Microparticle enlargement and altered surface proteins after air decompression are associated with inflammatory vascular injuries

Studies in a murine model have shown that decompression stress triggers a progressive elevation in the number of circulating annexin V-coated microparticles derived from leukocytes, erythrocytes, platelets, and endothelial cells. We noted that some particles appeared to be larger than anticipated, and size continued to increase for ≥24 h postdecompression. These observations led to the hypothesis that inert gas bubbles caused the enlargement and particle size could be reduced by hydrostatic pressure. After demonstrating pressure-induced particle size reduction, we hypothesized that annexin V-positive particle changes associated with decompression contributed to their proinflammatory potential. Intravenous injection of naive mice with particles isolated from decompressed mice, but not control mice, caused intravascular neutrophil activation; perivascular neutrophil sequestration and tissue injuries were documented as elevations of vascular permeability and activated caspase-3. These changes were not observed if mice were injected with particles that had been subjected to hydrostatic recompression or particles that had been emulsified by incubation with polyethylene glycol telomere B surfactant. Hydrostatic pressure and surfactant incubation also altered the pattern of proteins expressed on the surface of particles. We conclude that proinflammatory events and vascular damage are due to enlargement of annexin V-coated particles and/or changes in surface marker protein pattern associated with provocative decompression. Injection of annexin V-coated particles from decompressed mice will recapitulate the pathophysiological vascular changes observed following decompression stress.     

Hard shell gas-filled contrast enhancement particles for colour Doppler ultrasound imaging of tumors

Hollow hard shell particles of 200 nm and 2 micron diameter with a 10 nm thick porous silica shell have been synthesized using polystyrene templates and a sol-gel process. The template ensures than the hollow particles are monodispersed, while the charged silica surface ensures that they remain suspended in solution for weeks. When filled with perfluorocarbon gas, the particles behave as an efficient contrast agent for colour Doppler ultrasound imaging in human breast tissue. The silica shell provides unique properties compared to conventional soft shell particles employed as ultrasound contrast agents: uniform size control, strong adsorption to tissue and cells immobilizing particles at the tissue injection site, a long imaging lifetime, and a silica surface that can be easily modified with biotargeting ligands or small molecules to adjust the surface charge and polarity.     

Fabrication of PLGA nanoparticles with a fluidic nanoprecipitation system

Particle size is a key feature in determining performance of nanoparticles as drug carriers because it influences circulating half-life, cellular uptake and biodistribution. Because the size of particles has such a major impact on their performance, the uniformity of the particle population is also a significant factor. Particles comprised of the polymer poly(lactic-co-glycolic acid) (PLGA) are widely studied as therapeutic delivery vehicles because they are biodegradable and biocompatible. In fact, microparticles comprised of PLGA are already approved for drug delivery. Unfortunately, PLGA nanoparticles prepared by conventional methods usually lack uniformity. We developed a novel Fluidic NanoPrecipitation System (FNPS) to fabricate highly uniform PLGA particles. Several parameters can be fine-tuned to generate particles of various sizes.     

Size-Dependent Neurotoxicity of Aluminum Oxide Particles: a Comparison Between Nano- and Micrometer Size on the Basis of Mitochondrial Oxidative Damage

Aluminum nanoparticles (AlNPs) are among the most abundantly produced nanosized particles in the market. There is limited information about the potential harmful effects of aluminum oxide due to its particle size on human health. Considering the toxic effects of Al on brain as its target tissue, in this study, the toxicity of nanoparticles, microparticles, and ionic forms of Al on rat brain and isolated mitochondria was evaluated. Sixty male Wistar rats were divided into ten groups (six rats each), in which group I was the control, and the other groups were administered different doses of Al nanoparticles, Al microparticles (AlMP), and Al ionic forms (2, 4, and 8 mg/kg, i.p.) for 28 days. After 24 h, the animals were killed, brain tissue was separated, the mitochondrial fraction was isolated, and oxidative stress markers were measured. Also, mitochondrial function was assayed by MTT test. The results showed that all forms of Al particles induced ROS formation, lipid peroxidation, protein oxidation, glutathione depletion, mitochondrial dysfunction, and gait abnormalities in a dose-dependent manner. In addition, Al particles decreased mitochondrial membrane potential. These data indicated that oxidative stress might contribute to the toxicity effects of Al. Comparison of oxidative stress markers between all forms of Al revealed that the toxic effect of AlNP on brain tissue was substantially more than that caused by AlMP and bulk form. This study showed more neurotoxicity of AlNPs compared to other forms on brain oxidative damage that probably is due to more penetration into the brain.     

Polymer‐based microparticles in tissue engineering and regenerative medicine

Different types of biomaterials, processed into different shapes, have been proposed as temporary support for cells in tissue engineering (TE) strategies. The manufacturing methods used in the production of particles in drug delivery strategies have been adapted for the development of microparticles in the fields of TE and regenerative medicine (RM). Microparticles have been applied as building blocks and matrices for the delivery of soluble factors, aiming for the construction of TE scaffolds, either by fusion giving rise to porous scaffolds or as injectable systems for in situ scaffold formation, avoiding complicated surgery procedures. More recently, organ printing strategies have been developed by the fusion of hydrogel particles with encapsulated cells, aiming the production of organs in in vitro conditions. Mesoscale self‐assembly of hydrogel microblocks and the use of leachable particles in three‐dimensional (3D) layer‐by‐layer (LbL) techniques have been suggested as well in recent works. Along with innovative applications, new perspectives are open for the use of these versatile structures, and different directions can still be followed to use all the potential that such systems can bring. This review focuses on polymeric microparticle processing techniques and overviews several examples and general concepts related to the use of these systems in TE and RE applications. The use of materials in the development of microparticles from research to clinical applications is also discussed. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

New insight into formation of DNA-containing microparticles during PCR: the scaffolding role of magnesium pyrophosphate crystals

This work aims to study molecular mechanisms involved in the formation of DNA-containing microparticles and nanoparticles during PCR. Both pyrophosphate and Mg²⁺ ions proved to play an important role in the generation of DNA microparticles (MPs) with a unique and sophisticated structure in PCR with Taq polymerase. Thus, the addition of Tli thermostable pyrophosphatase to a PCR mixture inhibited this process and caused the destruction of synthesized DNA MPs. Thermal cycling of Na-pyrophosphate (Na-PPi)- and Mg²⁺-containing mixtures (without DNA polymerase and dNTPs) under the standard PCR regime yielded crystalline oval or lenticular microdisks and 3D MPs composed from magnesium pyrophosphate (Mg-PPi). As shown by scanning electron microscopy (SEM), the produced Mg-PPi microparticles consisted of intersecting disks or their segments. They were morphologically similar but simpler than DNA-containing MPs generated in PCR. The incorporation of dNTPs, primers, or dsDNA into Mg-pyrophosphate particles resulted in the structural diversification of 3D microparticles. Thus, the unusual and complex structure of DNA MPs generated in PCR is governed by the unique feature of Mg-pyrophosphate to form supramolecular particles during thermal cycling. We hypothesize the Mg-pyrophosphate particles that are produced during thermal cycling serve as scaffolds for amplicon DNA condensation.     

Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles

Background

Biolistic transfection is proving an increasingly popular method of incorporating DNA or RNA into cells that are difficult to transfect using traditional methods. The technique routinely uses 'microparticles', which are ~1 μm diameter projectiles, fired into tissues using pressurised gas. These microparticles are efficient at delivering DNA into cells, but cannot efficiently transfect small cells and may cause significant tissue damage, thus limiting their potential usefulness. Here we describe the use of 40 nm diameter projectiles - nanoparticles - in biolistic transfections to determine if they are a suitable alternative to microparticles.

Results

Examination of transfection efficiencies in HEK293 cells, using a range of conditions including different DNA concentrations and different preparation procedures, reveals similar behaviour of microparticles and nanoparticles. The use of nanoparticles, however, resulted in ~30% fewer damaged HEK293 cells following transfection. Biolistic transfection of mouse ear tissue revealed similar depth penetration for the two types of particles, and also showed that < 10% of nuclei were damaged in nanoparticle-transfected samples, compared to > 20% in microparticle-transfected samples. Visualising details of small cellular structures was also considerably enhanced when using nanoparticles.

Conclusions

We conclude that nanoparticles are as efficient for biolistic transfection as microparticles, and are more appropriate for use in small cells, when examining cellular structures and/or where tissue damage is a problem.     

Modeling immune complex-mediated autoimmune inflammation

A number of autoimmune diseases are thought to feature a particular type of self-sustaining inflammation, caused by the deposition of immune complexes (IC) in the inflamed tissue and a consequent activation of local effector cells. The persistence of this inflammation is due to a positive feedback loop, where autoantigen particles released as part of the tissue damage caused by the inflammation stimulate autoreactive B cells, leading to the formation of further immune complexes and their subsequent deposition. We present a mathematical model for the exploration of IC-mediated autoimmune inflammation and its clinical implications. We characterize the possible differences between normal individuals and those susceptible to such inflammation, and show that both random perturbations and bifurcations can lead to disease onset. Our model explains how defects in the mechanisms responsible for cellular debris clearance contribute to the development of disease, in agreement with empirical evidence. Moreover, we show that parameters governing the dynamics of immune complexes, such as their clearance rate, have an even stronger effect in determining the behavior of the system. We demonstrate the existence of hysteresis, implying that once IC-mediated autoimmune inflammation is triggered, its long-term suppression may be difficult to achieve. Our results can serve to guide the development of novel therapies to autoimmune diseases involving this type of inflammation.     

Integrating Dimension Reduction and Out-of-Sample Extension in Automated Classification of Ex Vivo Human Patellar Cartilage on Phase Contrast X-Ray Computed Tomography

Phase contrast X-ray computed tomography (PCI-CT) has been demonstrated as a novel imaging technique that can visualize human cartilage with high spatial resolution and soft tissue contrast. Different textural approaches have been previously investigated for characterizing chondrocyte organization on PCI-CT to enable classification of healthy and osteoarthritic cartilage. However, the large size of feature sets extracted in such studies motivates an investigation into algorithmic feature reduction for computing efficient feature representations without compromising their discriminatory power. For this purpose, geometrical feature sets derived from the scaling index method (SIM) were extracted from 1392 volumes of interest (VOI) annotated on PCI-CT images of ex vivo human patellar cartilage specimens. The extracted feature sets were subject to linear and non-linear dimension reduction techniques as well as feature selection based on evaluation of mutual information criteria. The reduced feature set was subsequently used in a machine learning task with support vector regression to classify VOIs as healthy or osteoarthritic; classification performance was evaluated using the area under the receiver-operating characteristic (ROC) curve (AUC). Our results show that the classification performance achieved by 9-D SIM-derived geometric feature sets (AUC: 0.96 ± 0.02) can be maintained with 2-D representations computed from both dimension reduction and feature selection (AUC values as high as 0.97 ± 0.02). Thus, such feature reduction techniques can offer a high degree of compaction to large feature sets extracted from PCI-CT images while maintaining their ability to characterize the underlying chondrocyte patterns.     

The role of membrane lipids in the induction of macrophage apoptosis by microparticles

Microparticles are membrane-derived vesicles that are released from cells during activation or cell death. These particles can serve as mediators of intercellular cross-talk and induce a variety of cellular responses. Previous studies have shown that macrophages undergo apoptosis after phagocytosing microparticles. Here, we have addressed the hypothesis that microparticles trigger this process via lipid pathways. In these experiments, microparticles induced apoptosis in primary macrophage cells or cell lines (RAW 264.7 or U937) with up to a 5-fold increase. Preincubation of macrophages with phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)BP) reduced the microparticle-induced apoptosis in a dose-dependent manner. PtdIns(3,5)BP is a specific inhibitor of the acid sphingomyelinase and thus can block the generation of pro-apoptotic ceramides. Similarly, the pre-incubation of macrophages with PtdIns(3,5)BP prevented microparticle-induced upregulation of caspase 8, which is a major target molecule of ceramide action in the apoptosis pathway. PtdIns(3,5)BP, however, had no effect on the spontaneous rate of apoptosis. To evaluate further signaling pathways induced by microparticles, the extracellular signal regulated kinase (ERK-) 1 was investigated. This kinase plays a role in activating phospholipases A2 which cleaves membrane phospholipids into arachidonic acid; microparticles have been suggested to be a preferred substrate for phospholipases A2. As shown in our experiments, microparticles strongly increased the amount of phosphorylated ERK1/2 in RAW 264.7 macrophages in a time-dependent manner, peaking 15 min after co-incubation. Addition of PD98059, a specific inhibitor of ERK1, prevented the increase in apoptosis of RAW 264.7 macrophages. Together, these data suggest that microparticles perturb lipid homeostasis of macrophages and thereby induce apoptosis. These results emphasize the importance of biolipids in the cellular cross-talk of immune cells. Based on the fact that in clinical situations with excessive cell death such as malignancies, autoimmune diseases and following chemotherapies high levels of circulating microparticles might modulate phagocytosing cells, a suppression of the immune response might occur due to loss of macrophages.     

Computational modelling and optimisation of soft tissue outcome in cranio-maxillofacial surgery planning

Cranio-maxillofacial (CMF) surgery operations are associated with rearrangement of facial hard and soft tissues, leading to dramatic changes in facial geometry. Often, correction of the aesthetical patient's appearance is the primary objective of the surgical intervention. Due to the complexity of the facial anatomy and the biomechanical behaviour of soft tissues, the result of the surgical impact cannot always be predicted on the basis of surgeon's intuition and experience alone. Computational modelling of soft tissue outcome using individual tomographic data and consistent numerical simulation of soft tissue mechanics can provide valuable information for surgeons during the planning stage. In this article, we present a general framework for computer-assisted planning of CMF surgery interventions that is based on the reconstruction of patient's anatomy from 3D computer tomography images and finite element analysis of soft tissue deformations. Examples from our clinical case studies that deal with the solution of direct and inverse surgical problems (i.e. soft tissue prediction, inverse implant shape design) demonstrate that the developed approach provides a useful tool for accurate prediction and optimisation of aesthetic surgery outcome.     

Grafting protein ligand monolayers onto the surface of microparticles for probing the accessibility of cell surface receptors.

Coupling of a specific ligand to vaccines or drugs can be a powerful aid to route these compounds to a certain target cell population. However, if the targeted receptor is buried in a glycocalyx, binding of the ligand may be sterically hindered or even abolished, especially when the ligand is attached to bulky payloads. The antigen-transporting M cells that cover the gut-associated lymphoid tissue have a less pronounced glycocalyx than neighboring enterocytes. Such architectural differences might provide a possibility for targeting micro- or nanoparticulate vaccines to the mucosal immune system. To investigate the influence of the glycocalyx on the accessibility of cell surface receptors, we developed a system where a monolayer of ligand molecules is coupled in spatially aligned manner onto the surface of microparticles. On the basis of fluorescent carboxylate-modified particles of 1 micron diameter, different synthetic strategies were tested. Particles were first modified to display aldehyde functions on their surface, then protein ligands were coupled via Schiff base formation. The performance of the particles was tested on cultured mouse fibroblasts using the B subunit of cholera toxin as ligand and the plasma membrane glycolipid ganglioside G(M1) as receptor. Cholera toxin B subunit-coated microparticles generated by one of our synthetic pathways exhibited specific binding to fibroblasts which could be blocked with soluble cholera toxin B subunit. As particles as small as 50 nm and any proteinaceous ligand may be used, this system provides a versatile means for monitoring receptor accessibilities in vitro and in vivo.     

Intra-lymph Node Injection of Biodegradable Polymer Particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g. size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.     

Microparticle delivery of Interleukin-7 to boost T-cell proliferation and survival

In HIV infections, homoeostasis of T cells is dysregulated such that there is a depletion of CD4⁺ T cells and a progressive loss of naïve CD4⁺ and CD8⁺ T cells. Methodologies that can improve the function of some or all of these cells will likely enhance immune responsiveness in HIV infection. Interleukin‐7 (IL‐7) is a cytokine that has been shown to be critical in homeostatic expansion of naïve CD8⁺ and CD4⁺ cells in lymphopenic hosts, as well as regulating effector T cell to memory T‐cell transition and memory T‐cell homeostasis. In animal studies and clinical trials, repeated injections of IL‐7 are used to boost both CD4⁺ and CD8⁺ cell counts. Daily injections, however, are painful, inconvenient, and provide a frequent route for pathogen entry. We developed a poly (D ,L ‐lactide‐co‐glycolide; PLGA) microparticle controlled release system to administer IL‐7 in which a single injection of microparticles can provide therapeutic delivery of IL‐7. IL‐7 encapsulated PLGA microparticles were first synthesized using a water/organic/water double emulsion method, release from the particles was then optimized using in vitro release studies and therapeutic effectiveness was finally studied in animal studies. These PLGA microparticles showed effective delivery of IL‐7 for 1 week in vitro. These results were translated to in vivo delivery as well, which was followed for 9 days. Controlled release of IL‐7 in mice demonstrated biological activity in both CD4⁺ and CD8⁺ T cells in mice, which was consistent with previously reported results using daily injections. Biotechnol. Bioeng. 2012; 109:1835–1843. © 2012 Wiley Periodicals, Inc.     

Dentin regeneration based on tooth tissue engineering: A review

Missing or damaged teeth due to caries, genetic disorders, oral cancer, or infection may contribute to physical and mental impairment that reduces the quality of life. Despite major progress in dental tissue repair and those replacing missing teeth with prostheses, clinical treatments are not yet entirely satisfactory, as they do not regenerate tissues with natural teeth features. Therefore, much of the focus has centered on tissue engineering (TE) based on dental stem/progenitor cells to create bioengineered dental tissues. Many in vitro and in vivo studies have shown the use of cells in regenerating sections of a tooth or a whole tooth. Tooth tissue engineering (TTE), as a promising method for dental tissue regeneration, can form durable biological substitutes for soft and mineralized dental tissues. The cell-based TE approach, which directly seeds cells and bioactive components onto the biodegradable scaffolds, is currently the most potential method. Three essential components of this strategy are cells, scaffolds, and growth factors (GFs). This study investigates dentin regeneration after an injury such as caries using TE and stem/progenitor cell-based strategies. We begin by discussing about the biological structure of a dentin and dentinogenesis. The engineering of teeth requires knowledge of the processes that underlie the growth of an organ or tissue. Then, the three fundamental requirements for dentin regeneration, namely cell sources, GFs, and scaffolds are covered in the current study, which may ultimately lead to new insights in this field.     

Plasma membrane microparticles in angiogenesis: role in ischemic diseases and in cancer

Microparticles are small fragments of the plasma membrane released by activated and/or apoptotic cells. In theory, all type of cells can shed microparticles representing a physiological process in the cell life. Mainly, microparticles generation has been studied in different cardiovascular pathologies due to the facility to obtain blood samples from individuals. Although microparticles have been considered as simply markers of several diseases, in the last decade, several studies support the hypothesis that they participate in the regulation of the cardiovascular system function by carrying biological messages between cells. Among the effects of microparticles, recent data show that they can be implicated in the modulation of neovascularization, an essential function of cells from cardiovascular system during either ischemic diseases or cancer development. Whereas during pathologies associated with ischemia an increase of neovascularization may have beneficial effects, anti-angiogenic strategies represent new approaches for manipulation of tumor development. Here, we give an overview of the mechanisms and factors involved in neovascularization, and finally, we look at the role and the consequences of the modulation of this process by microparticles in pathological situations.     

Selective cell targeting with light-absorbing microparticles and nanoparticles

We describe a new method for selective cell targeting based on the use of light-absorbing microparticles and nanoparticles that are heated by short laser pulses to create highly localized cell damage. The method is closely related to chromophore-assisted laser inactivation and photodynamic therapy, but is driven solely by light absorption, without the need for photochemical intermediates (particularly singlet oxygen). The mechanism of light-particle interaction was investigated by nanosecond time-resolved microscopy and by thermal modeling. The extent of light-induced damage was investigated by cell lethality, by cell membrane permeability, and by protein inactivation. Strong particle size dependence was found for these interactions. A technique based on light to target endogenous particles is already being exploited to treat pigmented cells in dermatology and ophthalmology. With exogenous particles, phamacokinetics and biodistribution studies are needed before the method can be evaluated against photodynamic therapy for cancer treatment. However, particles are unique, unlike photosensitizers, in that they can remain stable and inert in cells for extended periods. Thus they may be particularly useful for prelabeling cells in engineered tissue before implantation. Subsequent irradiation with laser pulses will allow control of the implanted cells (inactivation or modulation) in a noninvasive manner.     

Soft and hard tissue response to zirconium dioxide dental implants--a clinical study in man

Titanium dental implants have been used successfully in implantology for more than 40 years. Recent research, however, suggests that titanium might have more side effects than previously believed. Zirconia ceramics have been employed in orthopaedic surgery for approximately 30 years and were recently introduced into dentistry as a metal replacement for crown and bridge work as well as implant abutments. Zirconium dioxide has been shown in both in vitro and in vivo studies to have desirable osseointegrative properties. This clinical study shows that dental implants made from zirconia are a feasible alternative to titanium dental implants. In addition to excellent cosmetic results, zirconia implants allow a degree of osseointegration and soft tissue response that is superior to titanium dental implants.     

Imaging Liver Lesions Using Grating-Based Phase-Contrast Computed Tomography with Bi-Lateral Filter Post-Processing

X-ray phase-contrast imaging shows improved soft-tissue contrast compared to standard absorption-based X-ray imaging. Especially the grating-based method seems to be one promising candidate for clinical implementation due to its extendibility to standard laboratory X-ray sources. Therefore the purpose of our study was to evaluate the potential of grating-based phase-contrast computed tomography in combination with a novel bi-lateral denoising method for imaging of focal liver lesions in an ex vivo feasibility study. Our study shows that grating-based phase-contrast CT (PCCT) significantly increases the soft-tissue contrast in the ex vivo liver specimens. Combining the information of both signals – absorption and phase-contrast – the bi-lateral filtering leads to an improvement of lesion detectability and higher contrast-to-noise ratios. The normal and the pathological tissue can be clearly delineated and even internal structures of the pathological tissue can be visualized, being invisible in the absorption-based CT alone. Histopathology confirmed the presence of the corresponding findings in the analyzed tissue. The results give strong evidence for a sufficiently high contrast for different liver lesions using non-contrast-enhanced PCCT. Thus, ex vivo imaging of liver lesions is possible with a polychromatic X-ray source and at a spatial resolution of ∼100 µm. The post-processing with the novel bi-lateral denoising method improves the image quality by combining the information from the absorption and the phase-contrast images.     

Protein encapsulation via porous CaCO3 microparticles templating

Porous microparticles of calcium carbonate with an average diameter of 4.75 microm were prepared and used for protein encapsulation in polymer-filled microcapsules by means of electrostatic layer-by-layer assembly (ELbL). Loading of macromolecules in porous CaCO3 particles is affected by their molecular weight due to diffusion-limited permeation inside the particles and also by the affinity to the carbonate surface. Adsorption of various proteins and dextran was examined as a function of pH and was found to be dependent both on the charge of the microparticles and macromolecules. The electrostatic effect was shown to govern this interaction. This paper discusses the factors which can influence the adsorption capacity of proteins. A new way of protein encapsulation in polyelectrolyte microcapsules is proposed exploiting the porous, biocompatible, and decomposable microparticles from CaCO3. It consists of protein adsorption in the pores of the microparticles followed by ELbL of oppositely charged polyelectrolytes and further core dissolution. This resulted in formation of polyelectrolyte-filled capsules with protein incorporated in interpenetrating polyelectrolyte network. The properties of CaCO3 microparticles and capsules prepared were characterized by scanning electron microscopy, microelectrophoresis, and confocal laser scanning microscopy. Lactalbumin was encapsulated by means of the proposed technique yielding a content of 0.6 pg protein per microcapsule. Horseradish peroxidase saves 37% of activity after encapsulation. However, the thermostability of the enzyme was improved by encapsulation. The results demonstrate that porous CaCO3 microparticles can be applied as microtemplates for encapsulation of proteins into polyelectrolyte capsules at neutral pH as an optimal medium for a variety of bioactive material, which can also be encapsulated by the proposed method. Microcapsules filled with encapsulated material may find applications in the field of biotechnology, biochemistry, and medicine.