Advances in preparation and engineering of plant-derived extracellular vesicles for nutrition intervention.

Plant-derived extracellular vesicles (PLEVs), as a type of naturally occurring lipid bilayer membrane structure, represent an emerging delivery vehicle with immense potential due to their ability to encapsulate hydrophobic and hydrophilic compounds, shield them from external environmental stresses, control release, exhibit biocompatibility, and demonstrate biodegradability. This comprehensive review analyzes engineering preparation strategies for natural vesicles, focusing on PLEVs and their purification and surface engineering. Furthermore, it encompasses the latest advancements in utilizing PLEVs to transport active components, serving as a nanotherapeutic system. The prospects and potential development of PLEVs are also discussed. It is anticipated that this work will not only address existing knowledge gaps concerning PLEVs but also provide valuable guidance for researchers in the fields of food science and biomedical studies, stimulating novel breakthroughs in plant-based therapeutic options.

Core-shell nanofibers based on microalgae proteins/alginate complexes for enhancing survivability of probiotics.

In this study, a novel one-step coaxial electrospinning process is employed to fabricate shell-core structure fibers choosing Chlorella pyrenoidosa proteins (CP) as the core material. These nanofibers, serving as the wall material for probiotic encapsulation, aimed to enhance the stability and antioxidant activity of probiotics in food processing, storage, and gastrointestinal environments under sensitive conditions. Morphological analysis was used to explore the beads-on-a-string morphology and core-shell structure of the electrospun fibers. Probiotics were successfully encapsulated within the fibers (7.97 log CFU/g), exhibiting a well-oriented structure along the distributed fibers. Compared to free probiotics and uniaxial fibers loaded with probiotics, encapsulation within microalgae proteins/alginate core-shell structure nanofibers significantly enhanced the probiotic cells' tolerance to simulated gastrointestinal conditions (p < 0.05). Thermal analysis indicated that microalgae proteins/alginate core-shell structure nanofibers displayed superior thermal stability compared to uniaxial fibers. The introduction of CP resulted in a 50 % increase in the antioxidant capacity of probiotics-loaded microalgae proteins/alginate nanofibers compared to uniaxial alginate nanofibers, with minimal loss of viability (0.8 log CFU/g) after 28 days of storage at 4 °C. In summary, this dual-layer carrier holds immense potential in probiotic encapsulation and enhancing their resistance to harsh conditions.

From Nucleation to Fat Crystal Network: Effects of Stearic-Palmitic Sucrose Ester on Static Crystallization of Palm Oil.

Palm oil (PO), a semi-solid fat at room temperature, is a popular food ingredient. To steer the fat functionality, sucrose esters (SEs) are often used as food additives. Many SEs exist, varying in their hydrophilic-to-lipophilic balance (HLB), making them suitable for various food and non-food applications. In this study, a stearic-palmitic sucrose ester with a moderate HLB (6) was studied. It was found that the SE exhibited a complex thermal behavior consistent with smectic liquid crystals (type A). Small-angle X-ray scattering revealed that the mono- and poly-esters of the SE have different packings, more specifically, double and single chain-length packing. The polymorphism encountered upon crystallization was repeatable during successive heating and cooling cycles. After studying the pure SE, it was added to palm oil, and the crystallization behavior of the mixture was compared to that of pure palm oil. The crystallization conditions were varied by applying cooling at 20 °C/min (fast) and 1 °C/min (slow) to 0 °C, 20 °C or 25 °C. The samples were followed for one hour of isothermal time. Differential scanning calorimetry (DSC) showed that nucleation and polymorphic transitions were accelerated. Wide-angle X-ray scattering (WAXS) unraveled that the α-to-ß' polymorphic transition remained present upon the addition of the SE. SAXS showed that the addition of the SE at 0.5 wt% did not significantly change the double chain-length packing of palm oil, but it decreased the domain size when cooling in a fast manner. Ultra-small-angle X-ray scattering (USAXS) revealed that the addition of the SE created smaller crystal nanoplatelets (CNPs). The microstructure of the fat crystal network was visualized by means of polarized light microscopy (PLM) and cryo-scanning electron microscopy (cryo-SEM). The addition of the SE created a finer and space-filling network without the visibility of separate floc structures.

Intestinal-specific oral delivery of lactoferrin with alginate-based composite and hybrid CaCO3-hydrogel beads.

Sodium alginate hydrogel beads and sodium alginate/gellan gum composite hydrogel beads crosslinked by calcium chloride were prepared with different alginate concentrations (3-20 mg·mL-1). Additionally, a simple method for growing CaCO3in situ on the hydrogel to create novel inorganic-organic hybrid hydrogel beads was presented. FT-IR analysis revealed the involvement of hydrogen bonding and electrostatic interactions in bead formation. Swelling behavior in acidic conditions showed a maximum of 13 g/g for composite hydrogels and CaCO3-incorporated hybrid hydrogels. Lactoferrin encapsulation efficiency within these hydrogels ranged from 44.9 to 56.6%. In vitro release experiments demonstrated that these hydrogel beads withstand harsh gastric environments with <16% cumulative release of lactoferrin, achieving controlled release in intestinal surroundings. While composite sodium alginate/gellan gum beads exhibited slower gastrointestinal lactoferrin digestion, facile synthesis and pH responsiveness of CaCO3-incorporated hybrid hydrogel also provide new possibilities for future studies to construct a novel inorganic-organic synergetic system for intestinal-specific oral delivery.

Nanomedicine to aid immunogenic cell death (ICD)-based anticancer therapy.

Immunogenic cell death (ICD) is emerging as a key component of antitumor therapy that harnesses the immune system of the patient to combat cancer. In recent years, several efforts were made to improve the ICD-based therapies. Here, we discuss how nanomaterial-based strategies increase the efficacy of ICD and highlight their benefits and challenges.

Chlamydia psittaci infected cell studies by 4Pi Raman and atomic force microscopy.

Chlamydia psittaci is an avian bacterial pathogen that can cause atypical pneumonia in humans via zoonotic transmission. It is a Gram-negative intracellular bacterium that proliferates inside membrane bound inclusions in the cytoplasm of living eukaryotic cells. The study of such cells with C. psittaci inside without destroying them poses a significant challenge. We demonstrated in this work the utility of a combined multitool approach to analyze such complex samples. Atomic force microscopy was applied to obtain high-resolution images of the surface of infected cells upon entrance of bacteria. Atomic force microscopy scans revealed the morphological changes of the cell membrane of Chlamydia infected cells such as changes in roughness of cell membrane and the presence of micro vesicles. 4Pi Raman microscopy was used to image and probe the molecular composition of intracellular bacteria inside intact cells. Information about the structure of the inclusion produced by C. psittaci was obtained and it was found to have a similar molecular fingerprint as that of an intracellular lipid droplet but with less proteins and unsaturated lipids. The presented approach demonstrates complementarity of various microscopy-based approaches and might be useful for characterization of intracellular bacteria.

Advances in Nanoarchitectonics: A Review of "Static" and "Dynamic" Particle Assembly Methods.

Particle assembly is a promising technique to create functional materials and devices from nanoscale building blocks. However, the control of particle arrangement and orientation is challenging and requires careful design of the assembly methods and conditions. In this study, the static and dynamic methods of particle assembly are reviewed, focusing on their applications in biomaterial sciences. Static methods rely on the equilibrium interactions between particles and substrates, such as electrostatic, magnetic, or capillary forces. Dynamic methods can be associated with the application of external stimuli, such as electric fields, magnetic fields, light, or sound, to manipulate the particles in a non-equilibrium state. This study discusses the advantages and limitations of such methods as well as nanoarchitectonic principles that guide the formation of desired structures and functions. It also highlights some examples of biomaterials and devices that have been fabricated by particle assembly, such as biosensors, drug delivery systems, tissue engineering scaffolds, and artificial organs. It concludes by outlining the future challenges and opportunities of particle assembly for biomaterial sciences. This review stands as a crucial guide for scholars and professionals in the field, fostering further investigation and innovation. It also highlights the necessity for continuous research to refine these methodologies and devise more efficient techniques for nanomaterial synthesis. The potential ramifications on healthcare and technology are substantial, with implications for drug delivery systems, diagnostic tools, disease treatments, energy storage, environmental science, and electronics.

Three-dimensional co-culturing of stem cell-derived cardiomyocytes and cardiac fibroblasts reveals a role for both cell types in Marfan-related cardiomyopathy.

Pathogenic variants in the FBN1 gene, which encodes the extracellular matrix protein fibrillin-1, cause Marfan syndrome (MFS), which affects multiple organ systems, including the cardiovascular system. Myocardial dysfunction has been observed in a subset of patients with MFS and in several MFS mouse models. However, there is limited understanding of the intrinsic consequences of FBN1 variants on cardiomyocytes (CMs). To elucidate the CM-specific contribution in Marfan's cardiomyopathy, cardiosphere cultures of CMs and cardiac fibroblasts (CFs) are used. CMs and CFs were derived by human induced pluripotent stem cell (iPSC) differentiation from MFS iPSCs with a pathogenic variant in FBN1 (c.3725G>A; p.Cys1242Tyr) and the corresponding CRISPR-corrected iPSC line (Cor). Cardiospheres containing MFS CMs show decreased FBN1, COL1A2 and GJA1 expression. MFS CMs cultured in cardiospheres have fewer binucleated CMs in comparison with Cor CMs. 13% of MFS CMs in cardiospheres are binucleated and 15% and 16% in cardiospheres that contain co-cultures with respectively MFS CFs and Cor CFs, compared to Cor CMs, that revealed up to 23% binucleation when co-cultured with CFs. The sarcomere length of CMs, as a marker of development, is significantly increased in MFS CMs interacting with Cor CF or MFS CF, as compared to monocultured MFS CMs. Nuclear blebbing was significantly more frequent in MFS CFs, which correlated with increased stiffness of the nuclear area compared to Cor CFs. Our cardiosphere model for Marfan-related cardiomyopathy identified a contribution of CFs in Marfan-related cardiomyopathy and suggests that abnormal early development of CMs may play a role in the disease mechanism.

Gelatin-Based Hybrid Hydrogels as Matrices for Organoid Culture.

The application of liver organoids is very promising in the field of liver tissue engineering; however, it is still facing some limitations. One of the current major limitations is the matrix in which they are cultured. The mainly undefined and murine-originated tumor matrices derived from Engelbreth-Holm-Swarm (EHS) sarcoma, such as Matrigel, are still the standard culturing matrices for expansion and differentiation of organoids toward hepatocyte-like cells, which will obstruct its future clinical application potential. In this study, we exploited the use of newly developed highly defined hydrogels as potential matrices for the culture of liver organoids and compared them to Matrigel and two hydrogels that were already researched in the field of organoid research [i.e., polyisocyanopeptides, enriched with laminin-entactin complex (PIC-LEC) and gelatin methacryloyl (GelMA)]. The newly developed hydrogels are materials that have a physicochemical resemblance with native liver tissue. Norbornene-modified dextran cross-linked with thiolated gelatin (DexNB-GelSH) has a swelling ratio and macro- and microscale properties that highly mimic liver tissue. Norbornene-modified chondroitin sulfate cross-linked with thiolated gelatin (CSNB-GelSH) contains chondroitin sulfate, which is a glycosaminoglycan (GAG) that is present in the liver ECM. Furthermore, CSNB-GelSH hydrogels with different mechanical properties were evaluated. Bipotent intrahepatic cholangiocyte organoids (ICOs) were applied in this work and encapsulated in these materials. This research revealed that the newly developed materials outperformed Matrigel, PIC-LEC, and GelMA in the differentiation of ICOs toward hepatocyte-like cells. Furthermore, some trends indicate that an interplay of both the chemical composition and the mechanical properties has an influence on the relative expression of certain hepatocyte markers. Both DexNB-GelSH and CSNB-GelSH showed promising results for the expansion and differentiation of intrahepatic cholangiocyte organoids. The stiffest CSNB-GelSH hydrogel even significantly outperformed Matrigel based on ALB, BSEP, and CYP3A4 gene expression, being three important hepatocyte markers.

Optimization of hybrid gelatin-polysaccharide bioinks exploiting thiol-norbornene chemistry using a reducing additive.

Thiol-norbornene chemistry offers great potential in the field of hydrogel development, given its step growth crosslinking mechanism. However, limitations exist with regard to deposition-based bioprinting of thiol-containing hydrogels, associated with premature crosslinking of thiolated (bio)polymers resulting from disulfide formation in the presence of oxygen. More specifically, disulfide formation can result in an increase in viscosity thereby impeding the printing process. In the present work, hydrogels constituting norbornene-modified dextran (DexNB) combined with thiolated gelatin (GelSH) are selected as case study to explore the potential of incorporating the reducing agent tris(2-carboxyethyl)phosphine (TCEP), to prevent the formation of disulfides. We observed that, in addition to preventing disulfide formation, TCEP also contributed to premature, spontaneous thiol-norbornene crosslinking without the use of UV light as evidenced via1H-NMR spectroscopy. Herein, an optimal concentration of 25 mol% TCEP with respect to the amount of thiols was found, thereby limiting auto-gelation by both minimizing disulfide formation and spontaneous thiol-norbornene reaction. This concentration results in a constant viscosity during at least 24 h, a more homogeneous network being formed as evidenced using atomic force microscopy while retaining bioink biocompatibility as evidenced by a cell viability of human foreskin fibroblasts exceeding 70% according to ISO 10993-6:2016.

2D fibrillar osteoid niche mimicry through inclusion of visco-elastic and topographical cues in gelatin-based networks.

Given the clinical need for osteoregenerative materials incorporating controlled biomimetic and biophysical cues, a novel highly-substituted norbornene-modified gelatin was developed enabling thiol-ene crosslinking exploiting thiolated gelatin as cell-interactive crosslinker. Comparing the number of physical crosslinks, the degree of hydrolytic degradation upon modification, the network density and the chemical crosslinking type, the osteogenic effect of visco-elastic and topographical properties was evaluated. This novel network outperformed conventional gelatin-based networks in terms of osteogenesis induction, as evidenced in 2D dental pulp stem cell seeding assays, resulting from the presentation of both a local (substrate elasticity, 25-40 kPa) and a bulk (compressive modulus, 25-45 kPa) osteogenic substrate modulus in combination with adequate fibrillar cell adhesion spacing to optimally transfer traction forces from the fibrillar ECM (as evidenced by mesh size determination with the rubber elasticity theory) and resulting in a 1.7-fold increase in calcium production (compared to the gold standard gelatin methacryloyl (GelMA)).

Alginate-CaCO3 hybrid colloidal hydrogel with tunable physicochemical properties for cell growth.

Biomaterials composed of food polysaccharides are of great interest for future biomedical applications due to their great biocompatibility, tunable mechanical properties, and complex architectural designs that play a crucial role in the modulation of cell adhesion and proliferation. In this work, a facile approach was designed to obtain novel 3D alginate-CaCO3 hybrid hydrogel particles in situ. Controlling the gel concentration from 3 to 20 mg·mL-1 allows us to control the alginate-CaCO3 hydrogel particles' size and density (size variation from 1.86 to 2.34 mm and density from 1.22 to 1.29 mg/mm3). This variable also has a considerable influence on the mineralization process resulting in CaCO3 particles with varied sizes and amounts within the hydrogel beads. The measurements of Young's modulus showed that the inclusion of CaCO3 particles into the alginate hydrogel improved its mechanical properties, and Young's modulus of these hybrid hydrogel particles had a linear relationship with alginate content and hydrogel particle size. Cell experiments indicated that alginate-CaCO3 hybrid hydrogel particles can support osteoblastic cell proliferation and growth. In particular, the amount of hydroxyapatite deposition on the cell membrane significantly increased after the treatment of cells with hybrid hydrogel particles, up to 20-fold. This work offers a strategy for constructing inorganic particle-doped polysaccharide hybrid hydrogel scaffolds that provide the potential to support cell growth.

Mechanobiology of Ferroptotic Cancer Cells as a Novel "Eat-Me" Signal: Regulating Efferocytosis through Layer-by-Layer Coating.

The importance of the clearance of dead cells is shown to have a regulatory role for normal tissue homeostasis and for the modulation of immune responses. However, how mechanobiological properties of dead cells affect efferocytosis remains largely unknown. Here, it is reported that the Young's modulus of cancer cells undergoing ferroptosis is reduced. To modulate their Young's modulus a layer-by-layer (LbL) nanocoating is developed. Scanning electron and fluorescence microscopy confirm coating efficiency of ferroptotic cells while atomic force microscopy reveals encapsulation of the dead cells increases their Young's modulus dependent on the number of applied LbL layers which increases their efferocytosis by primary macrophages. This work demonstrates the crucial role of mechanobiology of dead cells in regulating their efferocytosis by macrophages which can be exploited for the development of novel therapeutic strategies for diseases where modulation of efferocytosis can be potentially beneficial and for the design of drug delivery systems for cancer therapy.

High-throughput mechano-cytometry as a method to detect apoptosis, necroptosis, and ferroptosis.

In recent years, the importance of the investigation of regulated cell death (RCD) has significantly increased and different methods are proposed for the detection of RCD including biochemical as well as fluorescence assays. Researchers have shown that early stages of cell death could be detected by using AFM. Although AFM offers a high single-cell resolution and sensitivity, the throughput (<100 cells/h) limits a broad range of biomedical applications of this technique. Here, a microfluidics-based mechanobiology technique, named shear flow deformability cytometry (sDC), is used to investigate and distinguish dying cells from viable cells purely based on their mechanical properties. Three different RCD modalities (i.e., apoptosis, necroptosis, and ferroptosis) are induced in L929sAhFas cells and analysed using sDC. Using machine learning on the extracted parameters, it was possible to predict the dead or viable state with 92% validation accuracy. A significant decrease in elasticity can be noticed for each of these RCD modalities by analysing the deformation of the dying cells. Analysis of morphological characteristics such as cell size and membrane irregularities also indicated significant differences in the RCD induced cells versus control cells. These results highlight the importance of mechanical properties during RCD and the significance of label-free techniques, such as sDC, which can be used to detect regulated cell death and can be further linked with sorting of live and dead cells.

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.

Cell Behavior Changes and Enzymatic Biodegradation of Hybrid Electrospun Poly(3-hydroxybutyrate)-Based Scaffolds with an Enhanced Piezoresponse after the Addition of Reduced Graphene Oxide.

This is the first comprehensive study of the impact of biodegradation on the structure, surface potential, mechanical and piezoelectric properties of poly(3-hydroxybutyrate) (PHB) scaffolds supplemented with reduced graphene oxide (rGO) as well as cell behavior under static and dynamic mechanical conditions. There is no effect of the rGO addition up to 1.0 wt% on the rate of enzymatic biodegradation of PHB scaffolds for 30 d. The biodegradation of scaffolds leads to the depolymerization of the amorphous phase, resulting in an increase in the degree of crystallinity. Because of more regular dipole order in the crystalline phase, surface potential of all fibers increases after the biodegradation, with a maximum (361 ± 5 mV) after the addition of 1 wt% rGO into PHB as compared to pristine PHB fibers. By contrast, PHB-0.7rGO fibers manifest the strongest effective vertical (0.59 ± 0.03 pm V-1 ) and lateral (1.06 ± 0.02 pm V-1 ) piezoresponse owing to a greater presence of electroactive ß-phase. In vitro assays involving primary human fibroblasts reveal equal biocompatibility and faster cell proliferation on PHB-0.7rGO scaffolds compared to pure PHB and nonpiezoelectric polycaprolactone scaffolds. Thus, the developed biodegradable PHB-rGO scaffolds with enhanced piezoresponse are promising for tissue-engineering applications.

A decade of developing applications exploiting the properties of polyelectrolyte multilayer capsules.

Transferring the layer-by-layer (LbL) coating approach from planar surfaces to spherical templates and subsequently dissolving these templates leads to the fabrication of polyelectrolyte multilayer capsules. The versatility of the coatings of capsules and their flexibility upon bringing in virtually any material into the coatings has quickly drawn substantial attention. Here, we provide an overview of the main developments in this field, highlighting the trends in the last decade. In the beginning, various methods of encapsulation and release are discussed followed by a broad range of applications, which were developed and explored. We also outline the current trends, where the range of applications is continuing to grow, including addition of whole new and different application areas.

Spontaneous shrinkage drives macromolecule encapsulation into layer-by-layer assembled biopolymer microgels.

HYPOTHESIS: Recently, the anomalous shrinkage of surface-supported hyaluronate/poly-l-lysine (HA/PLL) microgels (µ-gels), which exceeds that reported for any other multilayer-based systems, has been reported [1]. The current study investigates the capability of these unique µ-gels for the encapsulation and retention of macromolecules, and proposes the shrinkage-driven assembly of biopolymer-based µ-gels as a novel tool for one-step surface biofunctionalization. EXPERIMENTS: A set of dextrans (DEX) and their charged derivatives - carboxymethyl (CM)-DEX and diethylaminoethyl (DEAE)-DEX - has been utilized to evaluate the effects of macromolecular mass and net charge on µ-gel shrinkage and macromolecule entrapment. µ-gels formation on the surface of silicone catheters exemplifies their potential to tailor biointerfaces. FINDINGS: Shrinkage-driven µ-gel formation strongly depends on the net charge and mass content of encapsulated macromolecules. Inclusion of neutral DEX decreases the degree of shrinkage several times, whilst charged DEXs adopt to the backbone of oppositely charged polyelectrolytes, resulting in shrinkage comparable to that of non-loaded µ-gels. Retention of CM-DEX in µ-gels is significantly higher compared to DEAE-DEX. These insights into the mechanisms of macromolecular entrapment into biopolymer-based µ-gels promotes fundamental understanding of molecular dynamics within the multilayer assemblies. Organization of biodegradable µ-gels at biomaterial surfaces opens avenues for their further exploitation in a diverse array of bioapplications.

Gelatin-Based Hybrid Hydrogel Scaffolds: Toward Physicochemical Liver Mimicry.

There exists a clear need to develop novel materials that could serve liver tissue engineering purposes. Those materials need to be researched for the development of bioengineered liver tissue as an alternative to donor livers, as well as for materials that could be applied for scaffolds to develop an in vitro model for drug-induced liver injury (DILI) detection . In this paper, the hydrogels oxidized dextran-gelatin (Dexox-Gel) and norbornene-modified dextran-thiolated gelatin (DexNB-GelSH) were developed, and their feasibility toward processing via indirect 3D-printing was investigated with the aim to develop hydrogel scaffolds that physicochemically mimic native liver tissue. Furthermore, their in vitro biocompatibility was assessed using preliminary biological tests using HepG2 cells. Both materials were thoroughly physicochemically characterized and benchmarked to the methacrylated gelatin (GelMA) reference material. Due to inferior properties, Dexox-gel was not further processed into 3D-hydrogel scaffolds. This research revealed that DexNB-GelSH exhibited physicochemical properties that were in excellent agreement with the properties of natural liver tissue in contrast to GelMA. In combination with an equally good biological evaluation of DexNB-GelSH in comparison with GelMA based on an MTS proliferation assay and an albumin quantification assay, DexNB-GelSH can be considered promising in the field of liver tissue engineering.

Surface Modification with Particles Coated or Made of Polymer Multilayers.

The coating of particles or decomposable cores with polyelectrolytes via Layer-by-Layer (LbL) assembly creates free-standing LbL-coated functional particles. Due to the numerous functions that their polymers can bestow, the particles are preferentially selected for a plethora of applications, including, but not limited to coatings, cargo-carriers, drug delivery vehicles and fabric enhancements. The number of publications discussing the fabrication and usage of LbL-assembled particles has consistently increased over the last vicennial. However, past literature fails to either mention or expand upon how these LbL-assembled particles immobilize on to a solid surface. This review evaluates examples of LbL-assembled particles that have been immobilized on to solid surfaces. To aid in the formulation of a mechanism for immobilization, this review examines which forces and factors influence immobilization, and how the latter can be confirmed. The predominant forces in the immobilization of the particles studied here are the Coulombic, capillary, and adhesive forces; hydrogen bonding as well as van der Waal's and hydrophobic interactions are also considered. These are heavily dependent on the factors that influenced immobilization, such as the particle morphology and surface charge. The shape of the LbL particle is related to the particle core, whereas the charge was dependant on the outermost polyelectrolyte in the multilayer coating. The polyelectrolytes also determine the type of bonding that a particle can form with a solid surface. These can be via either physical (non-covalent) or chemical (covalent) bonds; the latter enforcing a stronger immobilization. This review proposes a fundamental theory for immobilization pathways and can be used to support future research in the field of surface patterning and for the general modification of solid surfaces with polymer-based nano- and micro-sized polymer structures.