Protein Encapsulation Efficiency Research Articles

Idebenone addition in protein‐loaded PLGA/VitE microspheres as neuroprotective IODDS

Purpose: Anti‐inflammatory and antioxidant evaluation of idebenone (IDB) in ARPE‐19 cell line and subsequent encapsulation in PLGA (polylactic‐co‐glycolic‐acid)/vitamin E microspheres (MS), together with neurotrophic factors, to confer additional neuroprotective activity and technological advantages.Methods: ARPE‐19, a human RPE cell line, were incubated for 24 with IDB (0.5‐150 μM) to determine cell viability by MTT assay. For anti‐inflammatory evaluation, cells were pre‐treated with IDB (10 μM) for 3 h and then, stimulated with LPS (10 μg/mL;24 h). Interleukin‐6 (IL‐6) and chemokine CCL2 levels were quantified by ELISA. To determine the antioxidant activity of IDB, the induction of intracellular reactive oxygen species (ROS) was investigated. Cells were 3 h pre‐treated with IDB (10 μM), posterior exposed to hydrogen peroxide (H2O2) (100 μM;6 h), and measured by a Cellular ROS Assay kit. The elaboration of the PLGA/vit E MS loaded with IDB and protein was carried out by the method of extraction‐evaporation of the solvent from an S/O/W emulsion. Microspheres were characterized by SEM, Confocal microscopy and LDS. The protein encapsulation efficiency and the % released in the first 24 h of in vitro release study was measured by fluorimetry.Results: IDB (10 μM) significantly suppressed LPS‐induced secretion of IL‐6 and CCL2 in ARPE‐19 cells. Moreover, pretreatment with the additive significantly attenuated the increased ROS levels in H2O2‐induced cells. Monodisperse spherical MSs were obtained with mean particle size suitable for intravitreal injection (29.41 ± 0.34 μm) and small superficial porous due to the presence of VitE. MSs showed a protein‐loading of 64.48 ± 1.99%. According to confocal images, protein was mainly distributed on the MS core. The initial protein release was 57.21 ± 10.89%.Conclusions: The inclusion of IDB in the PLGA/VitE MSs loaded with active proteins could increase the neuroprotective activity of the IODDS as well as improve its technological characteristics.

Ratio Variation of Maltodextrin and Gum Arabic as Encapsulant on White Jack Bean Tempe Protein Concentrate

The physical and chemical properties of encapsulated substance is directly influenced by the choice of the encapsulant. Different ratios of maltodextrin (MD) and Gum Arabic (GA) produce varying material properties. Therefore, this study aimed to obtain the optimal encapsulant ratio for white jack bean tempe protein concentrate based on physical and chemical characteristics. To achieve this, a descriptive method was employed along with One-Way ANOVA. The results showed that varying encapsulant ratio led to distinct protein content, moisture content, encapsulation efficiency, and yield. The treatment with maltodextrin and Gum Arabic ratio of 20:80 showed the highest effectiveness, with protein content, moisture content, encapsulation efficiency, and yield at 26.10 ± 0.45%, 7.93 ± 0.42%, 95.84 ± 0.71%, and 14.27%, respectively.

Process optimization and characterization of physicochemical properties of freeze-dried pineapple snacks enriched with propolis

ABSTRACT The propolis (PP) and linseed oil (LO) were encapsulated into the gum Arabic (GA) matrix along with whey protein concentrate (WPC) using the freeze-drying technique to form a hydrophilic matrix for production of functional pineapple snacks. The concentrations of the components in the matrix were optimized using response surface methodology (RSM) to maximize the protein content and encapsulation efficiency (EE). The encapsulation efficiency, protein content, color parameters (L*, a*, b* values), microstructure and texture properties of the snack containing optimum component concentrations were investigated. The glass transition temperature for the snack prepared with optimized ingredient concentration and components, oxidation temperature for linseed oil were estimated using a differential scanning calorimeter (DSC). The results revealed an encapsulation efficiency of 83.15% and a protein ratio of 31.53% for the developed matrix. Gum Arabic did not increase the encapsulation efficiency. The use of WPC has increased the encapsulation efficiency and protein content. Linolenic acid (50.804%), oleic acid (22.443%), and linoleic acid (14.012%) were the major fatty acids in the snack, owing to the highly encapsulated linseed oil and propolis. These result data support the freeze-dried pineapple snacks, which may be optimized for their component ratios and then used as a functional product suitable for consumption owing to its high nutritional value.

Investigation and Comparison of Active and Passive Encapsulation Methods for Loading Proteins into Liposomes.

In this work, four different active encapsulation methods, microfluidic (MF), sonication (SC), freeze-thawing (FT), and electroporation (EP), were investigated to load a model protein (bovine serum albumin-BSA) into neutral liposomes made from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC):cholesterol (Chol) and charged liposomes made from DSPC:Chol:Dioleoyl-3-trimethylammonium propane (DOTAP), DSPC:Chol:1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and DSPC:Chol:phosphatidylethanolamine (PE). The aim was to increase the protein encapsulation efficiency (EE%) by keeping the liposome size below 200 nm and the PDI value below 0.7, which warrants a nearly monodisperse preparation. Electroporation (100 V) yielded the best results in terms of EE%, with a dramatic increase in liposome size (>600 nm). The FT active-loading method, either applied to neutral or charged liposomes, allowed for obtaining suitable EE%, keeping the liposome size range below 200 nm with a suitable PDI index. Cationic liposomes (DSPC:Chol:DOTAP) loaded with the FT active method showed the best results in terms of EE% (7.2 ± 0.8%) and size (131.2 ± 11.4 nm, 0.140 PDI). In vitro release of BSA from AM neutral and charged liposomes resulted slower compared to PM liposomes and was affected by incubation temperature (37 °C, 4 °C). The empty charged liposomes tested for cell viability on Human Normal Dermal Fibroblast (HNDF) confirmed their cytocompatibility also at high concentrations (1010 particles/mL) and cellular uptake at 4 °C and 37 °C. It can be concluded that even if both microfluidic passive and active methods are more easily transferable to an industrial scale, the FT active-loading method turned out to be the best in terms of BSA encapsulation efficiencies, keeping liposome size below 200 nm.

Influence of Rapeseed Oil on Extruded Plant-Based Meat Analogues: Assessing Mechanical and Rheological Properties

Successfully replacing meat with plant-based options will require not only replicating the texture of muscular fibres, but also imitating the taste, aroma, and juiciness of meat as closely and realistically as possible. This study examines the impact of rapeseed oil on the textural properties of meat analogues. Pea protein and soy protein are chosen as model systems to assess the effect of rapeseed oil. Optical, mechanical, and rheological characterisation tests are conducted to investigate the oil droplet distribution, the gel strength, Young’s modulus, and the length of the LVE region. The hypothesis is that oil droplets will act as active fillers in the protein matrix, and thus, diminish the strength of the protein gel network. The results of this study show that rapeseed oil droplets act as inactive fillers, as they are not bound to both examined protein matrices. Soy protein extrudates display minimal changes, while pea protein extrudates are significantly affected by the addition of oil. For example, oil addition decreased the G′ in the LVE region of pea protein meat analogues by 50%, while soy protein samples showed no significant changes. Despite the similar interfacial activities of the investigated proteins, the decreased encapsulation efficiency of pea protein was confirmed by the higher amount of extracted oil from the respective extrudates.

Template-Based Porous Hydrogel Microparticles as Carriers for Therapeutic Proteins.

Hydrogels have been extensively researched for over 60 years for their limitless applications in biomedical research. In this study, porous hydrogel microparticles (PHMPs) made of poly(ethylene glycol) diacrylamide were investigated for their potential as a delivery platform for therapeutic proteins. These particles are made using hard calcium carbonate (CaCO3) templates, which can easily be dissolved under acidic conditions. After optimization of the synthesis processes, both CaCO3 templates and PHMPs were characterized using a wide range of techniques. Then, using an array of proteins with different physicochemical properties, the encapsulation efficiency of proteins in PHMPs was evaluated under different conditions. Strategies to enhance protein encapsulation via modulation of particle surface charge to increase electrostatic interactions and conjugation using EDC/NHS chemistry were also investigated. Conjugation of bovine serum albumin to PHMPs showed increased encapsulation and diminished release over time, highlighting the potential of PHMPs as a versatile delivery platform for therapeutic proteins such as enzymes or antibodies.

Challenges in estimating the encapsulation efficiency of proteins in polymersomes - Which is the best method?

Challenges in estimating the encapsulation efficiency of proteins in polymersomes - Which is the best method?

Liposome Formulation and In Vitro Testing in Non-Physiological Conditions Addressed to Ex Vivo Kidney Perfusion.

This work focuses on formulating liposomes to be used in isolated kidney dynamic machine perfusion in hypothermic conditions as drug delivery systems to improve preservation of transplantable organs. The need mainly arises from use of kidneys from marginal donors for transplantation that are more exposed to ischemic/reperfusion injury compared to those from standard donors. Two liposome preparation techniques, thin film hydration and microfluidic techniques, are explored for formulating liposomes loaded with two model proteins, myoglobin and bovine serum albumin. The protein-loaded liposomes are characterized for their size by DLS and morphology by TEM. Protein releases from the liposomes are tested in PERF-GEN perfusion fluid, 4 °C, and compared to the in vitro protein release in PBS, 37 °C. Fluorescent liposome uptake is analyzed by fluorescent microscope in vitro on epithelial tubular renal cell cultures and ex vivo on isolated pig kidney in hypothermic perfusion conditions. The results show that microfluidics are a superior technique for obtaining reproducible spherical liposomes with suitable size below 200 nm. Protein encapsulation efficiency is affected by its molecular weight and isoelectric point. Lowering incubation temperature slows down the proteins release; the perfusion fluid significantly affects the release of proteins sensitive to ionic media (such as BSA). Liposomes are taken up by epithelial tubular renal cells in two hours’ incubation time.

Optimizing zinc-HisTag coordination remote loading of proteins in PLGA microspheres

Metal-HisTag coordination remote loading (MHCRL) of proteins in PLGA microspheres was previously developed to provide a useful tool for discovery and preclinical development of controlled release protein formulations. Here we describe optimization of MHCRL, including (1) reducing thermal stress, (2) decreasing the complexity and duration of the procedure, (3) increasing loading capacity, (4) increasing the penetration depth of protein, and (5) improving the release profile. Directly encapsulating ZnCO3as a Zn2+source for HisTag coordination, rather than remotely loading Zn2+, increased the Zn content ∼6-fold. Microspheres with directly encapsulated ZnCO3more deeply encapsulated green fluorescent protein and more efficiently encapsulated human serum albumin at protein loading solutions concentrations ≥100 μg/mL than remotely loaded Zn2+microspheres. Tributyl acetylcitrate plasticized microspheres in terms of decreasingTg, but led to a decrease in protein encapsulation efficiency. As such, the plasticizer was not deemed useful. The loading/healing cycles were reduced in time and temperature from 48 h/42 h at 43 °C to 2 h/6h at 37 °C while maintaining strong encapsulation efficiency, resulting in significantly improved protein stability. Immunoreactive protein was slowly released for months following a modest burst release. The improved microspheres and shorter, low-temperature encapsulation could be a valuable asset to drug discovery scientists interested in controlled release of delicate and/or costly biologic candidates.

Synthesis and characterizations of sugar-glass nanoparticles mediated protein delivery system for tissue engineering application

The present work focuses on the synthesis and characterization of a sugar-glass nanoparticle (SGnP) based reservoir type protein delivery system pertinent to tissue engineering applications. The SGnP nanocarriers were prepared via inverse micelle of sodium bis(2-ethylhexyl) sulfosuccinate based on an anionic surfactant and subsequent flash-freezing technique. Initially, a total of five different grades of protein-free SGnPs have been prepared to examine the effects of systematic changes in starting concentrations of the aqueous phase, organic solvent, the molar ratio of water, and surfactant in controlling the size, shape, and uniformity of micelles. Evidently, the Fourier transform infrared (FTIR) and scanning electron microscope (SEM) results confirmed that the SGnP can be successfully prepared. Subsequently, SGnP based protein depot has been validated using bovine serum albumin (BSA), horseradish peroxidase (HRP) and growth and differentiation factor-5 (GDF-5). The particle size, morphology, protein encapsulation efficiency and in vitro release kinetics were assessed using SEM, FTIR, UV–visible spectroscopy and Bradford protein assays. Excellent encapsulation efficiency (93%–94%) and sustained release behaviour of BSA (∼22% protein release after 14 d) and GDF-5 proteins (∼29% protein release after 30 d) were exhibited by the optimal grades of SGnP constructs with an average particle size of 266 nm and 93 nm, respectively. Furthermore, FTIR, differential scanning calorimeter (DSC), polyacrylamide gel electrophoresis (PAGE) and NATIVE-PAGE studies results confirm successful encapsulation, stability and preserving the structural integrity of proteins placed into the core of the SGnP constructs. Evidently, a very high (93%) residual HRP enzyme activity signifies the capability of our SGnP system to protect the encapsulated proteins from process-related stresses. In vitro cytotoxicity and fluorescence cell morphology analyses using human adipose-derived mesenchymal stem cells affirmed good cytocompatibility of protein encapsulated SGnP. Overall, the study findings indicate SGnP nanocarrier-mediated protein delivery systems as a promising approach complementary to conventional techniques in tissue engineering and therapeutic applications.

Study in the stabilization of proteins encapsulated in PLGA delivery system: Effects of additives on protein encapsulation, release, and stability

Therapeutic proteins/peptides face severe stability impairment in biotechnology drug delivery systems prepared by the W/O/W solvent evaporation method. Screening of protein stabilizers for poly (D, l-lactide-co-glycolide) (PLGA) nanoparticles can improve the low encapsulation efficiency and undesirable release behavior of proteins caused by PLGA carriers, which is a crucial step to achieve a high proportion of continuous release of encapsulated protein in the target time and give full play to the therapeutic effect. Therefore, model proteins were selected for stability testing to simulate the specific destabilization pathways. Through the comprehensive study of protein conformation, recovery rate, encapsulation rate, and drug release behavior, the effects of additives on the stability of protein were synthetically characterized, and their action links and mechanisms were also further studied. The results showed that different denaturation environments were intertwined during the preparation and release of peptide-loaded NPs, jointly inducing protein denaturation and incomplete release behavior. After optimization with stabilizers, urea and lactose increased the encapsulation efficiency of proteins to more than 95% (P < 0.001). Except for the P188, all other stabilizers showed great potential for optimizing the release and stability of the encapsulated protein as a whole, increasing the release proportion of protein from 44% to 60 %-75% (P < 0.0001). The improvement of the drug release curve demonstrated the long-term stabilizing effect of the above-mentioned co-solubilizing additives on the therapeutic protein.

High throughput onion-like liposome formation with efficient protein encapsulation under flash antisolvent mixing

Achieving a high encapsulation efficiency and loading capacity of proteins in lecithin-based liposomes has always been a challenge. Here, we use Flash Nano-Precipitation (FNP) to produce liposomes and investigated the encapsulation of model protein (Bovine Serum Albumin, BSA). Through rapid turbulent mixing, we obtained liposomes with small size, low polydispersity, and good batch repeatability at a high production rate. We demonstrated that the bilayer of liposomes prepared solely using lecithin was defective, which led to the fusion, and increased size and polydispersity. When cholesterol was added to reach a lecithin-to-cholesterol molar ratio of 5:3, a compact bilayer formed to effectively inhibit liposome fusion. The encapsulation efficiency and loading capacity of BSA was as high as ∼ 68% and ∼ 6% in lecithin-cholesterol liposome, respectively, far exceeding the values reported in the literature. Further study by Quartz Crystal Microbalance with Dissipation (QCM-D) revealed that the highly effective encapsulation was due to the rapid mutual adsorption between BSA and defective/curved lecithin double layers during the liposome formation. Such rapid mutual adsorption leads to the layer-by-layer assembly and formation of onion-like compact liposome structure as revealed by Cryo-TEM. This simple FNP method provides a scalable manufacturing approach for liposomes with efficient protein encapsulation. The revealed adsorption mechanism between protein and lecithin bilayers could also serve as a guide for similar studies.

Enhancing Stability and Mucoadhesive Properties of Chitosan Nanoparticles by Surface Modification with Sodium Alginate and Polyethylene Glycol for Potential Oral Mucosa Vaccine Delivery.

Background: The present study aimed to fabricate surface-modified chitosan nanoparticles with two mucoadhesive polymers (sodium alginate and polyethylene glycol) to optimize their protein encapsulation efficiency, improve their mucoadhesion properties, and increase their stability in biological fluids. Method: Ionotropic gelation was employed to formulate chitosan nanoparticles and surface modification was performed at five different concentrations (0.05, 0.1, 0.2, 0.3, 0.4% w/v) of sodium alginate (ALG) and polyethylene glycol (PEG), with ovalbumin (OVA) used as a model protein antigen. The functional characteristics were examined by dynamic light scattering (DLS), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM)/scanning transmission electron microscopy (STEM). Stability was examined in the presence of simulated gastric and intestinal fluids, while mucoadhesive properties were evaluated by in vitro mucin binding and ex vivo adhesion on pig oral mucosa tissue. The impact of the formulation and dissolution process on the OVA structure was investigated by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism (CD). Results: The nanoparticles showed a uniform spherical morphology with a maximum protein encapsulation efficiency of 81%, size after OVA loading of between 200 and 400 nm and zeta potential from 10 to 29 mV. An in vitro drug release study suggested successful nanoparticle surface modification by ALG and PEG, showing gastric fluid stability (4 h) and a 96 h sustained OVA release in intestinal fluid, with the nanoparticles maintaining their conformational stability (SDS-PAGE and CD analyses) after release in the intestinal fluid. An in vitro mucin binding study indicated a significant increase in mucin binding from 41 to 63% in ALG-modified nanoparticles and a 27–49% increase in PEG-modified nanoparticles. The ex vivo mucoadhesion showed that the powdered particles adhered to the pig oral mucosa. Conclusion: The ALG and PEG surface modification of chitosan nanoparticles improved the particle stability in both simulated gastric and intestinal fluids and improved the mucoadhesive properties, therefore constituting a potential nanocarrier platform for mucosal protein vaccine delivery.

In‐Situ Encapsulation of Protein into Nanoscale Hydrogen‐Bonded Organic Frameworks for Intracellular Biocatalysis

AbstractHydrogen‐bonded organic frameworks (HOFs) are porous materials with great potential for biological applications. The self‐assembly of HOFs and biomacromolecules, however, is challenging. We report herein the self‐assembly of nanoscale HOFs (nHOFs) to encapsulate protein for intracellular biocatalysis. The self‐assembly of tetrakis(4‐amidiniumphenyl)methane and azobenzenedicarboxylate can encapsulate protein in situ to form protein@nHOFs under mild conditions. This strategy is applicable to proteins with different surface charge and molecular weight, showing a high protein encapsulation efficiency and minimal effect on protein activity. A cellular delivery study shows that the protein@TA‐HOFs can efficiently enter cells and retain enzyme activity for biochemical catalysis in living cells for neuroprotection. Our strategy paves new avenues for interfacing nHOFs with biological settings and sheds light on expanding nHOFs as a platform for biomacromolecule delivery and disease treatment.

CD44-Targeted and Enzyme-Responsive Photo-Cross-Linked Nanogels with Enhanced Stability for In Vivo Protein Delivery.

One of the biggest challenges of the protein delivery system is to realize stable and high protein encapsulation efficiency in blood circulation and rapid release of protein in the targeted tumor cells. To overcome these hurdles, we fabricated enzyme-responsive photo-cross-linked nanogels (EPNGs) through UV-triggered chemical cross-linking of cinnamyloxy groups in the side chain of PEGylation hyaluronic acid (HA) for CD44-targeted transport of cytochrome c (CC). The EPNGs showed high loading efficiency and excellent stability in different biological media. Notably, CC leakage effectively suppressed under physiological conditions but accelerated release in the presence of hyaluronidase, an overexpressed enzyme in tumor cells. Moreover, thiazolylblue tetrazolium bromide (MTT) results indicated that the vacant EPNGs showed excellent nontoxicity, while CC-loaded EPNGs exhibited higher killing efficiency to CD44-positive A549 cells than to CD44-negative HepG2 cells and free CC. Confocal images confirmed that CC-loaded EPNGs could effectively be internalized by CD44-mediated endocytosis pathway and rapidly escape from the endo/lysosomal compartment. Human lung tumor-bearing mice imaging assays further revealed that CC-loaded EPNGs actively target tumor locations. Remarkably, CC-loaded EPNGs also exhibited enhanced antitumor activity with negligible systemic toxicity. These results implied that these EPNGs have appeared as stable and promising nanocarriers for tumor-targeting protein delivery.

Protein-induced metamorphosis of unilamellar lipid vesicles to multilamellar hybrid vesicles

Protein encapsulation into nanocarriers has been extensively studied to improve the efficacy and stability of therapeutic proteins. However, the chemical modification of proteins or new synthetic carrier materials are essential to achieve a high encapsulation efficiency and structural stability of proteins, which hinders their clinical applications. New strategies to physically incorporate proteins into nanocarriers feasible for clinical uses are required to overcome the current limitation. Here we report the spontaneous protein-induced reorganization of ‘pre-formed’ unilamellar lipid vesicles to efficiently incorporate proteins within multilamellar protein-lipid hybrid vesicles without chemical modification. Epidermal growth factor (EGF) binds to the surface of cationic unilamellar lipid vesicles and induces layer-by-layer self-assembly of the vesicles. The protein is spontaneously entrapped in the interstitial layers of a multilamellar structure with extremely high loading efficiency, ~99%, through polyionic interactions as predicted by molecular dynamics simulation. The loaded protein exhibits much higher structural, chemical, and biological stability compared to free protein. The method is also successfully applied to several other proteins. This work provides a promising method for the highly efficient encapsulation of therapeutic proteins into multilamellar lipid vesicles without the use of specialized instruments, high energy, coupling agents, or organic solvents.

Development and Application of a Modified Method to Determine the Encapsulation Efficiency of Proteins in Polymer Matrices

A modified method to determine protein encapsulation efficiency in polymer matrices has been developed and applied to two proteins and two polymers to demonstrate its wide range of applicability. This study was pursued due to the wide variation in reported protein encapsulation efficiency of polymer-based microcapsules, even when the protein, the polymer, and the microcapsule manufacturing method were consistent. Hemoglobin (Hb) and bovine serum albumin (BSA) were chosen as model proteins and ethylcellulose and poly(lactic-co-glycolic acid) (PLGA) as model polymers. The polymer of the microcapsule was dissolved in dichloromethane/ethanol or dichloromethane/ethyl acetate for ethylcellulose or PLGA microcapsules, respectively. Liberated proteins were simultaneously precipitated, pelleted by centrifugation, isolated by decanting the polymer solution, redissolved in 10% w/v sodium dodecyl sulfate in 0.8N sodium hydroxide, and quantified using a modified Lowry assay. Blank microcapsules and exogenously added proteins demonstrated ≥ 93.8% recovery of proteins. The mean encapsulation efficiency of ethylcellulose or PLGA microcapsules was 52.4 or 76.9% for Hb and 86.4 or 74.7% for BSA, respectively. This demonstrates the effective use of centrifugation and the importance of an appropriate cosolvent system in the measure of encapsulation efficiency where one solvent dissolves the polymer while the other solvent quantitatively precipitates the liberated protein. It is evident that an alkaline solution of sodium dodecyl sulfate is efficient at quantitatively dissolving precipitated proteins. Remediation of problems observed with current methods and high reproducibility suggest that this modified method is generally applicable to the measure of protein encapsulation efficiency of polymer microcapsules.

A novel formulation of Mtb72F DNA vaccine for immunization against tuberculosis.

Objective(s): Mycobacterium tuberculosis (M. tuberculosis), an intracellular pathogen, causes 1.5 million deaths globally. Bacilli Calmette-Guérin (BCG) is commonly administered to protect people against M. tuberculosis infection; however, there are some obstacles with this first-generation vaccine. DNA vaccines, the third generation vaccines, can induce cellular immune responses for tuberculosis (TB) protection. In this study, optimized DNA vaccine (pcDNA3.1-Mtb72F) entrapped in poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) was used to achieve higher immunogenicity. Materials and Methods:Plasmid Mtb72F was formulated in PLGA NPs using double emulsion method in the presence of TB10.4 and/or CpG as an adjuvant. Female BALB/c mice were immunized either with NP-encapsulated Mtb72F or naked Mtb72F with or without each adjuvant, using the BCG-prime DNA boost regimen. Results:These NPs were approximately 250 nm in diameter and the nucleic acid and protein encapsulation efficiency were 80% and 25%, respectively. The NPs smaller than 200 nm are able to promote cellular rather than humoral responses. The immunization with the formulation consisting of Mtb72F DNA vaccine and TB10.4 entrapped in PLGA NPs showed significant immunogenicity and induced predominantly interferon-ɣ (IFN-ɣ) production and higher INF-ɣ/interleukin-4 (IL-4) ratio in the cultured spleen cells supernatant. Conclusion:PLGA NPs loaded with Mtb72F DNA-based vaccine with TB10.4 could be considered as a promising candidate for vaccination against TB. These results represent an excellent initial step toward development of novel vaccine for TB protection.

Modulation of Entrapment Efficiency and In Vitro Release Properties of BSA-Loaded Chitosan Microparticles Cross-Linked with Citric Acid as a Potential Protein-Drug Delivery System.

Microparticles, aimed for oral protein and peptide drug delivery, were prepared via emulsion cross-linking using citric acid as cross-linker and polyglycerol polyricinoleate as surfactant. A comparative study of the interaction between chitosan and citric acid and its effect on the resulting microparticle properties was performed using different chitosan-to-cross-linker mass ratios and pH-values during fabrication of the microparticles. Non-cross-linked and cross-linked microparticles were studied in terms of size (4–12 μm), zeta potential (−15.7 to 12.8 mV), erosion (39.7–75.6%), a model protein encapsulation efficiency (bovine serum albumin) (6.8–27.6%), and loading capacity (10.4–40%). Fourier transform infrared spectroscopy and X-ray diffraction confirmed the ionic interaction between the protonated amine groups of chitosan and the carboxylate ions of the cross-linking agent. Scanning electron microscopy revealed that the non-cross-linked microparticles had an uneven shape with wrinkled surfaces, while the cross-linked formulations were spherical in shape with smooth surfaces. On the basis of these data, the role of the surfactant and microparticle structure on the release mechanism was proposed. Control of the microparticle shape and release mechanisms is expected to be crucial in developing carriers for the controlled delivery of proteins and peptides.

Lipid and PLGA Microparticles for Sustained Delivery of Protein and Peptide Drugs.

Solid lipid particles have a great potential in sustained drug delivery, the lipid excipients are solid at room temperature with a slow degradation rate. Poly (D, L-lactic-coglycolic acid) (PLGA) has been successfully clinically applied for the sustained delivery of peptide drugs. A recent study showed the advantage of hybrid PLGA-lipid microparticles (MPs) over PLGA MPs for the sustained delivery of peptide drug in vivo. In this paper, we briefly present PLGA MPs, solid lipid MPs and PLGA lipid hybrid MP prepared by the double emulsion method and the spray drying method and discuss the effects of excipients on encapsulation efficiency of protein and peptide drugs in the MPs. The pros and cons of PLGA MPs, solid lipid MPs and PLGA lipid hybrid MP as carriers for sustained delivery of protein and peptide drugs are also discussed.