Protein Release Kinetics Research Articles

Identification and Characterization of Critical Processing Parameters in the Fabrication of Double-Emulsion Poly(lactic-co-glycolic) Acid Microparticles.

In the past several decades, polymeric microparticles (MPs) have emerged as viable solutions to address the limitations of standard pharmaceuticals and their corresponding delivery methods. While there are many preclinical studies that utilize polymeric MPs as a delivery vehicle, there are limited FDA-approved products. One potential barrier to the clinical translation of these technologies is a lack of understanding with regard to the manufacturing process, hindering batch scale-up. To address this knowledge gap, we sought to first identify critical processing parameters in the manufacturing process of blank (no therapeutic drug) and protein-loaded double-emulsion poly(lactic-co-glycolic) acid MPs through a quality by design approach. We then utilized the design of experiments as a tool to systematically investigate the impact of these parameters on critical quality attributes (e.g., size, surface morphology, release kinetics, inner occlusion size, etc.) of blank and protein-loaded MPs. Our results elucidate that some of the most significant CPPs impacting many CQAs of double-emulsion MPs are those within the primary or single-emulsion process (e.g., inner aqueous phase volume, solvent volume, etc.) and their interactions. Furthermore, our results indicate that microparticle internal structure (e.g., inner occlusion size, interconnectivity, etc.) can heavily influence protein release kinetics from double-emulsion MPs, suggesting it is a crucial CQA to understand. Altogether, this study identifies several important considerations in the manufacturing and characterization of double-emulsion MPs, potentially enhancing their translation.

Enhancing protein delivery for tissue regeneration: Development of AGR2-loaded hydrogels with controlled release properties

The growth factor Anterior Gradient 2 (AGR2) has been shown to have an effective role in tissue regeneration, but remained largely unexplored in localized tissue engineering applications. Alginate beads have been proven as safe carriers for protein encapsulation, but they suffer from fragility and uncontrolled protein release. For such alginate systems, little is known about how changes in concentrations and ion-crosslinking affect protein release and accumulation in 3-D matrices. To address these questions, an engineered interpenetrating polymer network (IPN) has been used to synthesize a novel hybrid system consisting of AGR2 loaded beads composed of calcium-crosslinked sodium alginate (SA) and carboxymethyl cellulose (CMC). These beads are embedded in films consisting of SA and polyvinyl alcohol (PVA), using a simple ion gelation technique. We assess protein release kinetics and accumulation within the hybrid system by varying polymer concentrations and cross-linking parameters. The IPN hybrid system maintains controlled release over two weeks, without an initial burst period. Through this approach efficicnt delivery of AGR2 is achieved which in turn effectively mediates cell migration and proliferation, resulting in excellent cell viability and complete wound closure. The described release system opens new perspectives in tissue engineering.

Injectable phase-separated tetra-armed poly(ethylene glycol) hydrogel scaffold allows sustained release of growth factors to enhance the repair of critical bone defects

With the rising prevalence of bone-related injuries, it is crucial to improve treatments for fractures and defects. Tissue engineering offers a promising solution in the form of injectable hydrogel scaffolds that can sustain the release of growth factors like bone morphogenetic protein-2 (BMP-2) for bone repair. Recently, we discovered that tetra-PEG hydrogels (Tetra gels) undergo gel-gel phase separation (GGPS) at low polymer content, resulting in hydrophobicity and tissue affinity. In this work, we examined the potential of a newer class of gel, the oligo-tetra-PEG gel (Oligo gel), as a growth factor-releasing scaffold. We investigated the extent of GGPS occurring in the two gels and assessed their ability to sustain BMP-2 release and osteogenic potential in a mouse calvarial defect model. The Oligo gel underwent a greater degree of GGPS than the Tetra gel, exhibiting higher turbidity, hydrophobicity, and pore formation. The Oligo gel demonstrated sustained protein or growth factor release over a 21-day period from protein release kinetics and osteogenic cell differentiation studies. Finally, BMP-2-loaded Oligo gels achieved complete regeneration of critical-sized calvarial defects within 28 days, significantly outperforming Tetra gels. The easy formulation, injectability, and capacity for sustained release makes the Oligo gel a promising candidate therapeutic biomaterial.

Thermosensitive Biodegradable Hydrogels for Local and Controlled Cerebral Delivery of Proteins: MRI-Based Monitoring of In Vitro and In Vivo Protein Release.

Hydrogels have been suggested as novel drug delivery systems for sustained release of therapeutic proteins in various neurological disorders. The main advantage these systems offer is the controlled, prolonged exposure to a therapeutically effective dose of the released drug after a single intracerebral injection. Characterization of controlled release of therapeutics from a hydrogel is generally performed in vitro, as current methods do not allow for in vivo measurements of spatiotemporal distribution and release kinetics of a loaded protein. Importantly, the in vivo environment introduces many additional variables and factors that cannot be effectively simulated under in vitro conditions. To address this, in the present contribution, we developed a noninvasive in vivo magnetic resonance imaging (MRI) method to monitor local protein release from two injected hydrogels of the same chemical composition but different initial water contents. We designed a biodegradable hydrogel formulation composed of low and high concentration thermosensitive polymer and thiolated hyaluronic acid, which is liquid at room temperature and forms a gel due to a combination of physical and chemical cross-linking upon injection at 37 °C. The in vivo protein release kinetics from these gels were assessed by MRI analysis utilizing a model protein labeled with an MR contrast agent, i.e. gadolinium-labeled albumin (74 kDa). As proof of principle, the release kinetics of the hydrogels were first measured with MRI in vitro. Subsequently, the protein loaded hydrogels were administered in male Wistar rat brains and the release in vivo was monitored for 21 days. In vitro, the thermosensitive hydrogels with an initial water content of 81 and 66% released 64 ± 3% and 43 ± 3% of the protein loading, respectively, during the first 6 days at 37 °C. These differences were even more profound in vivo, where the thermosensitive hydrogels released 83 ± 16% and 57 ± 15% of the protein load, respectively, 1 week postinjection. Measurement of volume changes of the gels over time showed that the thermosensitive gel with the higher polymer concentration increased more than 4-fold in size in vivo after 3 weeks, which was substantially different from the in vitro behavior where a volume change of 35% was observed. Our study demonstrates the potential of MRI to noninvasively monitor in vivo intracerebral protein release from a locally administered in situ forming hydrogel, which could aid in the development and optimization of such drug delivery systems for brain disorders.

Encapsulation of Milk Protein with Inulin for Improved Digestibility

Encapsulation is the packaging of bioactive compounds to isolate and control their release upon applying specific conditions. By using inulin as the wall material for milk protein encapsulation, it can improve the stability and reduce the rate of protein release which can be related to better digestibility. This study aims to find the best encapsulation parameters for milk protein encapsulation using inulin based on the encapsulation efficiency and evaluate the protein release by simulation of intestinal fluid in vitro. Protein encapsulation was prepared under various parameters including inulin concentration, stirring temperature and stirring speed. The encapsulation parameters were analysed by Response Surface Methodology to obtain the highest encapsulation efficiency, which was subsequently used for the analysis of particle size, zeta potential and protein release kinetics. The results showed that inulin concentration was the most significant in determining encapsulation efficiency, and the highest encapsulation efficiency (75.90%) was achieved at 0.47 % w/v of inulin concentration, stirring temperature of 35.17°C and stirring speed of 510.79 rpm. The inulin-encapsulated milk protein produced under the best encapsulation parameters had an average particle size value of 485.8 nm and zeta potential of -10.5 mV. Zero order kinetics model was most suited to describe the encapsulated milk protein release, indicating slow release of protein that may be associated with better digestibility.

Long acting injectables for therapeutic proteins

Biotherapeutic development presents a myriad of challenges in relation to delivery, in particular for protein therapeutics. Protein delivery is complicated due to hydrophilicity, size, rate of degradation in vivo, low permeation through biological barriers, pH and temperature sensitivity, as well as the need to conserve its quaternary structure to retain function. To preserve therapeutic levels in vivo, proteins require frequent administration due to their short half-lives. Formulation strategies combining proteins with lipid carriers for parenteral administration show potential for improving bioavailability, while preserving protein activity and bypassing the mucosal barriers of the body. Encapsulating protein in long acting injectable delivery systems can improve therapeutic indices by prolonging and controlling protein release and reducing the need for repeat interventions. Two lyotropic crystal forming lipids, monoolein and phytantriol, have been formulated to produce lipidic cubic phases and assessed for their use as long acting protein eluting injectables. Three soluble proteins, cytochrome c, glyceraldehyde-3-phosphate dehydrogenase and aldehyde dehydrogenase and one membrane protein, cytochrome c oxidase, were incorporated into bulk cubic phase formulations of each lipid system to comparatively assess protein release kinetics. The activity of the soluble proteins was measured upon release from a phytantriol bulk cubic phase and phytantriol cubosomes, produced using a liquid precursor method.

Fabrication Parameter-Dependent Physico-Chemical Properties of Thiolated Gelatin/PEGDA Interpenetrating Network Hydrogels.

The development of three-dimensional hydrogels using polymeric biomaterials is a key technology for tissue engineering and regenerative medicine. Successful tissue engineering requires the control and identification of the physicochemical properties of hydrogels. Interpenetrating network (IPN) hydrogel was developed using thiolated gelatin (GSH) and poly(ethylene glycol) diacrylate (PEGDA), with the aid of ammonium persulfate (APS) and N,N,N,N'-tetramethylethylenediamine (TEMED) as radical initiators. Each component was prepared in the following concentrations, respectively: 2.5 and 5% GSH (LG and HG), 12.5 and 25% PEGDA (LP and HP), 3% APS/1.5% TEMED (LI), and 4% APS/2% TEMED (HI). IPN hydrogel was fabricated by the mixing of GSH, PEGDA, and initiators in 5:4:1 volume ratios, and incubated at 37°C for 30min in the following 6 experimental formulations: (1) HG-LP-LI, (2) HG-LP-HI, (3) LG-HP-LI, (4) LG-HP-HI, (5) HG-HP-HI, and (6) HG-HP-LI. Herein, the physico-chemical characteristics of IPN hydrogels, including their morphological structures, hydrolytic degradation properties, mechanical properties, embedded protein release kinetics, and biocompatibility, were investigated. The characteristics of the hydrogel were significantly manipulated by the concentration of the polymer, especially the conversion between HP and LP, rather than the concentration of the initiator, and no hydrogel formulation exhibited any toxicity to fibroblast and HaCaTcells. We provide structural-physical relationships of the hydrogels by which means their physical properties could be conveniently controlled through component control, which could be versatilely utilized for various organizational engineering strategies.

High Loading Efficiency and Controlled Release of Bioactive Immunotherapeutic Proteins Using Vaterite Nanoparticles (Part. Part. Syst. Charact. 7/2021)

In article 2100012, Dähne and co-workers provide a framework for the development of a vaterite-based drug delivery system as a carrier for bioactive protein-based cancer therapeutics. In addition, a system to mimic (pH) conditions under body-like-flow rates is developed to predict protein release kinetics accurately.

Proteomic clustering reveals the kinetics of disease biomarkers in bovine and human models of post-traumatic osteoarthritis

ObjectivesIn this study, we apply a clustering method to proteomic data sets from bovine and human models of post-traumatic osteoarthritis (PTOA) to distinguish clusters of proteins based on their kinetics of release from cartilage and examined these groups for PTOA biomarker candidates. We then quantified the effects of dexamethasone (Dex) on the kinetics of release of the cartilage media proteome. DesignMass spectrometry was performed on sample medium collected from two separate experiments using juvenile bovine and human cartilage explants (3 samples/treatment condition) during 20- or 21-day treatment with inflammatory cytokines (TNF-α, IL-6, sIL-6R) with or without a single compressive mechanical injury. All samples were incubated with or without 100 ​nM Dex. Clustering was performed on the correlation between normalized averaged release vectors for each protein. ResultsOur proteomic method identified the presence of distinct clusters of proteins based on the kinetics of their release over three weeks of culture. Clusters of proteins with peak release after one to two weeks had biomarker candidates with increased release compared to control. Dex rescued some of the changes in protein release kinetics the level of control, and in all conditions except control, there was late release of immune-related proteins. ConclusionsWe demonstrate a clustering method applied to proteomic data sets to identify and validate biomarkers of early PTOA progression and explore the relationships between the release of spatially related matrix components. Dex restored the kinetics of release to many matrix components, but not all factors that contribute to cartilage homeostasis.

Property modelling of lysozyme‐crosslinker‐alginate complexes using latent variable methods

Statistical methods were used to provide insight into a polymer complex system composed of lysozyme and alginate to quantify the effects of such parameters as pH, and ionic composition of the mixing solution on the properties of the complexes including composition, particle diameter, and zeta potential. Various crosslinkers (calcium, barium, iron[III], and bovine serum albumin), were used with lysozyme for complex formation to investigate the effect of crosslinker charge density on protein release kinetics, modelled using ktn . Multivariate statistical analysis showed that the kinetic parameters associated with the release were, not surprisingly highly dependent on the ionic strength of the release media, with higher ionic strength leading to faster release. The release parameter k was also shown to depend on the protein properties (size, ionic strength) while n was slightly, but not statistically dependent on the charge density of the crosslinker demonstrating that the nature of the crosslinker had minimal impact on drug release. The multivariate statistical has the potential to be used for optimization of the complexes and prediction of physical properties and degradation rates.

Preparation of Immobilized Metal Affinity Nanofiber for Globulin Depletion

In this study, p-tert-butyl-calix[4]arene derivatives selectively were synthesized. Then, new nanofibers were obtained from this p-tert-butyl-Calix [4]arene amid derivatives by the electrospinning method. In this study, an IMA nanofiber system via iminodiacetic acid as ligand and metals as Cu2+ for protein purification was developed. Copper (II) ions were chelated with IDA on nanofiber surfaces to prepare an immobilized metal affinity nanofiber (IMAN) for globulin depletion by the study of protein loading and release kinetics in various pH values. The loading and release of the globulin were monitored via fluorescence spectroscopy. The maximum amount of loaded protein onto 2.25 cm2 of the CAL-IDA (CIDA) nanofiber surface was found to be 0.092 μg in 6.0 of pH for 60 min. Any release from protein-loaded nanofibers did not perform in any pH values. Loading of the protein to nanofibers was also clarified by SEM, TEM, EDX, AFM, and FT-IR analysis.

Protein encapsulation in functionalized sol-gel silica: Effect of the encapsulation method on the release kinetics and the activity

Over the last few years, bone repair has increasingly gained in importance. In recent years, considerable attention has been given to the administration of therapeutic biomolecules to promote tissue regeneration. The aim of this work is the study of the influence of the encapsulation method on the release kinetics of a model protein (i.e. Soybean Trypsin Inhibitor, STI) and on the preservation of its inhibitory activity. For this purpose, two alternative methods have been investigated: i) impregnation of presynthesized silica gels with the protein solution (i.e. impregnation method), ii) direct incorporation of the protein during the synthesis of the gel (i.e. in situ method). Regarding the impregnation method, a fast release of STI was observed when incubated in physiological conditions (i.e. burst followed by a plateau for the calcined samples or by a sustained release for the dried sample) while the in situ method allowed a better control of the release rate of this protein over the first 24 h. This difference in release kinetics could be explained in terms of the expected geometry of the pores (open versus closed porosity). Interestingly, no significant change in the activity of the protein was noticed for the silica synthesized via the in situ method, while a partial or total loss of inhibitory activity was measured after four weeks of incubation for the impregnated silica.

Construction of 3D fibrous PCL scaffolds by coaxial electrospinning for protein delivery.

In this study, a three-dimensional tablet-like porous scaffold, comprising core-shell fibers to host proteins inside the core, was developed. The fabrication method involved the novel combination of coaxial and wet electrospinning in a single setting. Poly (ε-caprolactone) was chosen as the based polymer and bovine serum albumin was used as a model protein. These 3D tablet-like scaffolds exhibited adequate porosity and suitable pore size for cell culture and cell infiltration, in addition to appropriate mechanical properties for cartilage tissue engineering. The effects of different parameters on the behavior of the system have been studied and the 3D scaffold based on the core-shell fiber was compared with that based on the matrix fiber. The core-shell structure showed superior performance in comparison to the matrix structure by sustaining protein release kinetics at least for 12days in PBS. The results from in vitro cell cytotoxicity study revealed that the presented scaffold was biocompatible and non-toxic. Coaxial electrospinning was shown to be a versatile technique in achieving the delivery of biochemical signals in a controlled manner for the regeneration of cartilage. These 3D tablet-like PCL scaffolds incorporated with protein solutions are engineered systems that closely mimic the characteristics of cartilage tissue.

Peptide gels for controlled release of proteins.

Treatment strategies in clinics have been shifting from small molecules to protein drugs due to the promising results of a highly specific mechanism of action and reduced toxicity. Despite their prominent roles in disease treatment, delivery of the protein therapeutics is challenging due to chemical instability, immunogenicity and biological barriers. Peptide hydrogels with spatiotemporally tunable properties have shown an outstanding potential to deliver complex protein therapeutics, maintain drug efficacy and stability over time, mimicking the extracellular matrix, and responding to external stimuli. In this review, we present recent advances in peptide hydrogel design strategies, protein release kinetics and mechanisms for protein drug delivery in cellular engineering, tissue engineering, immunotherapy and disease treatments.

Nanostructured degradable macroporous hydrogel scaffolds with controllable internal morphologies via reactive electrospinning

Creating micro/nanostructured hydrogels with tunable morphologies under cell-friendly processing conditions would enable rational engineering of hydrogel scaffolds for targeted biomedical applications. Herein, an all-aqueous single-step reactive electrospinning method is applied to prepare hydrogel networks with controlled morphologies on both the nanoscale and the microscale. Hydrazide and aldehyde-functionalized poly(oligo ethylene glycol methacrylate) (POEGMA) are co-spun from a double barrel syringe together with poly(ethylene oxide) (PEO) as an electrospinning aid. By varying the concentrations and molecular weights of PEO and/or POEGMA, various morphologies from pure fibers to beaded fibers to bead network morphologies with tunable bead sizes can be fabricated, all of which remain monolithically stable in water due to the dynamic covalent crosslinks formed within the gel structure. The rates and magnitudes of swelling, degradation, and mechanics of the resulting scaffolds can be tuned by independently controlling gel morphologies on the nanoscale (i.e. crosslink density within the gel) and the microscale (i.e. the network structure formed), with an atypical independence of swelling relative to the mechanics and degradation rate observed. Furthermore, the internal morphology of the networks is demonstrated to systematically alter both the cell responses within the scaffolds and the rate of protein release from the scaffolds, with small fibers showing optimal cell proliferation, bead networks exhibiting the slowest protein release kinetics and very high maintained cell viabilities post-electrospinning, and beaded fibers showing intermediate properties. Statement of SignificanceControlling the internal structure of hydrogels is critical to successfully applying hydrogels in biomedical applications such as tissue engineering or cell/drug delivery. However, current techniques to fabricate hydrogel scaffolds typically require additives or gelation processes that are poorly compatible with cells and/or require multi-step processes. In this paper, we describe the fabrication of hydrogel scaffolds with tunable feature sizes (from nanometer to micrometer scale) and structures (from all fibers to bead/fiber mixtures to a new “bead network” morphology) using a reactive electrospinning strategy leveraging dynamic hydrazone crosslinking. We show single-step cell/protein loading and systematic control over cell proliferation and protein release kinetics by systematically manipulating the scaffold morphologies and feature sizes, allowing facile customization of scaffold properties for targeted applications.

High-Throughput Synthesis, Analysis, and Optimization of Injectable Hydrogels for Protein Delivery.

The development of in situ-gelling hydrogels that can enable prolonged protein release is increasingly important due to the emergence of a growing number of protein-based therapeutics. Herein, we describe a high-throughput strategy to fabricate, characterize, and subsequently optimize hydrazone-cross-linked in situ-gelling hydrogels for protein delivery. Hydrogels are fabricated using an automated high-throughput robot to mix a variety of thermoresponsive, nonthermoresponsive, charged, neutral, naturally sourced, and synthetic polymers functionalized with hydrazide or aldehyde groups, generating in situ-gelling hydrogels with well-defined compositions within a 96-well plate. High-throughput characterization strategies are subsequently developed to enable on-plate analysis of hydrogel swelling, mechanics, degradation, transparency, and protein (ovalbumin) release kinetics that yield results consistent with those collected using traditional bulk hydrogel analysis techniques. Dynamic regression and latent variable modeling are then applied to fit performance statistics to the collected data set; subsequently, numerical optimization is used to identify mixtures of precursor polymers that exhibit targeted combinations of minimal burst release, maximum total protein release, minimum release rate, and maximum transparency (the latter of particular relevance for ophthalmic protein delivery applications). Given the rapid throughput of the protocols developed (i.e., 126 hydrogels can be synthesized and screened in quadruplicate within hours), this approach offers particular promise for accelerating the identification of injectable hydrogel compositions relevant for both protein delivery as well as other biomedical applications for which clearly predefined materials properties are required.

Electrostatically Driven Guanidinium Interaction Domains that Control Hydrogel-Mediated Protein Delivery In Vivo.

Protein biologics are an important class of drugs, but the necessity for frequent parenteral administration is a major limitation. Drug-delivery materials offer a potential solution, but protein-material adsorption can cause denaturation, which reduces their effectiveness. Here, we describe a new protein delivery platform that limits direct contact between globular protein domains and material matrix, yet from a single subcutaneous administration can be tuned for long-term drug release. The strategy utilizes complementary electrostatic interactions made between a suite of designed interaction domains (IDs), installed onto the terminus of a protein of interest, and a negatively charged self-assembled fibrillar hydrogel. These intermolecular interactions can be easily modulated by choice of ID to control material interaction and desorption energies, which allows regulation of protein release kinetics to fit desired release profiles. Molecular dynamics studies provided a molecular-level understanding of the mechanisms that govern release and identified optimal binding zones on the gel fibrils that facilitate strong ID–material interactions, which are crucial for sustained release of protein. This delivery platform can be easily loaded with cargo, is shear-thin syringe implantable, provides improved protein stability, is capable of a diverse range of in vitro release rates, and most importantly, can accomplish long-term control over in vivo protein delivery.

Bioactivity of catalase loaded into vaterite CaCO3 crystals via adsorption and co-synthesis

Protein therapy gained a reputation of the most direct and safe approach for treating various diseases, yet biodegradation and loss of bioactivity of fragile therapeutic proteins limit their wide medical use. Recently, a new hard templating technology using decomposable mesoporous vaterite CaCO3 crystals became extremely popular strategy for formulation of protein nano(micro)-vectors. This study deciphers how protein bioactivity depends on protein loading/release for this technology utilizing catalase as a promising antioxidant therapeutic agent. Catalase has been loaded into CaCO3 using two approaches: i) passive - via adsorption (ADS) into pre-formed crystals and ii) active - via co-synthesis (COS) in the pH range 8–10. Crystal morphology, protein secondary structure and enzymatic bioactivity, and protein retention upon washing are assessed. The activity reduction (∼70% for COS and ∼20% for ADS) is caused by both protein exposure to an alkaline medium and protein aggregation induced by Ca2+. The aggregation significantly governs protein release kinetics. Catalase loading into the crystals is pH-independent and van der Waals interactions dominate over the electrostatics, while catalase activity strongly depends on pH. This study implicates the prime role of loading/release mechanism in the preservation of protein bioactivity and guide for the control over the retention of protein bioactivity.

Enzymatic crosslinked gelatin 3D scaffolds for bone tissue engineering

Bone tissue engineering is an emerging medical field that has been developed in recent years to address pathologies with limited ability of bones to regenerate. Here we report the fabrication and characterization of microbial transglutaminase crosslinked gelatin-based scaffolds designed for serving as both cell substrate and growth factor release system. In particular, morphological, biomechanical and biological features have been analyzed. The enzyme ratio applied during the fabrication of the scaffolds affects the swelling capacity and the mechanical properties of the final structure. The developed systems are not cytotoxic according to the biocompatibility tests. The biological performance of selected formulations was studied using L-929 fibroblasts, D1 MSC and MG63 osteoblasts. Moreover, scaffolds allowed efficient osteogenic differentiation and signaling of MSCs. MSC cultured on the scaffolds not only presented lower proliferative and stemness profile, but also increased expression of osteoblast-related genes (Col1a1, Runx2, Osx). Furthermore, the in vitro release kinetics of vascular endothelial growth factor (VEGF) and bone morphogenetic protein −2 (BMP-2) from the scaffolds were also investigated. The release of the growth factors produced from the scaffolds followed a first order kinetics. These results highlight that the scaffolds designed and developed in this work may be suitable candidates for bone tissue regeneration purposes.

Lactoferrin-Loaded Alginate Microparticles to Target Clostridioides difficile Infection

Some forms of bovine lactoferrin (bLf) are effective in delaying Clostridioides difficile growth and preventing toxin production. However, therapeutic use of bLf may be limited by protein stability issues. The objective of this study was to prepare and evaluate colon-targeted, pH-triggered alginate microparticles loaded with bioactive bLf and to evaluate their anti-C difficile defense properties in vitro. Different forms of metal-bound bLf were encapsulated in alginate microparticles using an emulsification or internal gelation method. The microparticles were coated with chitosan to control protein release. In vitro drug release studies were conducted in pH-simulated gastrointestinal conditions to investigate the release kinetics of encapsulated protein. No significant release of metal-bound bLf was observed at acidic pH; however, on reaching simulated colonic pH, most of the encapsulated lactoferrin was released. The application of bLf (5 mg/mL) delivered from alginate microparticles to human intestinal epithelial cells significantly reduced the cytotoxic effects of toxins A and B as well as bacterial supernatant on Caco-2 and Vero cells, respectively. These results are the first to suggest that alginate-bLf microparticles show protective effects against C difficile toxin-mediated epithelial damage and impairment of barrier function in human intestinal epithelial cells. The future potential of lactoferrin-loaded alginate microparticles against C difficile deserves further study.