, liposomes containing PPE were able to significantly attenuate these alterations, which could not be observed when worms were treated with free PPE. Furthermore, we could evidence that liposomes were ingested by worms through their labelling to uranin fluorescence dye. Through total phenolic compounds quantification, we estimated an entrapment efficacy of PPE into liposomes of 87.7%. The high levels of phenolic compounds present in PPE, as previously described by our group, indicate that these antioxidants may interfere in worms lipid metabolism, which may occur through many and intricated mechanisms. Although the use of conventional liposomes for human consumption may not be pragmatic, its application for oral delivery of a hydrophilic substance in C. elegans was absolutely critical for our experimental design and has proven to be efficient.

Microrheological characterization of covalent adaptable hydrogels for applications in oral delivery.

The feasibility of a covalent adaptable hydrogel (CAH) as an oral delivery platform is explored using μ2rheology, microrheology in a microfluidic device. CAH degradation is initiated by physiologically relevant pHs, including incubation at a single pH and consecutively at different pHs. At a single pH, we determine CAH degradation can be tuned by changing the pH, which can be exploited for controlled release. We calculate the critical relaxation exponent, which defines the gel-sol transition and is independent of the degradation pH. We mimic the changing pH environment through part of the gastrointestinal tract (pH 4.3 to 7.4 or pH 7.4 to 4.3) in our microfluidic device. We determine that dynamic material property evolution is consistent with degradation at a single pH. However, the time scale of degradation is reduced by the history of degradation. These investigations inform the design of this material as a new vehicle for targeted delivery.

Solubilization of vardenafil HCl in lipid-based formulations enhances its oral bioavailability in vivo: A comparative study using Tween - 20 and Cremophor - EL

The aim of the present investigation was to develop and characterize a promising oral drug delivery system for vardenafil HCl trihydrate (VDN), a poorly soluble drug. For this, self-microemulsifying drug delivery system (SMEDDS) was prepared. Capmul MCM C8 showing highest solubilization capacity was selected as oil. Self-microemulsifying capacity of two surfactants viz. Cremophor EL and Tween 20 were compared for the selected oil phase. Comparing the size of the reconstituted emulsions obtained by small angle neutron scattering (SANS) and Cryo-TEM with that from dynamic light scattering (DLS), it was found that actual core size of oil globule was much smaller than the hydrodynamic diameter. Owing to more number of carbons, Cremophor EL based system was more viscous than Tween 20 VDN SMEDDS. In vitro dissolution and ex vivo permeation study showed superior performance of SMEDDS over drug suspension. In vitro toxicity study using MTT dye on CaCO2 cell line indicated safety of the drug and formulation. Endocytosis and perinuclear localization of SMEDDS system was qualitatively confirmed by cellular uptake study using Coumarin 6 fluorescent dye. In vivo performance was assessed by cryo-sectioning of intestinal tissue and pharmacokinetics. Compared to Cremophor-EL VDN SMEDDS, Tween 20 VDN SMEDDS showed 2.8-fold increase in bioavailability owing to smaller globule size, enhanced dissolution and permeation ability. The encouraging results of SMEDDS suggests its potential application for oral delivery of VDN for treatment of erectile dysfunction.

In vitro permeability enhancement of curcumin across Caco-2 cells monolayers using electrospun xanthan-chitosan nanofibers

Xanthan-Chitosan (X-Ch) polysaccharides nanofibers were prepared using electrospinning processing as an encapsulation and delivery system of curcumin (Cu). The X-Ch-Cu nanofibers remained stable in aqueous HBSS medium at pH 6.5 and pH 7.4, mainly due to the ability of oppositely charged xanthan-chitosan polyelectrolytes to form ionically associated electrospun nanofibers. The xanthan-chitosan-curcumin nanofibers were incubated with Caco-2 cells, and the cell viability, transepithelial transport and permeability properties across cell monolayers were investigated. After 24 h of incubation, the exposure of Caco-2 cell monolayers to X-Ch-Cu nanofibers resulted in a cell viability of ∼80%. A 3.4-fold increase of curcumin permeability was observed when the polyphenol was loaded into X-Ch nanofibers, compared to the free curcumin. This increased in vitro transepithelial permeation of curcumin without compromising cellular viability was induced by interactions upon contact between the nanofibers and the Caco-2 cells, leading to the opening of the tight junctions. The results obtained revealed that X-Ch nanofibers can be used for oral delivery applications of poorly water-soluble compounds at the gastrointestinal tract.

Preparation and Characterization of an Oral Vaccine Formulation Using Electrosprayed Chitosan Microparticles.

Chitosan particles loaded with the antigen ovalbumin (OVA) and the adjuvant Quil-A were produced by electrospray, using mixtures of water/ethanol/acetic acid as a solvent. Three different chitosans designed as HMC+70, HMC+85, and HMC+90 (called as 705010, 855010, and 905010) were tested and its efficacy to be used in oral vaccine delivery applications was investigated. The morphology, size, and zeta potential of the produced particles were investigated, together with the encapsulation efficiency and release of OVA from the three chitosan formulations. Moreover, the mucoadhesion and cytotoxicity of the chitosan microparticles was examined. All the three formulations with OVA and Quil-A were in the micrometer size range and had a positive zeta potential between 46 and 75mV. Furthermore, all the three formulations displayed encapsulation efficiencies above 80% and the release of OVA over a period of 80h was observed to be between 38 and 47%. None of the developed formulations exhibited high mucoadhesive properties, either cytotoxicity. The formulation prepared with HMC+70, OVA, and Quil-A had the highest stability within 2h in buffer solution, as measured by dynamic light scattering. The electrosprayed formulation consisting of HMC+70 with OVA and Quil-A showed to be the most promising as an oral vaccine system.

Alginate hydrogel beads as a carrier of low density lipoprotein/pectin nanogels for potential oral delivery applications

Alginate hydrogel beads have been extensively investigated as drug delivery systems due to promising gastric environment stability. In the present study, alginate hydrogel beads were prepared with Ca2+ or Fe3+ to serve as the loading vehicles for egg yolk low density lipoprotein (LDL)/pectin nanogels. Scanning electron microscope was carried out to confirm the successful incorporation of nanogels into the beads. The FT-IR spectra and swelling ratio analyses proved that incorporation of nanogels did not affect the physicochemical properties of the hydrogel beads. The developed hydrogel beads exhibited pH dependent release of curcumin pre-encapsulated in nanogels, with significant retention of curcumin in gastric condition compared to curcumin encapsulated in nanogels or alginate beads alone. Hydrogel beads prepared with low viscous alginate and Ca2+ showed limited swelling property and more sustained release of curcumin in simulated gastrointestinal conditions, compared to the beads prepared with high viscous alginate and Fe3+. Gradual dissociation of nanogels from the beads during incubation in simulated intestinal fluid was studied with transmission electron microscope. Our study demonstrated the promising potential of alginate beads as a carrier to protect LDL-based nanogels from destabilization in gastric condition, thus expanding their applications as oral delivery system.

Toxicological assessment of mesoporous silica particles in the nematode Caenorhabditis elegans

Here we report the toxicological evaluation of mesoporous silica particles (MSPs) in the nematode C. elegans. Specifically, we have investigated the effect of bare micro- (M0) and nano-sized (N0) MSPs, and their corresponding functionalized particles with a starch derivative (Glu-N) (M1 and N1, respectively) on C. elegans ageing parameters. The toxicity of MSPs, their impact on C. elegans lifespan, movement capacity, progeny and ability to survive upon exposure to acute oxidative stress were assessed. This study demonstrated that both size particles assayed (M0 and N0), labeled with rhodamine and monitored through fluorescence microscopy, are ingested by the nematode. Moreover, toxicity assays indicated that bare nano-sized particles (N0) have a negative impact on the C. elegans lifespan, reducing mobility and progeny production. By contrast, micro-sized particles (M0) proved innocuous for the nematodes. Furthermore, functionalization of nanoparticles with starch derivative reduced their toxicity in C. elegans. Thus, oral intake of N1 comparatively increased the mean lifespan and activity rates as well as resistance to oxidative stress. The overall findings presented here demonstrate the influence of MSP size and surface on their potential toxicity in vivo and indicate the silica-based mesoporous particles to be a potential support for encapsulation in oral delivery applications. Furthermore, the good correlation obtained between healthy aging variables and viability (mean lifespan) validates the use of C. elegans as a multicellular organism for nanotoxicology studies of MSPs.

The formation of zein-chitosan complex coacervated particles: Relationship to encapsulation and controlled release properties

The complex coacervation between zein and chitosan (CS) as well as the relationship with the controlled release properties of their complex nanoparticles were studied. The factors influencing the nanoparticle formation between zein and CS, including solid to liquid ratio, zein to CS ratio and pH, were systematically investigated. The isothermal titration calorimetry (ITC) showed that zein-CS interaction was spontaneous exothermic process. The pH the higher was, the stronger the interaction between zein and CS. The mean particle sizes of ZCNPs were increased by enhanced turbidity between zein and CS (from 90.89 nm to 1368.77 nm). The morphology study showed that spherical particles and coacervate were obtained with the increased interaction between zein and CS. The release profiles of curcumin in vitro indicated that slight burst effect followed by slow release was observed after interacting CS. The ZCNPs at pH 4.0 exhibited smaller particle size (162.07 nm), more stable ζ-potential (49.7 mV), higher encapsulation efficiency (94.67%) and slower release rate. In conclusion, the stronger the interaction was, the lower the curcumin released from the nanoparticles in vitro, and the ZCNPs at pH 4.0 had better potential in oral delivery application.

PH-Dependent silica nanoparticle dissolution and cargo release.

The dissolution of microporous silica nanoparticles (NP) in aqueous environments of different biologically relevant pH was studied in order to assess their potential as drug delivery vehicles. Silica NPs, loaded with fluorescein, were prepared using different organosilane precursors (tetraethoxysilane, ethyl triethoxysilane or a 1:1 molar ratio of both) and NP dissolution was evaluated in aqueous conditions at pH 4, pH 6 and pH 7.4. These conditions correspond to the acidity of the intracellular environment (late endosome, early endosome, cytosol respectively) and gastrointestinal tract ('fed' stomach, duodenum and jejunum respectively). All NPs degraded at pH 6 and pH 7.4, while no dissolution was observed at pH 4. NP dissolution could be clearly visualised as mesoporous hollows and surface defects using electron microscopy, and was supported by UV-vis, fluorimetry and DLS data. The dissolution profiles of the NPs are particularly suited to the requirements of oral drug delivery, whereby NPs must resist degradation in the harsh acidic conditions of the stomach (pH 4), but dissolve and release their cargo in the small intestine (pH 6-7.4). Particle cores made solely of ethyl triethoxysilane exhibited a 'burst release' of encapsulated fluorescein at pH 6 and pH 7.4, whereas NPs synthesised with tetraethoxysilane released fluorescein in a more sustained fashion. Thus, by varying the organosilane precursor used in NP formation, it is possible to modify particle dissolution rates and tune the release profile of encapsulated fluorescein. The flexible synthesis afforded by silica NPs to achieve pH-responsive dissolution therefore makes this class of nanomaterial an adaptable platform that may be well suited to oral delivery applications.

Surface-modified PLGA nanoparticles with chitosan for oral delivery of tolbutamide

The main purpose of present study was to develop novel chitosan-modified polylactic-co-glycolicacid nanoparticles (CS@PLGA NPs) for improving the bio-availability of tolbutamide (TOL). The TOL-loaded CS@PLGA NPs (TOL-CS@PLGA NPs) were fabricated with the solvent evaporation method. The cargo-free CS@PLGA NPs showed a diameter of 228.3±2.5nm monitored with a laser light particlesizer, and the transmission electron microscope (TEM) photographs revealed their “core-shell” structures. The Zeta potential of the original PLGA NPs and the cargo-free CS@PLGA NPs was measured to be −20.2±3.21mV and 24.2±1.1mV, respectively. The changes in Zeta potential indicated the CS chains were coated on the surfaces of the original PLGA NPs. The thermal gravity analysis (TGA) curves suggested that the CS chains improved the thermostability of the original PLGA NPs. The results of cells viability indicated the cargo-free CS@PLGA NPs were nontoxicity. The in vitro release profiles suggested that TOL-CS@PLGA NPs could release TOL in pH 7.4 phosphate buffer solution (PBS) at a sustained manner. Streptozotocin (STZ) was employed to build the diabetic rat models. The physiological changes in the islet β cells confirmed the obtaining of diabetic rats. After treatment by gavage, the TOL-CS@PLGA NPs showed an excellent hypoglycemic effect. Therefore, the TOL-CS@PLGA NPs had a potential application in oral delivery of TOL.

A novel and organic solvent-free preparation of solid lipid nanoparticles using natural biopolymers as emulsifier and stabilizer

In this work, a new and novel organic solvent-free and synthetic surfactant-free method was reported to fabricate stable solid lipid nanoparticles (SLNs) from stearic acid, sodium caseinate (NaCas) and pectin, as well as water. Melted stearic acid was directly emulsified into an aqueous phase containing NaCas and pectin, followed by pH adjustment and thermal treatment to induce the formation of a compact and dense polymeric coating which stabilized SLNs. The preparation procedures and formulations were comprehensively optimized. The inter- and intra-molecular interactions among three ingredients were characterized by fluorescence and Fourier transform infrared spectroscopies. The stability of as-prepared SLNs was evaluated under simulated gastrointestinal conditions, and compared with traditional SLNs prepared with organic solvents. Our results revealed that the SLNs prepared from this organic solvent-free method had superior physicochemical properties over the traditional SLNs, including smaller size and better stability. Furthermore, redispersible SLNs powders were obtained by nano spray drying, but only the SLNs prepared by organic solvent-free method had sub-micron scale, uniform and spherical morphology. The organic solvent-free preparation method was proved to be a promising approach to prepare stable and uniform SLNs for potential oral delivery applications.

Drug Delivery: Plant‐Based Hollow Microcapsules for Oral Delivery Applications: Toward Optimized Loading and Controlled Release (Adv. Funct. Mater. 31/2017)

Drug Delivery: Plant‐Based Hollow Microcapsules for Oral Delivery Applications: Toward Optimized Loading and Controlled Release (Adv. Funct. Mater. 31/2017)

Preparation of lipid nanoparticles with high loading capacity and exceptional gastrointestinal stability for potential oral delivery applications

Exploitation of lipid nanoparticles for oral delivery of nutrients and drugs is limited by their poor stability under gastrointestinal tract and low loading capacity, unless a high concentration of synthetic surfactants is formulated. The main objective of present study is to design a series of new formulations for solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) that are suitable for potential oral delivery applications, using natural biopolymers, i.e. sodium caseinate (NaCas) as emulsifier and pectin as coating, with minimal addition of a synthetic surfactant, Tween 80. Effects of pectin coating, concentration of Tween 80, thermal treatment (80°C for 30min), as well as two chemical cross-linkers on the particulate characteristics, stability, encapsulation efficiency, controlled release and drying feasibility were comprehensively investigated. The intermolecular interactions and cross-linking reactions were studied using Fourier transform infrared spectroscopy. Tween 80 at 0.15% (w/v) together with 0.15% (w/v) NaCas was proved effective to obtain stable cross-linked pectin-coated SLN (PSLN) under 200nm with high loading capacity for curcumin, while NLC prepared under the same condition failed to pass storage stability test. The 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) cross-linked PSLN exhibited superior characteristics than glutaradehyde (GA) cross-linked PSLN, especially for the stability and controlled release under simulated gastrointestinal conditions, with curcumin studied as a model compound. The feasibility of both nano spray drying and freeze-drying technologies were both investigated to transform of colloidal lipid nanoparticles into dry powders. Our results demonstrated a novel strategy to prepare small and homogenous SLN with exceptional GI stability and high loading capacity as a potential oral delivery system.

Size-Dependent Translocation of Nanoemulsions via Oral Delivery.

The in vivo translocation of nanoemulsions (NEs) was tracked by imaging tools with an emphasis on the size effect. To guarantee the accurate identification of NEs in vivo, water-quenching environment-responsive near-infrared fluorescent probes were used to label NEs. Imaging evidence confirmed prominent digestion in the gastrointestinal tract and oral absorption of integral NEs that survive digestion by enteric epithelia in a size-dependent way. In general, reducing particle size leads to slowed in vitro lipolysis and in vivo digestion, a prolonged lifetime in the small intestine, increased enteric epithelial uptake, and enhanced transportation to various organs. Histological examination revealed a pervasive distribution of smaller NEs (80 nm) into enterocytes and basolateral tissues, whereas bigger ones (550, 1000 nm) primarily adhered to villi surfaces. Following epithelial uptake, NEs are transported through the lymphatics with a fraction of approximately 3-6%, suggesting a considerable contribution of the lymphatic pathway to overall absorption. The majority of absorbed NEs were found 1 h post administration in the livers and lungs. A similar size dependency of cellular uptake and transmonolayer transport was confirmed in Caco-2 cell lines as well. In conclusion, the size-dependent translocation of integral NEs was confirmed with an absolute bioavailability of at least 6%, envisioning potential applications in oral delivery of labile entities.

Plant‐Based Hollow Microcapsules for Oral Delivery Applications: Toward Optimized Loading and Controlled Release

Efficient oral administration of protein‐based therapeutics faces significant challenges due to degradation from the highly acidic conditions present in the stomach and proteases present in the digestive tract. Herein, investigations into spike‐covered sunflower sporopollenin exine capsules (SECs) for oral protein delivery using bovine serum albumin (BSA) as a model drug are reported and provide significant insights into the optimization of SEC extraction, SEC loading, and controlled release. The phosphoric‐acid‐based SEC extraction process is optimized. Compound loading is shown to be driven by the evacuation of air bubbles from SEC cavities through the porous SEC shell wall, and vacuum loading is shown to be the optimal loading method. Three initial BSA‐loading proportions are evaluated, leading to a practical loading efficiency of 22.3 ± 1.5 wt% and the determination that the theoretical maximum loading is 46.4 ± 2.5 wt%. Finally, an oral delivery formulation for targeted intestinal delivery is developed by tableting BSA‐loaded SECs and enteric coating. BSA release is inhibited for 2 h in simulated gastric conditions followed by 100% release within 8 h in simulated intestinal conditions. Collectively, these results indicate that sunflower SECs provide a versatile platform for the oral delivery of therapeutics.

Caseinate-zein-polysaccharide complex nanoparticles as potential oral delivery vehicles for curcumin: Effect of polysaccharide type and chemical cross-linking

The formation of protein-polysaccharide complex nanoparticles was investigated using caseinate (NaCas), zein, and three polysaccharides, i.e. pectin, carboxymethyl cellulose (CMC) and gum arabic. Their complexation behavior was examined by dynamic light scattering, Fourier transform infrared and fluorescence spectroscopy. Chemical cross-linker, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide/N-hydroxysuccinimide, were exploited to covalently bridge proteins and polysaccharide to improve the stability of complex nanoparticles in simulated gastrointestinal (GI) fluids. Curcumin was studied as a model lipophilic nutrient to explore the encapsulation and delivery applications. The polysaccharide type significantly affected the cross-linking efficiency which led to distinctive physical stability under simulated GI conditions and completely different morphology of powders obtained by nano spray drying, as well as their redispersibility in water. Compared to gum arabic, pectin and CMC were more favorable to form cross-linked nanoparticles with smaller size (160–210 nm), uniform distribution, spherical shape, higher encapsulation efficiency (up to 80%), slower release and much improved antioxidant activity, as well as exceptional redispersibility after drying. Our study suggested that chemical cross-linking process and selection of polysaccharide should be considered when developing desirable protein-polysaccharide complex nanoparticles that could maintain their integrity under simulated GI conditions for oral delivery applications.

Zein/caseinate/pectin complex nanoparticles: Formation and characterization

In this study, pectin was used as coating material to form zein/caseinate/pectin complex nanoparticles through pH adjustment and heating treatment for potential oral delivery applications. The preparation conditions were studied by applying heating treatment at different pHs, either the isoelectric point of zein (pH 6.2) or caseinate (pH 4.6), or consecutively at both pHs. The particulate characteristics, including particle size, polydispersity index, and zeta potential were monitored for complex nanoparticles formed under different preparation conditions. The complex nanoparticles generally exhibited particle size smaller than 200nm with narrow distribution, spherical shape, and strong negative charge. Fourier transform infrared and fluorescence spectroscopy revealed that hydrophobic interactions and hydrogen bonds were involved in the formation of complex nanoparticles, in addition to electrostatic interactions. Fresh colloidal dispersion and freeze-dried powders varied in their morphology, depending on their preparation conditions. Our results suggested that heating pH and sequence significantly affected the morphology of complex nanoparticles, and pectin coating exerted stabilization effect under simulated gastrointestinal conditions. The present study provides insight into the formation of protein/polysaccharide complex nanoparticles under different preparation conditions.