Nanoformulated Delivery Systems of Essential Nutraceuticals and Their Applications

Malnutrition and poor diet constitute the number one driver of the global burden of disease. Undernutrition is responsible for up to 50% of all deaths in children under the age of 5. In South Africa, 25% of the country’s children suffer from undernutrition. This increases the risk of child mortality as well as contracting infectious diseases. It also affects the physical and intellectual development of the children. The greatest drawback in malnutrition is the deficiency of essential nutraceuticals involved in important biological functions. Innovative technologies such as nanoformulated products are needed for food and agriculture in order to enhance the children’s health. The evaluation and application of various nanoformulated delivery systems will be explored for improving the stability and bioavailability of essential nutraceuticals for consumers

Protein-polysaccharide complexes for improved protein delivery within CaCO3 microparticles

Introduction: The controlled delivery of proteins within calcium carbonate (CaCO3) particles is currently widely investigated due to accessibility, low cost, safety, pH-sensitive properties, high surface area and high porosity. The success of these carriers has been driven by the ionic interactions between proteins and particles making the encapsulation of proteins highly dependent on the pH of reaction solutions and the isoelectric point of the protein. This poses a great limitation on the successful loading of proteins into microparticles. In this study we explored the use of polysaccharide-protein complexes to enhance the encapsulation of otherwise poorly encapsulated proteins in CaCO3 microparticles. Methods: A chitin binding domain (ChBD) was inserted on the protein β-lactamase to form a chimeric protein. A protein-polysaccharide complex was formed between the protein and hyaluronic acid (HA) owing to the intrinsic affinity of the ChBD to HA. The chimeric protein was then loaded into CaCO3 microparticles using super critical CO2 technology aided by the templating effect of HA on CaCO3. The microparticles were characterised for size, surface charge, polymorphism and protein loading. Bioactivity and stability of the encapsulated β-lactamase was characterised by kinetic reaction with nitrocefin. A thrombin cleavage site was inserted onto the gene sequence of the protein to achieve release of the protein from the microparticles by proteases mediation using thrombin. Results: Vaterite CaCO3 microparticles of sizes ranging between 6 and 8 µm were produced. The presence of the ChBD on the β-lactamase increased the encapsulation of the protein by 6 fold when complexed with HA. Thrombin mediated release increased the release of the protein from the microparticles within 36 hours from <25% to 87%. The protein-polysaccharide complex demonstrated success in encapsulation of the protein while retaining up to 81% activity of the protein and allowing controlled release by proteases. Conclusion: Protein-polysaccharide complexation demonstrates an excellent approach for the delivery of sensitive biomacromolecules which can otherwise be complicated due to electrostatic hindrances. Future prospects include using the methods we have developed for encapsulation of therapeutic proteins and using calcium carbonate as a carrier and scaffold for example in bone regeneration

Improved encapsulation of proteins within calcium carbonate microparticles by means of protein-polysaccharide complexes

The controlled delivery of proteins within calcium carbonate particles is currently widely investigated. The success of these carriers has been driven by the ionic interactions between proteins and particles making the encapsulation of proteins highly dependent on the pH of reaction solutions and the isoelectric point of the protein. This poses a great limitation on the successful loading of proteins into microparticles. In this study we explored the use of polysaccharide-protein complexes to enhance the encapsulation of otherwise poorly encapsulated proteins in CaCO3 microparticles. A chitin binding domain (ChBD) was inserted on the protein β-lactamase to form a chimeric protein. A protein-polysaccharide complex was formed between the protein and hyaluronic acid (HA) owing to the intrinsic affinity of the ChBD to HA. The chimeric protein was then loaded into calcium carbonate (CaCO3) microparticles using super critical CO2 technology aided by the templating effect of HA on CaCO3. The microparticles were characterised for size, surface charge, poly-morphism and protein loading. Biochemical stability of the encapsulated β-lactamase was characterized by kinetic reaction with nitrocefin. The presence of the ChBD on the β-lactamase increased the encapsulation of the protein by 6 fold when complexed with HA. Thrombin mediated release increased the release of the protein from the microparticles within 36 hours from <25% to 87%. The protein-polysaccharide complex proved successful in enhancing the encapsulation of protein while retaining up to 81% activity and allowing controlled release of the protein by proteases

Protein-polysaccharide complexes, a tool for protein delivery in CaCO3 microparticles

INTRODUCTION The controlled delivery of proteins within calcium carbonate (CaCO3) particles is currently widely investigated. The success of these carriers has been driven by the ionic interactions between proteins and particles making the encapsulation of proteins highly dependent on the pH of reaction solutions and the isoelectric point of the protein.1 This poses a great limitation on the successful loading of proteins into microparticles. In this study, we explored the use of polysaccharide-protein interactions to strongly enhance the encapsulation of proteins in CaCO3 microparticles. EXPERIMENTAL METHODS Previously, Vandevenne and colleagues2 inserted a human chitin binding domain (ChBD) that has intrinsic affinity for hyaluronic acid (HA) into β-lactamase (BlaP). This generated chimeric protein, named BlaPChBD, was shown to be fully bifunctional. In this study this hybrid protein (BlapChBD) was associated to HA and successfully loaded into CaCO3 microparticles using super critical CO2 technology aided by the templating effect of HA on CaCO3. Furthermore, thrombin cleavage sites were engineered on both sides of the inserted ChBD in the chimeric BlaP so that release of the protein from the microparticles could be easily achieved by protease cleavage. The microparticles were characterised for size, surface charge, poly-morphism and protein loading and in-vitro release assays were performed. RESULTS AND DISCUSSION The presence of ChBD inserted into the β-lactamase increased the encapsulation of the protein by 6-fold when complexed with HA (Fig. 1). In addition, we also showed that the encapsulated BlaP remains stable during this process using kinetic reaction of β-lactam hydrolysis. Our data showed that vaterite CaCO3 microparticles of sizes ranging between 4 and 7 µm were produced. We were also able to demonstrate that thrombin cleavage increased the release of the protein from the microparticles within 36 hours from <25% to 87% (Fig. 2). In conclusion, the presence of ChBD successfully improved the encapsulation yield of the protein while retaining up to 81% of its activity. CONCLUSION Protein-polysaccharide complexation demonstrates an excellent approach for the delivery of sensitive biomacromolecules which can otherwise be complicated due to electrostatic hindrances. Future prospects include using the methods we have developed for encapsulation of therapeutic proteins and using calcium carbonate as a carrier and scaffold in bone regeneration for example

Design of new delivery systems for therapeutic proteins based on calcium carbonate microspheres

There exists a constant need for delivery systems that are biocompatible, offer bioactives protection from premature degradation and allow for targeted delivery and controlled release. Calcium carbonate (CaCO3) is one such system that has gained great favour for employment in the biomedical field due to possibilities of controlling size, morphology and crystalline forms of particles by tuning the synthesis conditions. CaCO3 has demonstrated ability to increase safety, stability and overall efficiency of protein therapeutics. The aim of the present work was to assess the significance of polysaccharide-protein complexes in enhancing the encapsulation of proteins in CaCO3 microspheres. A Chitin Binding Domain (ChBD), reported to have affinity for hyaluronic acid, was inserted on β-lactamase enzyme to develop a chimeric protein. The chimeric protein retained the activity of the enzyme and the binding properties and was encapsulated in CaCO3 microspheres by a super critical CO2 (ScCO2) process using hyaluronic acid as a templating agent. The particles were characterised in terms of size, zeta potential, morphology and protein loading. The results obtained confirmed the affinity of the ChBD to hyaluronic acid towards the production of stable, vaterite microparticles. Protein assays demonstrated that the ChBD enhanced the encapsulation of protein by up to 10 fold. Confocal images also suggested high encapsulation of the chimeric protein compared to native protein. Thus the production of polysaccharide-protein complexes seems effective in enhancing the encapsulation of proteins in CaCO3 microparticles using the ScCO2 process. Moreover this method will further be used to enhance encapsulation of therapeutic proteins such as growth factors for bone and cartilage regeneration

The use of protein-polysaccharide complexes to enhance protein delivery in CaCO3 microparticles

The role of protein therapeutics in modern medicine has increased considerably due to their potential to perform complex functions in the body that cannot be mimicked by simple chemical compounds. To increase the safety, stability and efficacy of protein therapeutics; there exists a need for delivery systems that are biocompatible, prevent premature degradation of bioactives and allow targeted delivery and controlled release. Calcium carbonate (CaCO3) has gained great favour for employment in protein delivery due to possibilities of controlling size, morphology and crystalline forms of particles by tuning the synthesis conditions. The aim of the present work was to assess the significance of polysaccharide-protein complexes in enhancing the encapsulation of proteins in CaCO3 microspheres. A Chitin Binding Domain (ChBD), reported to have affinity for hyaluronic acid, was inserted on β-lactamase enzyme to develop a chimeric protein. The chimeric protein retained the activity of the enzyme and the binding properties and was encapsulated in CaCO3 microspheres by a super critical CO2 (ScCO2) process using hyaluronic acid as a templating agent. The particles were characterised in terms of size, zeta potential, morphology and protein loading. The results obtained confirmed the affinity of the ChBD to hyaluronic acid towards the production of stable, vaterite microparticles. Protein assays demonstrated that the ChBD enhanced the encapsulation of protein by up to 10 fold. Confocal images also suggested high encapsulation of the chimeric protein compared to native protein. Thus the production of polysaccharide-protein complexes seems effective in enhancing the encapsulation of proteins in CaCO3 microparticles using the ScCO2 process

In vivo uptake and acute immune response to orally administered chitosan and PEG coated PLGA nanoparticles

Nanoparticulate drug delivery systems offer great promise in addressing challenges of drug toxicity, poor bioavailability and non-specificity for a number of drugs. Much progress has been reported for nano drug delivery systems for intravenous administration, however very little is known about the effects of orally administered nanoparticles. Furthermore, the development of nanoparticulate systems necessitates a thorough understanding of the biological response post exposure. This study aimed to elucidate the in vivo uptake of chitosan and polyethylene glycol (PEG) coated Poly, DL, lactic-co-glycolic Acid (PLGA) nanoparticles and the immunological response within 24 h of oral and peritoneal administration. These PLGA nanoparticles were administered orally and peritoneally to female Balb/C mice, they were taken up by macrophages of the peritoneum. When these particles were fluorescently labelled, intracellular localisation was observed. The expression of pro-inflammatory cytokines IL-2, IL-6, IL-12p70 and TNF-α in plasma and peritoneal lavage was found to remain at low concentration in PLGA nanoparticles treated mice as well as ZnO nanoparticles during the 24 hour period. However, these were significantly increased in lipopolysaccharide (LPS) treated mice. Of these pro-inflammatory cytokines, IL-6 and IL-12p70 were produced at the highest concentration in the positive control group. The anti-inflammatory cytokines IL-10 and chemokines INF-γ, IL-4, IL-5 remained at normal levels in PLGA treated mice. IL-10 and INF-γ were significantly increased in LPS treated mice. MCP-1 was found to be significantly produced in all groups in the first hours, except the saline treated mice. These results provide the first report to detail the induction of cytokine production by PLGA nanoparticles engineered for oral applications.South African Department of Science and Technolog

Effects of protein binding on the biodistribution of PEGylated PLGA nanoparticles post oral administration

The surface of nanoparticles is often functionalised with polymeric surfactants, in order to increase systemic circulation time. This has been investigated mainly for intravenously administered nanoparticles. This study aims to elucidate the effect of surface coating with various concentrations of polymeric surfactants (PEG and Pluronics F127) on the in vitro protein binding as well as the tissue biodistribution, post oral administration, of PLGA nanoparticles. The in vitro protein binding varied depending on the polymeric surfactant used. However, in vivo, 1% PEG and 1% Pluronics F127 coated particles presented similar biodistribution profiles in various tissues over seven days. Furthermore, the percentage of PEG and Pluronics coated particles detected in plasma was higher than that of uncoated PLGA particles, indicating that systemic circulation time can also be increased with oral formulations. The difference in the in vitro protein binding as a result of the different poloxamers used versus similar in vivo profiles of these particles indicates that in vitro observations for nanoparticles cannot represent or be correlated to the in vivo behaviour of the nanoparticles. Our results therefore suggest that more studies have to be conducted for oral formulations to give a better understanding of the kinetics of the particles

Protein–polysaccharide complexes for enhanced protein delivery in hyaluronic acid templated calcium carbonate microparticles

International audienceThe controlled delivery of proteins within calcium carbonate (CaCO 3) particles is currently widely investigated. The success of these carriers is driven by ionic interactions between the encapsulated proteins and the particles. This poses a great limitation on the successful loading of proteins that have no ionic affinity to CaCO 3. In this study, we explored the use of polysaccharide–protein interactions to strongly enhance the encapsulation of proteins in CaCO 3 microparticles. Previously, Vandevenne and colleagues inserted a human chitin binding domain (ChBD) that has intrinsic affinity for hyaluronic acid (HA) into a b-lactamase (BlaP). This generated chimeric protein, named BlaPChBD, was shown to be fully bifunctional. In this study we showed that this hybrid protein can associate with HA and be successfully loaded into vaterite CaCO 3 microparticles using supercritical CO 2 (ScCO 2) technology aided by the templating effect of HA on CaCO 3. The presence of ChBD inserted into BlaP increased the encapsulation of the protein by 6-fold when complexed with HA. Furthermore, thrombin cleavage sites were engineered on both sides of the inserted ChBD in the chimeric BlaP to achieve release of the protein from the micro-particles by protease cleavage. Our results showed that thrombin cleavage increased the release of the protein from the microparticles within 36 hours from o20% to 87%. In conclusion, the presence of ChBD successfully improved the encapsulation yield of the protein while retaining up to 82% of its activity and efficient release of the protein from the microparticles was achieved by protease cleavage