Microencapsulation Of Proteins Research Articles

Stability and structural properties of bee pollen protein hydrolysate microencapsulated using maltodextrin and whey protein concentrate

In this research, the bee pollen protein hydrolysate was microencapsulated by spray drying using maltodextrin (MD), whey protein concentrate (WPC) and a mixture of both compounds. For this purpose, the bee pollen was hydrolysed by alcalase (enzyme concentration of 1.5%) at 50 °C and pH 8 during 3.95 h, and then freeze-dried. The hydrolysed protein and wall materials were used in ratio of 1:10 (w/w). The wall materials included maltodextrin 2%, WPC 2%, as well as maltodextrin and WPC mixtures with 3:1 ratio. The resulting capsules were exposed to UV radiation for 48 h to accelerate the oxidation. The results showed that the capsule prepared using maltodextrin and WPC mixture showed the highest DPPH radical scavenging during exposure to UV radiation. Based on the FTIR spectroscopy results, the wall containing maltodextrin and WPC mixture showed the best performance in maintaining the chemical structure of hydrolysed protein. The SEM results indicated that the microcapsules prepared with WPC and maltodextrin mixture as wall material showed uniform and smoother wall than those prepared with maltodextrin alone. Finally, it was found that the maltodextrin and WPC mixture was the best wall with an appropriate protective capability for the microencapsulation of hydrolysed proteins and their protection against UV radiation.

Emulsion based microencapsulation of proteins in poly(L-lactic acid) films and membranes for the controlled release of drugs

The aim of this study was to develop micro- or macro-porous substrates of poly(L-lactic acid) (PLLA) for the sustained release of water-soluble proteins. A model protein, bovine serum albumin (BSA), was incorporated into the hydrophobous PLLA matrix by emulsifying an aqueous BSA solution into an organic PLLA solution. The work focused on the effect of the fabrication procedure on substrates' microstructure and protein delivery kinetics. The films were prepared by solvent casting or freeze-extraction, after which the protein release profiles from both matrices were compared. A homogeneous distribution of BSA in the PLLA-films and membranes was obtained. In vitro release of the protein from the freeze-extraction membranes was more significant than the release from the solvent cast films. BSA was found to be released from the freeze-extraction membranes in two stages: an initial fast release followed by a sustained release. The release from the solvent cast films was more moderated and accompanied by the polymer degradation.These materials can find applications in wound healing by local delivery of proteins known to have a positive effect on the regeneration of basal lamina. They can also be used to produce macroporous scaffolds able to deliver growth factors during bone or cartilage regeneration.

Study of stability and biophysical characterization of ranibizumab and aflibercept

The anti-vascular endothelial growth factor (VEGF) agents such as ranibizumab (Lucentis®) and aflibercept (EyLea®) are currently used as monthly or bimonthly intravitreal injections to treat potentially retinal diseases such as wet age-related macular degeneration (AMD) or diabetic macular edema (DME). Because of the complications associated with repeated intra-vitreal injections, there is considerable interest in developing a sustained delivery system. The purpose of this study was to examine the stability of both therapeutic proteins under physiological conditions as well as when incorporated into drug delivery systems (DDS). First, thermotropic properties in physiological conditions and at different pH values were evaluated by differential scanning calorimetry (DSC) to determine the protein denaturation temperature. Second, the effects of pH and incubation time on conformational changes and aggregation were evaluated by circular dichroism (CD), steady-state tryptophan fluorescence spectroscopy, and size-exclusion chromatography (SEC). Also, the ability of both proteins to bind to VEGF was tested in the aforementioned experimental conditions for up to 30 days. Finally, we investigated the stability of both proteins after a rapid screening method that simulates the first homogenizing step during the protein microencapsulation process. This method allowed the development of stable ranibizumab and aflibercept formulations that may be useful to entrap these proteins into microparticles selecting the most convenient organic solvent and protein stabilizers.

Bioerodable PLGA-Based Microparticles for Producing Sustained-Release Drug Formulations and Strategies for Improving Drug Loading.

Poly(lactic-co-glycolic acid) (PLGA) is the most widely used biomaterial for microencapsulation and prolonged delivery of therapeutic drugs, proteins and antigens. PLGA has excellent biodegradability and biocompatibility and is generally recognized as safe by international regulatory agencies including the United States Food and Drug Administration and the European Medicines Agency. The physicochemical properties of PLGA may be varied systematically by changing the ratio of lactic acid to glycolic acid. This in turn alters the release rate of microencapsulated therapeutic molecules from PLGA microparticle formulations. The obstacles hindering more widespread use of PLGA for producing sustained-release formulations for clinical use include low drug loading, particularly of hydrophilic small molecules, high initial burst release and/or poor formulation stability. In this review, we address strategies aimed at overcoming these challenges. These include use of low-temperature double-emulsion methods to increase drug-loading by producing PLGA particles with a small volume for the inner water phase and a suitable pH of the external phase. Newer strategies for producing PLGA particles with high drug loading and the desired sustained-release profiles include fabrication of multi-layered microparticles, nanoparticles-in-microparticles, use of hydrogel templates, as well as coaxial electrospray, microfluidics, and supercritical carbon dioxide methods. Another recent strategy with promise for producing particles with well-controlled and reproducible sustained-release profiles involves complexation of PLGA with additives such as polyethylene glycol, poly(ortho esters), chitosan, alginate, caffeic acid, hyaluronic acid, and silicon dioxide.

Evaluation of alginate-whey protein microcapsules for intestinal delivery of lipophilic compounds in pigs.

In animal care and management, there is an increasing demand for convenient methods of oral delivery of bioactive compounds to specific segments of an animal's gastrointestinal tract. The objective of this study was to test the suitability of microcapsules made with alginate and whey proteins of two different sizes (250 and 800 µm; containing 72 and 76 g kg(-1) of carvacrol respectively) for intestinal delivery of carvacrol in pigs. Encapsulated carvacrol was completely released from the microcapsules after 5 h incubation in simulated intestinal fluids or 6 h in (ex vivo) ileal digesta, whereas release in simulated gastric fluid was minimal. Tests with growing pigs showed over 95% of unencapsulated carvacrol was absorbed or metabolized in the stomach and the duodenum. Encapsulation effectively minimized carvacrol absorption in the stomach (P < 0.05), and increased carvacrol recovery in the small intestine (P < 0.05). Encapsulated carvacrol was completely released from both small and large size capsules within the gastrointestinal tract of pigs. Larger size microcapsules showed a slower in vitro release and greater in vivo recovery of carvacrol in the small intestine (P < 0.05) than the smaller ones. This study indicates alginate-whey protein microencapsulation is a feasible approach for targeted oral delivery of hydrophobic compounds to pig intestines; increasing capsule size increased delivery of carvacrol to the end of the small intestine. © 2015 Her Majesty the Queen in Right of Canada Journal of the Science of Food and Agriculture © 2015 Society of Chemical Industry.

Microencapsulation of single-cell protein from various microalgae species

&lt;p class="BasicParagraph" align="center"&gt;&lt;strong&gt;ABSTRACT&lt;/strong&gt;&lt;/p&gt;&lt;p class="BasicParagraph" align="center"&gt;&lt;strong&gt; &lt;/strong&gt;&lt;/p&gt;&lt;p class="Pa2"&gt;The objective of the research was to evaluate nutritional values of microencapsulated diet made from single cell protein of microalgae. Complete randomized design was applied using three different types of microalgae for inclusion trials i.e. (A) &lt;em&gt;Nannochloropsis &lt;/em&gt;sp., (B) &lt;em&gt;Chlorella &lt;/em&gt;sp., and (C) &lt;em&gt;Spirulina &lt;/em&gt;sp. with five replications respectively. Microencapsulated diet was produced by a modification method based on thermal cross-linking with stable temperature. Phytoplankton was cultured in sea water for which fertilized by a modification of Walne and Guillard fertilizer. The results showed that the highest value of nutrition content was &lt;em&gt;Spirulina &lt;/em&gt;sp. and the average composition of protein, crude lipid, carbohydrate, ash, nitrogen free extract, and water content was 34.80%, 0.30%, 18.53%, 20.09%, 26.29%, and 13.32%, respectively. Organoleptically, microcapsule showed that the color of capsule was dark green and smell fresh phytoplankton.&lt;/p&gt;&lt;p class="Default"&gt; &lt;/p&gt;Keywords: microcapsule, single-cell protein, thermal cross-linking, microalgae, phytoplankton&lt;br /&gt;&lt;p class="BasicParagraph"&gt; &lt;/p&gt;&lt;p class="BasicParagraph"&gt; &lt;/p&gt;&lt;p class="BasicParagraph" align="center"&gt;&lt;strong&gt;ABSTRAK&lt;/strong&gt;&lt;/p&gt;&lt;p class="BasicParagraph"&gt;&lt;strong&gt; &lt;/strong&gt;&lt;/p&gt;&lt;p class="Pa2"&gt;Tujuan penelitian adalah mengevaluasi kandungan nutrisi pakan mikrokapsul protein sel tunggal (&lt;em&gt;single cell protein&lt;/em&gt;) yang berasal dari berbagai jenis mikroalga (fitoplankton). Rancangan percobaan yang digunakan adalah rancangan acak lengkap, dengan perlakuan inklusi mikrokapsul dari jenis fitoplankton (A) &lt;em&gt;Nannochloropsis &lt;/em&gt;sp., (B) &lt;em&gt;Chlorella &lt;/em&gt;sp., dan (C) &lt;em&gt;Spirulina &lt;/em&gt;sp., masing-masing diulang lima kali. Pembuatan mikrokapsul dilakukan dengan menggunakan modifikasi metode dasar &lt;em&gt;thermal cross-linking&lt;/em&gt;, serta menerapkan teknik pengeringan suhu konstan. Proses pembuatan mikrokapsul protein diawali dengan kultur fitoplankton jenis &lt;em&gt;Nannochloropsis &lt;/em&gt;sp., &lt;em&gt;Chlorella &lt;/em&gt;sp., dan &lt;em&gt;Spirulina &lt;/em&gt;sp. Kultur dilakukan di dalam laboratorium menggunakan media air laut dan modifikasi pupuk Walne dan Guillard. Hasil penelitian menunjukkan bahwa kandungan nutrisi tertinggi terdapat pada jenis mikrokapsul protein sel tunggal yang berasal dari &lt;em&gt;Spirulina &lt;/em&gt;sp., dengan rata-rata komposisi kandungan protein 34,80%, lemak 0,30%, karbohidrat 18,53%, abu 20,09%, dan BETN 26,29%. Mikrokapsul berwarna hijau tua dan aroma fitoplankton segar.&lt;/p&gt;&lt;p class="Default"&gt; &lt;/p&gt;Kata kunci: mikrokapsul, protein sel tunggal, &lt;em&gt;thermal cross-linking&lt;/em&gt;, mikroalga, fitoplankton

Critical attributes of formulation and of elaboration process of PLGA-protein microparticles

Low drug loading, burst effect during release and drug inactivation account for the main drawbacks of protein microencapsulation in poly(d,l-lactic-co-glycolic) acid (PLGA) matrix by the water-in oil-in water (W/O/W) solvent evaporation method. Thus, the current study was set to invest the critical attributes of formulation and of elaboration process which determine protein loading into microparticles as well as its further release, using albumin as protein model.NaCl concentration in the external aqueous phase, poly(vinyl alcohol) (PVA) concentration and mostly viscosity of both the internal aqueous phase and the organic phase were critical attributes for improving drug loading, with polymer molecular weight and hydrophobicity likewise directly related to albumin loading. In such a way, when using 0.5% PVA as internal aqueous phase the highest albumin loading was achieved. Optimized microparticles exhibited a sustained in vitro release of albumin over 130 days. The influence of the microencapsulation process on albumin stability and biological activity was evaluated by carrying out cell proliferation assays on PC12 cells with albumin released from microparticles. Such assay demonstrated that the microencapsulation procedure optimized in this study did not affect the biological stability of the microencapsulated protein.

A biomimetic approach to active self-microencapsulation of proteins in PLGA

A biomimetic approach to organic solvent-free microencapsulation of proteins based on the self-healing capacity of poly (dl)-lactic-co-glycolic acid (PLGA) microspheres containing glycosaminoglycan-like biopolymers (BPs), was examined. To screen BPs, aqueous solutions of BP [high molecular weight dextran sulfate (HDS), low molecular weight dextran sulfate (LDS), chondroitin sulfate (CS), heparin (HP), hyaluronic acid (HA), chitosan (CH)] and model protein lysozyme (LYZ) were combined in different molar and mass ratios, at 37°C and pH7. The BP-PLGA microspheres (20–63μm) were prepared by a double water–oil–water emulsion method with a range of BP content, and trehalose and MgCO3 to control microclimate pH and to create percolating pores for protein. Biomimetic active self-encapsulation (ASE) of proteins [LYZ, vascular endothelial growth factor165 (VEGF) and fibroblast growth factor (FgF-20)] was accomplished by incubating blank BP-PLGA microspheres in low concentration protein solutions at ~24°C, for 48h. Pore closure was induced at 42.5°C under mild agitation for 42h. Formulation parameters of BP-PLGA microspheres and loading conditions were studied to optimize protein loading and subsequent release. LDS and HP were found to bind >95% LYZ at BP:LYZ>0.125 w/w, whereas HDS and CS bound >80% LYZ at BP:LYZ of 0.25–1 and <0.33, respectively. HA-PLGA microspheres were found to be not ideal for obtaining high protein loading (>2% w/w of LYZ). Sulfated BP-PLGA microspheres were capable of loading LYZ (~2–7% w/w), VEGF (~4% w/w), and FgF-20 (~2% w/w) with high efficiency. Protein loading was found to be dependent on the loading solution concentration, with higher protein loading obtained at higher loading solution concentration within the range investigated. Loading also increased with content of sulfated BP in microspheres. Release kinetics of proteins was evaluated in-vitro with complete release media replacement. Rate and extent of release were found to depend upon volume of release (with non-sink conditions observed <5ml release volume for ~18mg loaded BP-PLGA microspheres), ionic strength of release media and loading solution concentration. HDS-PLGA formulations were identified as having ideal loading and release characteristics. These optimal microspheres released ~73–80% of the encapsulated LYZ over 60days, with >90% of protein being enzymatically active. Nearly 72% of immunoreactive VEGF was similarly released over 42days, without significant losses in heparin binding affinity in the release medium.

One-Step Production of Protein-Loaded PLGA Microparticles via Spray Drying Using 3-Fluid Nozzle

The aim of this study was to investigate the potential of using a spray-dryer equipped with a 3-fluid nozzle to microencapsulate protein drugs into polymeric microparticles. Lysozyme and PLGA were used as a model protein and a model polymer, respectively. The effects of process and formulation variables, such as i) the type of organic solvent, ii) the feeding rate ratio of the outer PLGA-containing feed solution to inner lysozyme-containing feed solution, and iii) the mass ratio of PLGA to protein, on the properties (morphology, internal structure, protein surface enrichment and release profiles) of the spray dried microparticles were investigated to understand protein microencapsulation and particle formation mechanisms. The spherical, condensed microparticles were obtained with D50 of 1.07-1.60μm and Span in the range of 0.82-1.23. The lysozyme surface content decreased upon different organic solvents used as follows: acetonitrile > acetone > dichloromethane. Additionally, the lysozyme surface enrichment decreased slightly when increasing the feeding rate ratio of the outer feed solution to the inner feed solution from 4:1 to 10:1. Furthermore, it was observed that there was a correlation between the degree of burst release and the lysozyme surface enrichment, whereas the lysozyme loading content had no substantial impact on the release kinetics. The present work demonstrates the potential of spray dryer equipped with a 3-fluid nozzle in microencapsulation of proteins into PLGA matrices with different characteristics by varying process and formulation parameters.

Use of Protease Inhibitors in Composite Polyelectrolyte Microparticles in Order to Increase the Bioavailability of Perorally Administered Encapsulated Proteins

Protein protease inhibitors (aprotinin, soybean Bowman–Birk inhibitor, and Kunitz soybean trypsin inhibitor) possessing different specificity with respect to trypsin, chymotrypsin, and elastase were encapsulated together with a cargo protein in polyelectrolyte microparticles using layer-by-layer (LbL) deposition techniques. The most efficient inclusion of the inhibitors occurred at the formation stage of the insoluble protein complex with the polyanion. Simultaneous immobilization of the inhibitor and protein did not influence the physicochemical properties of the microparticles, specifically their pH-sensitive behavior under conditions modeling the passage through various parts of the human gastrointestinal tract after peroral administration. The most effective protection against the action of proteolytic enzymes of pancreatic juice and the small intestine was achieved for simultaneous release of cargo protein and inhibitor from the microparticles. Soybean Bowman–Birk inhibitor, which is most similar to insulin with respect to physicochemical properties, in addition to the soybean extract enriched with protease inhibitors were the most suitable agents for protection of human insulin or its rapidly acting analogs (lispro and aspart). These findings suggested that simultaneous microencapsulation of both protein and protein protease inhibitor was a promising way to increase the protein bioavailability upon peroral administration of polyelectrolyte microparticles.

Microencapsulation of protein into biodegradable matrix: a smart solution cross-linking technique

Sustained-release albumin microspheres (MSs) can be obtained by chemically cross-linking albumin. However, a significant challenge is preventing the cross-linking of the active pharmaceutical (protein or small molecule) ingredient (API) with the MS matrix. To prevent cross-linking of the API with the albumin matrix, a smart “solution cross-linking-microencapsulation” method was developed which involves cross-linking albumin solution with glutaraldehyde first, neutralizing any excess glutaraldehyde with sodium bisulphite, followed by the addition of API and finally spray drying. Using lysozyme as model API, MS formulations FL1 and FL2 were prepared and characterized. Physicochemical characterization using FT-IR and bioactivity evaluation indicate that microencapsulated API did not undergo any significant change in its native structure and the bioactivity was preserved during the formulation processing. Preliminary immunogenicity potential of the cross-linked albumin matrix determined by in vivo studies did not show any significant increase in antigen-specific serum-IgG levels, implying safety and biocompatibility of the cross-linked albumin matrix.

Hoffmeister Ion Series Protected Bee Venom Proteins from Damages Induced by Microencapsulation Process

PLGA (poly-lactide-co-glycolide) microspheres have more than 30 years of use in drug delivery, but there are some limitations related to protein stability during the process of microencapsulation. During the W1/O phase (in the W1/O/W2 microencapsulation process), hydrophobic interfaces are expanded where interfacial adsorption occurs followed by protein unfolding and aggregation. These problems have limited their usage as vaccine carriers. The Hoffmeister series ions were added to BV (bee venom) proteins to obtain their stability during microencapsulation. A correlation was established between the salts used in aqueous solutions and changes in BV solubility and conformations. The BV α-helical content was stable without protein aggregation or hydrophobic exposition, and a small change at the S-S dihedral angles. BV aggregation was avoided by 20 % in the presence of 40 mM MgCl2, (chaotropic salt) with preserved biological activity. The aim of this work was achieved, which was to obtain the maximum BV stability and can be considered a biotechnological breakthrough because a simple solution like salt addition avoided heterologous proteins usage to reach protein stability. Keywords: Nanomedicine, venom immunotherapy, protein stability, Protein microencapsulation, vaccine carriers, vaccine adjuvants, Drug Content Assay

Hoffmeister Ion Series Protected Bee Venom Proteins from Damages Induced by Microencapsulation Process

PLGA (poly-lactide-co-glycolide) microspheres have more than 30 years of use in drug delivery, but there are some limitations related to protein stability during the process of microencapsulation. During the W1/O phase (in the W1/O/W2 microencapsulation process), hydrophobic interfaces are expanded where interfacial adsorption occurs followed by protein unfolding and aggregation. These problems have limited their usage as vaccine carriers. The Hoffmeister series ions were added to BV (bee venom) proteins to obtain their stability during microencapsulation. A correlation was established between the salts used in aqueous solutions and changes in BV solubility and conformations. The BV α-helical content was stable without protein aggregation or hydrophobic exposition, and a small change at the S-S dihedral angles. BV aggregation was avoided by 20 % in the presence of 40 mM MgCl2, (chaotropic salt) with preserved biological activity. The aim of this work was achieved, which was to obtain the maximum BV stability and can be considered a biotechnological breakthrough because a simple solution like salt addition avoided heterologous proteins usage to reach protein stability. Keywords: Nanomedicine, venom immunotherapy, protein stability, Protein microencapsulation, vaccine carriers, vaccine adjuvants, Drug Content Assay

Drug and Protein Encapsulation by Emulsification: Technology Enhancement Using Foam Formulations

AbstractSignificant progress has been made in developing formulations for protein and drug encapsulation and delivery. The most frequently used method is the emulsion/solvent removal technology, where microencapsulation of proteins in polymeric matrices can be easily achieved with a simple stirrer setup. However, it remains a challenge to produce protein‐encapsulated formulations with high encapsulation efficiencies. The emulsion/solvent removal technique and the relevant formulation and process parameters that govern the protein encapsulation processes are reviewed. A new encapsulation method of using foam as a delivery medium during the preparation of protein‐loaded microparticles is proposed. The foam characteristics of direct relevance to protein encapsulation are discussed. The unique properties of foam could enhance intermolecular interactions and access to internal pore surfaces, thus making them superior compared to traditional methods employing aqueous liquid phases during protein encapsulation.

Micro- et nanotechnologies pour l’administration de médicaments au niveau cérébral

As far as micromedicines are concerned, we are interested in the microencapsulation of recombinant proteins, to generate microcarriers upon which living cells can be adsorbed, a highly challenging technology. The whole system forms a Pharmacologically Active Microcarrier (PAM) to be used in cell therapy in the context of neurodegenerative diseases. More precisely, the PAMs are used for tissue engineering, they will increase cell survival time as well as the differentiation and integration of grafted cells following transplants in animals, these micromedicines can also activate the regenerative potential of adult stem cells such as the MIAMI cells. Within the domain of nanomedicines, we are pursuing the development of lipid nanocapsules that act as biomimetic nanovectors resembling lipoproteins. We are studying systematically the biodistribution profiles of these nanomedicines depending on their route of administration, local or systemic. In particular, we are trying to define the essential physicochemical parameters of these nanovectors that, after administration, control the targeting of tumours. In the same way, we are trying to understand how these nanomedicines cross biological barriers and how they interact with cells. In terms of preclinical applications, we are focusing on glioblastomas. The route of administration can be systemic or local. The most promising results in terms of survival of tumour-bearing animals were obtained by infusing radioactive nanocapsules intratumourally, in order to achieve an in-situ radiotherapy approach.

Conference Report: Pharmaceutical Development of Biologics: fundamentals, Challenges and Recent Advances

The 46th Arden Conference, held in West Point, NY, USA, March 2011, focused on development of protein therapeutics, comprising preformulation, formulation, manufacturing, advanced delivery systems, protein characterization/analysis, and regulatory landscape. The sessions of preformulation and formulation development consisted of nine lectures discussing protein stability implications and characterization during purification, freeze-drying and manufacturing. The session on advanced drug delivery encompassed two new sustained-release microsphere formulations (protein microencapsulation by annealing of premade porous PLGA microspheres and aqueous-aqueous emulsion for preformulating proteins to solvent-resistant particles), two transdermal insulins (patching after thermal ablation of skin and phase-transition hydrogel microneedle patch), and a responsible hydrogel system for intra-ear delivery. The sessions on analytical technologies and regulatory landscape both focused on challenges for biosimilars.

Influence of l-cysteine, oxygen and relative humidity upon survival throughout storage of probiotic bacteria in whey protein-based microcapsules

The survival rates of Lactobacilus acidophilus Ki, Lactobacillus paracasei L26 and Bifidobacterium animalis BB-12 were studied after whey protein microencapsulation via spray-drying, with or without l-cysteine-HCl, and storage up to 6 months at 5 °C and 22 °C, with variation in relative air humidity and oxygen levels. Lb. paracasei L26 was the least susceptible to storage conditions: above 10 6 cfu g −1 were recorded by 180 d at 22 °C, irrespective of relative humidity, and the presence/absence of oxygen and l-cysteine. Higher relative humidity, higher temperature and longer storage periods were deleterious to survival of both B. animalis BB-12 and Lb. acidophilus Ki; the effect of l-cysteine-HCl was dependent on the probiotic strain. The effect of overhead oxygen was not significant upon any probiotic strain studied. Whey protein microcapsules containing l-cysteine-HCl protected probiotic cultures from simulated gastrointestinal conditions.

A study of enzyme and protein microencapsulation--some factors affecting the low apparent enzymic activity yields.

Microencapsulation of aqueous solutions of enzymes, alpha-chymotrypsin (EC 3.4.21.1) and histidase (EC 4.3.1.3), with semipermeable polyamide membranes resulted in a loss of enzymic activity. The low yields (less than 40%) found with both enzymes were typical of others reported in the literature. The activity of broken histidase-containing microcapsules was greater than that of the microcapsules before breaking, and this was interpreted as being due to a simple diffusional restriction on the substrate and product. The maximum of the apparent pH-activity curve of alpha-chymotrypsin was found to be shifted one unit to more alkaline pH when the enzyme was encapsulated. This phenomenon was explained in terms of the hydrogen ion concentration in the microenvironment surrounding the enzyme being different from that in the bulk solution. Microencapsulation of aqueous solutions of enzymes is accomplished by in situ polymerization reactions at the interface of a water-in-oil emulsion. 125I-labelled proteins (albumin and fibrinogen) were encapsulated under similar conditions to determine the efficiency of the microencapsulation process. About one third of these proteins was lost during the overall preparation procedure and a further fraction was attached to the membranes of the microcapsules.

Surface functionalization of polyketal microparticles with nitrilotriacetic acid–nickel complexes for efficient protein capture and delivery

Microparticle drug delivery systems have been used for over 20 years to deliver a variety of drugs and therapeutics. However, effective microencapsulation of proteins has been limited by low encapsulation efficiencies, large required amounts of protein, and risk of protein denaturation. In this work, we have adapted a widely used immobilized metal affinity protein purification strategy to non-covalently attach proteins to the surface of microparticles. Polyketal microparticles were surface modified with nitrilotriacetic acid–nickel complexes which have a high affinity for sequential histidine tags on proteins. We demonstrate that this high affinity interaction can efficiently capture proteins from dilute solutions with little risk of protein denaturation. Proteins that bound to the Ni–NTA complex retain activity and can diffuse away from the microparticles to activate cells from a distance. In addition, this surface modification can also be used for microparticle targeting by tethering cell-specific ligands to the surface of the particles, using VE-Cadherin and endothelial cells as a model. In summary, we show that immobilized metal affinity strategies have the potential to improve targeting and protein delivery via degradable polymer microparticles.

Hoffmeister Series Ions Protect Diphtheria Toxoid from Structural Damages at Solvent/Water Interface

During the W1/O phase (in the W1/O/W2 process) of protein microencapsulation within poly-lactide-co-glycolide (PLGA), hydrophobic interfaces are expanded where interfacial adsorption occurs followed by protein unfolding and aggregation. Spectroscopic and immunological techniques were used to ascertain the effects of the Hoffmeister series ions on Diphtheria toxoid (Dtxd) stability during the W1/O phase. A correlation was established between salts used in aqueous solutions and the changes in Dtxd solubility and conformation. The Dtxd α-helical content was quite stable thus leading to the conclusion that encapsulation was followed by protein aggregation, with minor exposition of hydrophobic residues and a small change at the S-S dihedral angle. Dtxd aggregation is 95% avoided by the chaotropic SCN-. This was used to prepare a stable Dtxd and immunologically recognized/PLGA formulation in the presence of 30 mM SNC-. The recovery increased by 10.42% or 23.2% when microencapsulation was within the -COOMe or -COOH (12kDa) PLGA, respectively. In conclusion, the aim of this work was achieved, which was to obtain the maximum of Dtxd stability after contact with CH2Cl2 to begin its PLGA microencapsulation within ideal conditions. This was a technological breakthrough because a simple solution like salt addition avoided heterologous proteins usage.