The spherical vaterite CaCO3 microcrystals are nowadays widely used as sacrificial templates for fabrication of various microcarriers made of biopolymers (e.g., proteins, nucleic acids, enzymes) due to porous structure and mild template elimination conditions. Here, we demonstrated for the first time that polymer microcarriers with tuned internal nanoarchitecture can be designed by employing the CaCO3 crystals of controlled porosity. The layer-by-layer deposition has been utilized to assemble shell-like (hollow) and matrix-like (filled) polymer capsules due to restricted and free polymer diffusion through the crystal pores, respectively. The crystal pore size in the range of few tens of nanometers can be adjusted without any additives by variation of the crystal preparation temperature in the range 7-45 °C. The temperature-mediated growth mechanism is explained by the Ostwald ripening of nanocrystallites forming the crystal secondary structure. Various techniques including SEM, AFM, CLSM, Raman microscopy, nitrogen adsorption-desorption, and XRD have been employed for crystal and microcapsule analysis. A three-dimensional model is introduced to describe the crystal internal structure and predict the pore cutoff and available surface for the pore diffusing molecules. Inherent biocompatibility of CaCO3 and a possibility to scale the porosity in the size range of typical biomacromolecules make the CaCO3 crystals extremely attractive tools for template assisted designing tailor-made biopolymer-based architectures in 2D to 3D targeted at drug delivery and other bioapplications.

Formulation of proteins into particulate form is a principal strategy to achieve controlled and targeted delivery, as well as to protect fragile protein molecules. Control over size, mechanical properties, and surface area (porosity) of particulate proteins has been successfully achieved by hard templating under mild conditions using porous CaCO3 microspheres. A crucial step in this approach, which determines protein content, is the loading of proteins into the CaCO3 microspheres. In this study, the adsorption of different proteins into the microspheres has been investigated. Proteins differing in characteristics such as molecular weight and charge have been employed: catalase (Cat), insulin (Ins), aprotinin (Apr), and protamine (Pro). Thermodynamics of adsorption equilibria have been studied, together with quantitative and qualitative analysis of protein loading and distribution in the microspheres. Protein interaction with the CaCO3 microspheres is not limited by the diffusion of protein molecules (protein dimensions are significantly smaller than microsphere pores) but is determined by the protein affinity for the microsphere surface. Cat and Ins bind much more strongly to the microspheres than Apr and Pro, which can be explained by electrostatic attractive forces. Protein binding/release and protein biological activity have been investigated as a function of pH. It is shown that pH variation during the adsorption process plays a principal role and defines not only the amount of protein adsorbed/released but also protein biological activity. Protein adsorption and microsphere elimination (by EDTA) do not affect protein bioactivity. In addition to applications for protein particle/capsule formulations, the findings of this study might help in understanding protein interactions with carbonate minerals such as calcium carbonate, which is used as a natural material for multiple applications.

Encapsulation of model proteins (catalase, insulin, aprotinin) into multilayer dextran sulphate/protamin capsules by templating on CaCO3 microparticles is investigated employing: (i) PRE-loading into CaCO3 particles by adsorption or co-synthesis and (ii) POST-loading into performed capsules. Protein encapsulation is governed by both its size and electrostatic interactions with the carbonate microparticles and multilayer shell. PRE-loading enables improved encapsulation compared to POST-loading (catalase content in capsules 630 and 70 mg · g(-1)). Bioactivity of encapsulated protein is not affected by interaction with multilayers but may be reduced at slightly alkaline pH due to CaCO3 hydrolysis. This study might help to successfully encapsulate fragile bio-macromolecules into multilayer capsules.

Polymeric scaffolds serve as valuable supports for biological cells since they offer essential features for guiding cellular organization and tissue development. The main challenges for scaffold fabrication are i) to tune an internal structure and ii) to load bio-molecules such as growth factors and control their local concentration and distribution. Here, a new approach for the design of hollow polymeric scaffolds using porous CaCO3 particles (cores) as templates is presented. The cores packed into a microfluidic channel are coated with polymers employing the layer-by-layer (LbL) technique. Subsequent core elimination at mild conditions results in formation of the scaffold composed of interconnected hollow polymer microspheres. The size of the cores determines the feature dimensions and, as a consequence, governs cellular adhesion: for 3T3 fibroblasts an optimal microsphere size is 12 μm. By making use of the carrier properties of the porous CaCO3 cores, the microspheres are loaded with BSA as a model protein. The scaffolds developed here may also be well suited for the localized release of bio-molecules using external triggers such as IR-light.

signifi cantly precise control over the scaffold architecture. Well-defi ned internal structure (porosity) determines both cellular infi ltration into the scaffold and its material properties. Pore size and distribution ensuring diffusion of nutrients into the scaffold and removal of metabolic products is of high importance. [ 4,11 ] Ionically crosslinked polymer gels such as alginate hydrogels have been extensively developed as scaffolds for tissue engineering. [12][13][14][15] The adjustable kinetics of the alginate gel degradation at neutral pH [ 1,16,17 ] gives an option to use the gels for multiple applications as wound dressings, anti-adhesive, and repair materials. [ 14,[18][19][20][21] Characteristics of alginate gels (hydration, softness, porosity, swelling in water, etc.) can be adjusted using a certain fabrication approach and by variation of interactions between charged polyanionic groups and crosslinking counterions; [ 13,17,[22][23][24][25] these interactions are known to be strongly affected by ionic strength, molecule length and structure, solvent pH, salt concentration, and temperature conditions. [26][27][28][29][30][31] This allows the fabrication of alginate scaffolds possessing desired properties corresponding to a certain tissue, for instance cartilage, [ 12 ] or dura mater. [ 21 ] Porous alginate gels can provide space and mechanical support to seed biological cells for tissue formation, [ 1,13,32 ] also allowing a controlled release of gel-laden drugs (peptides and proteins) trapped within the alginate network. [ 14,24,25,33,34 ] To achieve loading of both biological cells and bioactive molecules, the internal structure of hydrogels has to be controlled on the scale of nano-and micrometers. Adjustment of the gel geometry has been demonstrated on substrate surfaces using different micropatterning techniques including a light-addressable electrolytic system, [ 14 ] electrodeposition, [ 15 ] electrochemical patterning, [ 35 ] or the benchtop method using Nylon mesh. [ 11 ] The internal structure of alginate gels has been patterned with regular tube-like pores, [ 16,36 ] interconnected ordered honeycomb pores, [ 12 ] and sponge-like isotropic pores. [37][38][39] However, most of the approaches for alginate gel fabrication lack a precise control over the microstructure and require special equipment. Encapsulation of molecules of interest with controlled spatial distribution is rather complicated or even impossible. There is a need to develop a simple approach for design of a scaffold with welldefi ned internal structure providing both a space to culture cells and cavities to host/release encapsulated (bio)active molecules.Fabrication of porous alginate hydrogels with a well-controlled architecture useful for tissue engineering is still a challenge. Here, CaCO 3 -based templating is utilized to design stable alginate gels with controlled pore dimensions in the range of 5-50 µm. The mechanism of pore formation is studied considering two factors affecting the pore size: i) osmotic pressure generat...

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Poly( N -isopropylacrylamide) (pNIPAM) hydrogels have broad potential applications as drug delivery vehicles because of their thermoresponsive behavior. pNIPAM loading/release performances are directly affected by the gel network structure. Therefore, there is a need with the approaches for accurate design of 3D pNIPAM assemblies with the structure ordered at the nanoscale. This study demonstrates size-selective spontaneous loading of macromolecules (dextrans 10−500 kDa) into pNIPAM microgels by microgel heating from 22 to 35 °C (microgels collapse and trap dextrans) followed by the dextran release upon further cooling down to 22 °C (microgels swell back) . This temperature-mediated behavior is fully reversible. The structure of pNIPAM microgels was tailored via hard templating and cross-linking of the hydrogel using sacrificial mesoporous cores of vaterite CaCO 3 microcrystals. In addition, the fabrication of hollow thermoresponsive pNIPAM microshells has been demonstrated, utilizing vaterite microcrystals that had narrower pores. The proposed approach for heating-triggered encapsulation and cooling-triggered release into/from pNIPAM microgels may pave the ways for applications of pNIPAM hydrogels for skin and transdermal cooling-responsive drug delivery in the future.

Loading of therapeutic proteins into mesoporous vaterite crystals is driven by inter-protein interactions in bulk solution and inside the crystals.