Nanoarchitectonics of Bactericidal Coatings Based on CaCO3-Nanosilver Hybrids.

Antimicrobial coatings provide protection against microbes colonization on surfaces. This can prevent the stabilization and proliferation of microorganisms. The ever-increasing levels of microbial resistance to antimicrobials are urging the development of alternative types of compounds that are potent across broad spectra of microorganisms and target different pathways. This will help to slow down the development of resistance and ideally halt it. The development of composite antimicrobial coatings (CACs) that can host and protect various antimicrobial agents and release them on demand is an approach to address this urgent need. In this work, new CACs based on microsized hybrids of calcium carbonate (CaCO3) and silver nanoparticles (AgNPs) were designed using a drop-casting technique. Polyvinylpyrrolidone and mucin were used as additives. The CaCO3/AgNPs hybrids contributed to endowing colloidal stability to the AgNPs and controlling their release, thereby ensuring the antibacterial activity of the coatings. Moreover, the additives PVP and mucin served as a matrix to (i) control the distribution of the hybrids, (ii) ensure mechanical integrity, and (iii) prevent the undesired release of AgNPs. Scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) techniques were used to characterize the 15 µm thick CAC. The antibacterial activity was determined against Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), and Pseudomonas aeruginosa, three bacteria responsible for many healthcare infections. Antibacterial performance of the hybrids was demonstrated at concentrations between 15 and 30 µg/cm2. Unloaded CaCO3 also presented bactericidal properties against MRSA. In vitro cytotoxicity tests demonstrated that the hybrids at bactericidal concentrations did not affect human dermal fibroblasts and human mesenchymal stem cell viability. In conclusion, this work presents a simple approach for the design and testing of advanced multicomponent and functional antimicrobial coatings that can protect active agents and release them on demand.

Hierarchy of hybrid materials. Part-II: The place of organics-on-inorganics in it, their composition and applications.

Hybrid materials or hybrids incorporating organic and inorganic constituents are emerging as a very potent and promising class of materials due to the diverse but complementary nature of their properties. This complementarity leads to a perfect synergy of properties of the desired materials and products as well as to an extensive range of their application areas. Recently, we have overviewed and classified hybrid materials describing inorganics-in-organics in Part-I (Saveleva, et al., Front. Chem., 2019, 7, 179). Here, we extend that work in Part-II describing organics-on-inorganics, i.e., inorganic materials modified by organic moieties, their structure and functionalities. Inorganic constituents comprise of colloids/nanoparticles and flat surfaces/matrices comprise of metallic (noble metal, metal oxide, metal-organic framework, magnetic nanoparticles, alloy) and non-metallic (minerals, clays, carbons, and ceramics) materials; while organic additives can include molecules (polymers, fluorescence dyes, surfactants), biomolecules (proteins, carbohydtrates, antibodies and nucleic acids) and even higher-level organisms such as cells, bacteria, and microorganisms. Similarly to what was described in Part-I, we look at similar and dissimilar properties of organic-inorganic materials summarizing those bringing complementarity and composition. A broad range of applications of these hybrid materials is also presented whose development is spurred by engaging different scientific research communities.

Hybrid Mucin-Vaterite Microspheres for Delivery of Proteolytic Enzyme Chymotrypsin.

While the enteral delivery of proteolytic enzymes is widely established for combating many diseases as an alternative to antibiotic treatment, their local delivery only emerges as administration route enabling sustained release in a controlled manner on site. The latest requires the development of drug delivery systems suitable for encapsulation and preservation of enzymatic proteolytic activity. This study proposes hybrid microspheres made of mucin and biodegradable porous crystals of calcium carbonate (CC) as the carriers for chymotrypsin (CTR) delivery. CTR is impregnated into CC and hybrid CC/mucin (CCM) microspheres by means of sorption without any chemical modification. The loading of the CC with mucin enhances CTR retention on hybrid microspheres (adsorption capacity of ≈8.7 mg g-1  vs 4.7 mg g-1 ), recharging crystal surface due to the presence of mucin and diminishing the average pore diameter of the crystals from 25 to 8 nm. Mucin also retards recrystallization of vaterite into nonporous calcite improving stability of CCM microspheres upon storage. Proteolytic activity of CTR is preserved in both CC and CCM microspheres, being pH dependent. Temperature-induced inactivation of CTR significantly diminishes by CTR encapsulation into CC and CCM microspheres. Altogether, these findings indicate promises of hybrid mucin-vaterite microspheres for mucosal application of proteases.

Mesoporous One-Component Gold Microshells as 3D SERS Substrates.

Surface-enhanced Raman scattering (SERS) is a powerful analytical tool for label-free analysis that has found a broad spectrum of applications in material, chemical, and biomedical sciences. In recent years, a great interest has been witnessed in the rational design of SERS substrates to amplify Raman signals and optionally allow for the selective detection of analytes, which is especially essential and challenging for biomedical applications. In this study, hard templating of noble metals is proposed as a novel approach for the design of one-component tailor-made SERS platforms. Porous Au microparticles were fabricated via dual ex situ adsorption of Au nanoparticles and in situ reduction of HAuCl4 on mesoporous sacrificial microcrystals of vaterite CaCO3. Elimination of the microcrystals at mild conditions resulted in the formation of stable mesoporous one-component Au microshells. SERS performance of the microshells at very low 0.4 µW laser power was probed using rhodamine B and bovine serum albumin showing enhancement factors of 2 × 108 and 8 × 108, respectively. The proposed strategy opens broad avenues for the design and scalable fabrication of one-component porous metal particles that can serve as superior SERS platforms possessing both excellent plasmonic properties and the possibility of selective inclusion of analyte molecules and/or SERS nanotags for highly specific SERS analysis.

Biopolymer-Based Multilayer Capsules and Beads Made via Templating: Advantages, Hurdles and Perspectives.

One of the undeniable trends in modern bioengineering and nanotechnology is the use of various biomolecules, primarily of a polymeric nature, for the design and formulation of novel functional materials for controlled and targeted drug delivery, bioimaging and theranostics, tissue engineering, and other bioapplications. Biocompatibility, biodegradability, the possibility of replicating natural cellular microenvironments, and the minimal toxicity typical of biogenic polymers are features that have secured a growing interest in them as the building blocks for biomaterials of the fourth generation. Many recent studies showed the promise of the hard-templating approach for the fabrication of nano- and microparticles utilizing biopolymers. This review covers these studies, bringing together up-to-date knowledge on biopolymer-based multilayer capsules and beads, critically assessing the progress made in this field of research, and outlining the current challenges and perspectives of these architectures. According to the classification of the templates, the review sequentially considers biopolymer structures templated on non-porous particles, porous particles, and crystal drugs. Opportunities for the functionalization of biopolymer-based capsules to tailor them toward specific bioapplications is highlighted in a separate section.

Which Biopolymers Are Better for the Fabrication of Multilayer Capsules? A Comparative Study Using Vaterite CaCO3 as Templates.

The polymer layer-by-layer assembly is accounted among the most attractive approaches for the design of advanced drug delivery platforms and biomimetic materials in 2D and 3D. The multilayer capsules can be made of synthetic or biologically relevant (e.g., natural) polymers. The biopolymers are advantageous for bioapplications; however, the design of such "biocapsules" is more challengeable due to intrinsic complexity and lability of biopolymers. Until now, there are no systematic studies that report the formation mechanism for multilayer biocapsules templated upon CaCO3 crystals. This work evaluates the structure-property relationship for 16 types of capsules made of different biopolymers and proposes the capsule formation mechanism. The capsules have been fabricated upon mesoporous cores of vaterite CaCO3, which served as a sacrificial template. Stable capsules of polycations poly-l-lysine or protamine and four different polyanions were successfully formed. However, capsules made using the polycation collagen and dextran amine underwent dissolution. Formation of the capsules has been correlated with the stability of the respective polyelectrolyte complexes at increased ionic strength. All formed capsules shrink upon core dissolution and the degree of shrinkage increased in the series of polyanions: heparin sulfate < dextran sulfate < chondroitin sulfate < hyaluronic acid. The same trend is observed for capsule adhesiveness to the glass surface, which correlates with the decrease in polymer charge density. The biopolymer length and charge density govern the capsule stability and internal structure; all formed biocapsules are of a matrix-type, other words are microgels. These findings can be translated to other biopolymers to predict biocapsule properties.

Cooling-Triggered Release from Mesoporous Poly(N-isopropylacrylamide) Microgels at Physiological Conditions.

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 CaCO3 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.

CaCO3 crystals as versatile carriers for controlled delivery of antimicrobials.

CaCO3 crystals have been known for a long time as naturally derived and simply fabricated nano(micro)-sized materials able to effectively host and release various molecules. This review summarises the use of CaCO3 crystals as versatile carriers to host, protect and release antimicrobials, offering a strong tool to tackle antimicrobial resistance, a serious global health problem. The main methods for the synthesis of CaCO3 crystals with different properties, as well as the approaches for the loading and release of antimicrobials are presented. Finally, prospects to utilize the crystals in order to improve the therapeutic outcome and combat antimicrobial resistance are highlighted. Ultimately, this review intends to provide an in-depth overview of the application of CaCO3 crystals for the smart and controlled delivery of antimicrobial agents and aims at identifying the advantages and drawbacks as well as guiding future works, research directions and industrial applications.

Layer-By-Layer Assemblies of Biopolymers: Build-Up, Mechanical Stability and Molecular Dynamics.

Rapid development of versatile layer-by-layer technology has resulted in important breakthroughs in the understanding of the nature of molecular interactions in multilayer assemblies made of polyelectrolytes. Nowadays, polyelectrolyte multilayers (PEM) are considered to be non-equilibrium and highly dynamic structures. High interest in biomedical applications of PEMs has attracted attention to PEMs made of biopolymers. Recent studies suggest that biopolymer dynamics determines the fate and the properties of such PEMs; however, deciphering, predicting and controlling the dynamics of polymers remains a challenge. This review brings together the up-to-date knowledge of the role of molecular dynamics in multilayers assembled from biopolymers. We discuss how molecular dynamics determines the properties of these PEMs from the nano to the macro scale, focusing on its role in PEM formation and non-enzymatic degradation. We summarize the factors allowing the control of molecular dynamics within PEMs, and therefore to tailor polymer multilayers on demand.

Inter-protein interactions govern protein loading into porous vaterite CaCO3 crystals.

The fast development of protein therapeutics has resulted in a high demand for advanced delivery carriers that can effectively host therapeutic proteins, preserve their bioactivity and release them on demand. Accordingly, vaterite CaCO3 crystals have attracted special attention as sacrificial templates for protein encapsulation in micro- and nanoparticles (capsules and beads, respectively) under mild biofriendly conditions. This study aimed to better understand the mechanism of protein loading into crystals as a primary step for protein encapsulation. The loading of three therapeutic proteins (250 kDa catalase, 5.8 kDa insulin, and 6.5 kDa aprotinin) was investigated for crystals with different porosities. However, unexpectedly, the protein loading capacity was not consistent with the protein molecular weight. It solely depends on the inter-protein interactions in the bulk solution in the presence of crystals and that inside the crystals. The smallest protein aprotinin aggregates in the bulk (its aggregate size is about 100 nm), which prohibits its loading into the crystals. Insulin forms hexamers in the bulk, which can diffuse into the crystal pores but tend to aggregate inside the pores, suppressing protein diffusion inward. Catalase, the largest protein tested, does not form any aggregates in the bulk and diffuses freely into the crystals; however, its diffusion into small pores is sterically restricted. These findings are essential for the encapsulation of protein therapeutics by means of templating based on CaCO3 crystals and for the engineering of protein-containing microparticles having desired architectures.

Editorial for the Special Issue on Self-Assembly of Polymers.

The self-assembly of polymers is a powerful tool for producing various functional materials with a high precision from nano- to macroscale [...].

Bio-friendly encapsulation of superoxide dismutase into vaterite CaCO3 crystals. Enzyme activity, release mechanism, and perspectives for ophthalmology.

Mesoporous vaterite CaCO3 crystals are nowadays one of the most popular vectors for loading of fragile biomolecules like proteins due to biocompatibility, high loading capacity, cost effective and simple loading procedures. However, recent studies reported the reduction of bioactivity for protein encapsulation into the crystals in water due to rather high alkaline pH of about 10.3 caused by the crystal hydrolysis. In this study we have investigated how to retain the bioactivity and control the release rate of the enzyme superoxide dismutase (SOD) loaded into the crystals via co-synthesis. SOD is widely used as an antioxidant in ophthalmology and its formulations with high protein content and activity as well as opportunities for a sustained release are highly desirable. Here we demonstrate that SOD co-synthesis can be done at pH 8.5 in a buffer without affecting crystal morphology. The synthesis in the buffer allows reaching the high loading efficiency of 93%, high SOD content (24 versus 15 w/w % for the synthesis in water), and order of magnitude higher activity compared to the synthesis in water. The enormous SOD concentration into crystals of 10-2 M is caused by the entrapment of SOD aggregates into the crystal pores. The SOD released from crystals at physiologically relevant ionic strength fully retains its bioactivity. As found by fitting the release profiles using zero-order and Baker-Lonsdale models, the SOD release mechanism is governed by both the SOD aggregate dissolution and by the diffusion of SOD molecules thorough the crystal pores. The latest process contributes more in case of the co-synthesis in the buffer because at higher pH (co-synthesis in water) the unfolded SOD molecules aggregate stronger. The release is bi-modal with a burst (ca 30%) followed by a sustained release and a complete release due to the recrystallization of vaterite crystals to non-porous calcite crystals. The mechanism of SOD loading into and release from the crystals as well as perspectives for the use of the crystals for SOD delivery in ophthalmology are discussed. We believe that together with a fundamental understanding of the vaterite-based protein encapsulation and protein release, this study will help to establish a power platform for a mild and effective encapsulation of fragile biomolecules like proteins at bio-friendly conditions.

Porous Alginate Scaffolds Assembled Using Vaterite CaCO3 Crystals.

Formulation of multifunctional biopolymer-based scaffolds is one of the major focuses in modern tissue engineering and regenerative medicine. Besides proper mechanical/chemical properties, an ideal scaffold should: (i) possess a well-tuned porous internal structure for cell seeding/growth and (ii) host bioactive molecules to be protected against biodegradation and presented to cells when required. Alginate hydrogels were extensively developed to serve as scaffolds, and recent advances in the hydrogel formulation demonstrate their applicability as "ideal" soft scaffolds. This review focuses on advanced porous alginate scaffolds (PAS) fabricated using hard templating on vaterite CaCO3 crystals. These novel tailor-made soft structures can be prepared at physiologically relevant conditions offering a high level of control over their internal structure and high performance for loading/release of bioactive macromolecules. The novel approach to assemble PAS is compared with traditional methods used for fabrication of porous alginate hydrogels. Finally, future perspectives and applications of PAS for advanced cell culture, tissue engineering, and drug testing are discussed.

Mucin adsorption on vaterite CaCO3 microcrystals for the prediction of mucoadhesive properties.

Porous vaterite CaCO3 crystals are widely used as containers for drug loading and as sacrificial templates to assemble polymer-based nano- and micro-particles at mild conditions. Special attention is paid nowadays to mucosal delivery where the glycoprotein mucin plays a crucial role as a main component of a mucous. In this work mucoadhesive properties of vaterite crystals have been tested by investigation of mucin binding to the crystals as a function of (i) time, (ii) glycoprotein concentration, (iii) adsorption conditions and (iv) degree of mucin desialization. Mucin adsorption follows Bangham equation indicating that diffusion into crystal pores is the rate-limiting step. Mucin strongly binds to the crystals (ΔG = -35 ±â€¯4 kJ mol-1) via electrostatic and hydrophobic interactions forming a gel and thus giving the tremendous mucin mass content in the crystals of up to 16%. Despite strong intermolecular mucin-mucin interactions, pure mucin spheres formed after crystal dissolution are unstable. However, introduction of protamine, actively used for mucosal delivery, makes the spheres stable via additional electrostatic bonding. The results of this work indicate that the vaterite crystals are extremely promising carriers for mucosal drug delivery and for development of test-systems for the analysis of the mucoadhesion.

Self-Assembled Mucin-Containing Microcarriers via Hard Templating on CaCO3 Crystals.

Porous vaterite crystals of CaCO3 are extensively used for the fabrication of self-assembled polymer-based microparticles (capsules, beads, etc.) utilized for drug delivery and controlled release. The nature of the polymer used plays a crucial role and discovery of new perspective biopolymers is essential to assemble microparticles with desired characteristics, such as biocompatibility, drug loading efficiency/capacity, release rate, and stability. Glycoprotein mucin is tested here as a good candidate to assemble the microparticles because of high charge due to sialic acids, mucoadhesive properties, and a tendency to self-assemble, forming gels. Mucin loading into the crystals via co-synthesis is twice as effective as via adsorption into preformed crystals. Desialylated mucin has weaker binding to the crystals most probably due to electrostatic interactions between sialic acids and calcium ions on the crystal surface. Improved loading of low-molecular-weight inhibitor aprotinin into the mucin-containing crystals is demonstrated. Multilayer capsules (mucin/protamine)3 have been made by the layer-by-layer self-assembly. Interestingly, the deposition of single mucin layers (mucin/water)3 has also been proven, however, the capsules were unstable, most probably due to additional (to hydrogen bonding) electrostatic interactions in the case of the two polymers used. Finally, approaches to load biologically-active compounds (BACs) into the mucin-containing microparticles are discussed.

Internal Structure of Matrix-Type Multilayer Capsules Templated on Porous Vaterite CaCO3 Crystals as Probed by Staining with a Fluorescence Dye.

Multilayer capsules templated on decomposable vaterite CaCO3 crystals are widely used as vehicles for drug delivery. The capsule represents typically not a hollow but matrix-like structure due to polymer diffusion into the porous crystals during multilayer deposition. The capsule formation mechanism is not well-studied but its understanding is crucial to tune capsule structure for a proper drug release performance. This study proposes new approach to noninvasively probe and adjust internal capsule structure. Polymer capsules made of poly(styrene-sulfonate) (PSS) and poly(diallyldimethylammonium chloride) (PDAD) have been stained with fluorescence dye rhodamine 6G. Physical-chemical aspects of intermolecular interactions required to validate the approach and adjust capsule structure are addressed. The capsules consist of a defined shell (typically 0.5⁻2 µm) and an internal matrix of PSS-PDAD complex (typically 10⁻40% of a total capsule volume). An increase of ionic strength and polymer deposition time leads to the thickening of the capsule shell and formation of a denser internal matrix, respectively. This is explained by effects of a polymer conformation and limitations in polymer diffusion through the crystal pores. We believe that the design of the capsules with desired internal structure will allow achieving effective encapsulation and controlled/programmed release of bioactives for advanced drug delivery applications.

A "Cell-Friendly" Window for the Interaction of Cells with Hyaluronic Acid/Poly-l-Lysine Multilayers.

Polyelectrolyte multilayers assembled from hyaluronic acid (HA) and poly-l-lysine (PLL) are most widely studied showing excellent reservoir characteristics to host molecules of diverse nature; however, thick (HA/PLL)n films are often found cell repellent. By a systematic study of the adhesion and proliferation of various cells as a function of bilayer number "n" a correlation with the mechanical and chemical properties of films is developed. The following cell lines have been studied: mouse 3T3 and L929 fibroblasts, human foreskin primary fibroblasts VH-Fib, human embryonic kidney HEK-293, human bone cell line U-2-OS, Chinese hamster ovary CHO-K and mouse embryonic stem cells. All cells adhere and spread well in a narrow "cell-friendly" window identify in the range of n = 12-15. At n < 12, the film is inhomogeneous and at n > 15, the film is cell repellent for all cell lines. Cellular adhesion correlates with the mechanical properties of the films showing that softer films at higher "n" number exhibiting a significant decrease of the Young's modulus below 100 kPa are weakly adherent to cells. This trend cannot be reversed even by coating a strong cell-adhesive protein fibronectin onto the film. This indicates that mechanical cues plays a major role for cell behavior, also in respect to biochemical ones.

Binding Mechanism of the Model Charged Dye Carboxyfluorescein to Hyaluronan/Polylysine Multilayers.

Biopolymer-based multilayers become more and more attractive due to the vast span of biological application they can be used for, e.g., implant coatings, cell culture supports, scaffolds. Multilayers have demonstrated superior capability to store enormous amounts of small charged molecules, such as drugs, and release them in a controlled manner; however, the binding mechanism for drug loading into the multilayers is still poorly understood. Here we focus on this mechanism using model hyaluronan/polylysine (HA/PLL) multilayers and a model charged dye, carboxyfluorescein (CF). We found that CF reaches a concentration of 13 mM in the multilayers that by far exceeds its solubility in water. The high loading is not related to the aggregation of CF in the multilayers. In the multilayers, CF molecules bind to free amino groups of PLL; however, intermolecular CF-CF interactions also play a role and (i) endow the binding with a cooperative nature and (ii) result in polyadsorption of CF molecules, as proven by fitting of the adsorption isotherm using the BET model. Analysis of CF mobility in the multilayers by fluorescence recovery after photobleaching has revealed that CF diffusion in the multilayers is likely a result of both jumping of CF molecules from one amino group to another and movement, together with a PLL chain being bound to it. We believe that this study may help in the design of tailor-made multilayers that act as advanced drug delivery platforms for a variety of bioapplications where high loading and controlled release are strongly desired.

Biodegradation-Resistant Multilayers Coated with Gold Nanoparticles. Toward a Tailor-made Artificial Extracellular Matrix.

Polymer multicomponent coatings such as multilayers mimic an extracellular matrix (ECM) that attracts significant attention for the use of the multilayers as functional supports for advanced cell culture and tissue engineering. Herein, biodegradation and molecular transport in hyaluronan/polylysine multilayers coated with gold nanoparticles were described. Nanoparticle coating acts as a semipermeable barrier that governs molecular transport into/from the multilayers and makes them biodegradation-resistant. Model protein lysozyme (mimics of ECM-soluble signals) diffuses into the multilayers as fast- and slow-diffusing populations existing in an equilibrium. Such a composite system may have high potential to be exploited as degradation-resistant drug-delivery platforms suitable for cell-based applications.

Controlling the Vaterite CaCO3 Crystal Pores. Design of Tailor-Made Polymer Based Microcapsules by Hard Templating.

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.