Insights into spheroids formation in cellulose nanofibrils and Matrigel hydrogels using AFM-based techniques.

The recent FDA decision to eliminate animal testing requirements emphasises the role of cell models, such as spheroids, as regulatory test alternatives for investigations of cellular behaviour, drug responses, and disease modelling. The influence of environment on spheroid formation are incompletely understood, leading to uncertainty in matrix selection for scaffold-based 3D culture. This study uses atomic force microscopy-based techniques to quantify cell adhesion to Matrigel and cellulose nanofibrils (CNF), and cell-cell adhesion forces, and their role in spheroid formation of hepatocellular carcinoma (HepG2) and induced pluripotent stem cells (iPS(IMR90)-4). Results showed different cell behaviour in CNF and Matrigel cultures. Both cell lines formed compact spheroids in CNF but loose cell aggregates in Matrigel. Interestingly, the type of cell adhesion protein, and not the bond strength, appeared to be a key factor in the formation of compact spheroids. The gene expression of E- and N-cadherins, proteins on cell membrane responsible for cell-cell interactions, was increased in CNF culture, leading to formation of compact spheroids while Matrigel culture induced integrin-laminin binding and downregulated E-cadherin expression, resulting in looser cell aggregates. These findings enhance our understanding of cell-biomaterial interactions in 3D cultures and offer insights for improved 3D cell models, culture biomaterials, and applications in drug research.

Toward Corneal Limbus In Vitro Model: Regulation of hPSC-LSC Phenotype by Matrix Stiffness and Topography During Cell Differentiation Process.

A functional limbal epithelial stem cells (LSC) niche is a vital element in the regular renewal of the corneal epithelium by LSCs and maintenance of good vision. However, little is known about its unique structure and mechanical properties on LSC regulation, creating a significant gap in development of LSC-based therapies. Herein, the effect of mechanical and architectural elements of the niche on human pluripotent derived LSCs (hPSC-LSC) phenotype and growth is investigated in vitro. Specifically, three formulations of polyacrylamide gels with different controlled stiffnesses are used for culture and characterization of hPSC-LSCs from different stages of differentiation. In addition, limbal mimicking topography in polydimethylsiloxane is utilized for culturing hPSC-LSCs at early time point of differentiation. For comparison, the expression of selected key proteins of the corneal cells is analyzed in their native environment through whole mount staining of human donor corneas. The results suggest that mechanical response and substrate preference of the cells is highly dependent on their developmental stage. In addition, data indicate that cells may carry possible mechanical memory from previous culture matrix, both highlighting the importance of mechanical design of a functional in vitro limbus model.

Tuning the water interactions of cellulose nanofibril hydrogels using willow bark extract.

Cellulose nanofibrils (CNFs) are increasingly used as precursors for foams, films and composites, where water interactions are of great importance. In this study, we used willow bark extract (WBE), an underrated natural source of bioactive phenolic compounds, as a plant-based modifier for CNF hydrogels, without compromising their mechanical properties. We found that the introduction of WBE into both native, mechanically fibrillated CNFs and TEMPO-oxidized CNFs increased considerably the storage modulus of the hydrogels and reduced their swelling ratio in water up to 5-7 times. A detailed chemical analysis revealed that WBE is composed of several phenolic compounds in addition to potassium salts. Whereas the salt ions reduced the repulsion between fibrils and created denser CNF networks, the phenolic compounds - which adsorbed readily on the cellulose surfaces - played an important role in assisting the flowability of the hydrogels at high shear strains by reducing the flocculation tendency, often observed in pure and salt-containing CNFs, and contributed to the structural integrity of the CNF network in aqueous environment. Surprisingly, the willow bark extract exhibited hemolysis activity, which highlights the importance of more thorough investigations of biocompatibility of natural materials. WBE shows great potential for managing the water interactions of CNF-based products.

Biobased Nanomaterials─The Role of Interfacial Interactions for Advanced Materials.

This review presents recent advances regarding biomass-based nanomaterials, focusing on their surface interactions. Plant biomass-based nanoparticles, like nanocellulose and lignin from industry side streams, hold great potential for the development of lightweight, functional, biodegradable, or recyclable material solutions for a sustainable circular bioeconomy. However, to obtain optimal properties of the nanoparticles and materials made thereof, it is crucial to control the interactions both during particle production and in applications. Herein we focus on the current understanding of these interactions. Solvent interactions during particle formation and production, as well as interactions with water, polymers, cells and other components in applications, are addressed. We concentrate on cellulose and lignin nanomaterials and their combination. We demonstrate how the surface chemistry of the nanomaterials affects these interactions and how excellent performance is only achieved when the interactions are controlled. We furthermore introduce suitable methods for probing interactions with nanomaterials, describe their advantages and challenges, and introduce some less commonly used methods and discuss their possible applications to gain a deeper understanding of the interfacial chemistry of biobased nanomaterials. Finally, some gaps in current understanding and interesting emerging research lines are identified.

Affinity of Keratin Peptides for Cellulose and Lignin: A Fundamental Study toward Advanced Bio-Based Materials.

Keratin is a potential raw material to meet the growing demand for bio-based materials with special properties. Keratin can be obtained from feathers, a by-product from the poultry industry. One approach for keratin valorization is to use the protein to improve the properties of already existing cellulose and lignin-based materials to meet the requirements for replacing fossil-based plastics. To ensure a successful combination of keratin with lignocellulosic building blocks, keratin must have an affinity to these substrates. Hence, we used quartz crystal microbalance with a dissipation monitoring (QCM-D) technique to get a detailed understanding of the adsorption of keratin peptides onto lignocellulosic substrates and how the morphology of the substrate, pH, ionic strength, and keratin properties affected the adsorption. Keratin was fractionated from feathers with a scalable and environmentally friendly deep eutectic solvent process. The keratin fraction used in the adsorption studies consisted of different sized keratin peptides (about 1-4 kDa), which had adopted a random coil conformation as observed by circular dichroism (CD). Measuring keratin adsorption to different lignocellulosic substrates by QCM-D revealed a significant affinity of keratin peptides for lignin, both as smooth films and in the form of nanoparticles but only a weak interaction between cellulose and keratin. Systematic evaluation of the effect of surface, media, and protein properties enabled us to obtain a deeper understanding of the driving force for adsorption. Both the structure and size of the keratin peptides appeared to play an important role in its adsorption. The keratin-lignin combination is an attractive option for advanced material applications. For improved adsorption on cellulose, modifications of either keratin or cellulose would be required.

Biological activity of multicomponent bio-hydrogels loaded with tragacanth gum.

Producing hydrogels capable of mimicking the biomechanics of soft tissue remains a challenge. We explore the potential of plant-based hydrogels as polysaccharide tragacanth gum and antioxidant lignin nanoparticles in bioactive multicomponent hydrogels for tissue engineering. These natural components are combined with TEMPO-oxidized cellulose nanofibrils, a material with known shear thinning behavior. Hydrogels presented tragacanth gum (TG) concentration-dependent rheological properties suitable for extrusion 3D printing. TG enhanced the swelling capacity up to 645% and the degradation rate up to 1.3%/day for hydrogels containing 75% of TG. Young's moduli of the hydrogels varied from 5.0 to 11.6 kPa and were comparable to soft tissues like skin and muscle. In vitro cell viability assays revealed that the scaffolds were non-toxic and promoted proliferation of hepatocellular carcinoma HepG2 cells. Therefore, the plant-based hydrogels designed in this work have a significant potential for tissue engineering.

Synthesis of an Azide- and Tetrazine-Functionalized [60]Fullerene and Its Controlled Decoration with Biomolecules.

Bingel cyclopropanation between Buckminster fullerene and a heteroarmed malonate was utilized to produce a hexakis-functionalized C60 core, with azide and tetrazine units. This orthogonally bifunctional C60 scaffold can be selectively one-pot functionalized by two pericyclic click reactions, that is, inverse electron-demand Diels-Alder and azide-alkyne cycloaddition, which with appropriate ligands (monosaccharides, a peptide and oligonucleotides tested) allows one to control the assembly of heteroantennary bioconjugates.

Experimental and Simulation Study of the Solvent Effects on the Intrinsic Properties of Spherical Lignin Nanoparticles.

Spherical lignin nanoparticles (LNPs) fabricated via nanoprecipitation of dissolved lignin are among the most attractive biomass-derived nanomaterials. Despite various studies exploring the methods to improve the uniformity of LNPs or seeking more application opportunities for LNPs, little attention has been given to the fundamental aspects of the solvent effects on the intrinsic properties of LNPs. In this study, we employed a variety of experimental techniques and molecular dynamics (MD) simulations to investigate the solvent effects on the intrinsic properties of LNPs. The LNPs were prepared from softwood Kraft lignin (SKL) using the binary solvents of aqueous acetone or aqueous tetrahydrofuran (THF) via nanoprecipitation. The internal morphology, porosity, and mechanical properties of the LNPs were analyzed with electron tomography (ET), small-angle X-ray scattering (SAXS), atomic force microscopy (AFM), and intermodulation AFM (ImAFM). We found that aqueous acetone resulted in smaller LNPs with higher uniformity compared to aqueous THF, mainly ascribing to stronger solvent-lignin interactions as suggested by MD simulation results and confirmed with aqueous 1,4-dioxane (DXN) and aqueous dimethyl sulfoxide (DMSO). More importantly, we report that both LNPs were compact particles with relatively homogeneous density distribution and very low porosity in the internal structure. The stiffness of the particles was independent of the size, and the Young's modulus was in the range of 0.3-4 GPa. Overall, the fundamental understandings of LNPs gained in this study are essential for the design of LNPs with optimal performance in applications.

Cytokeratin 5 determines maturation of the mammary myoepithelium.

At invasion, transformed mammary epithelial cells expand into the stroma through a disrupted myoepithelial (ME) cell layer and basement membrane (BM). The intact ME cell layer has thus been suggested to act as a barrier against invasion. Here, we investigate the mechanisms behind the disruption of ME cell layer. We show that the expression of basal/ME proteins CK5, CK14, and α-SMA altered along increasing grade of malignancy, and their loss affected the maintenance of organotypic 3D mammary architecture. Furthermore, our data suggests that loss of CK5 prior to invasive stage causes decreased levels of Zinc finger protein SNAI2 (SLUG), a key regulator of the mammary epithelial cell lineage determination. Consequently, a differentiation bias toward luminal epithelial cell type was detected with loss of mature, α-SMA-expressing ME cells and reduced deposition of basement membrane protein laminin-5. Therefore, our data discloses the central role of CK5 in mammary epithelial differentiation and maintenance of normal ME layer.

Effect of laminin, polylysine and cell medium components on the attachment of human hepatocellular carcinoma cells to cellulose nanofibrils analyzed by surface plasmon resonance.

The development of in vitro cell models that mimic cell behavior in organs and tissues is an approach that may have remarkable impact on drug testing and tissue engineering applications in the future. Plant-based, chemically unmodified cellulose nanofibrils (CNF) hydrogel is a natural, abundant, and biocompatible material that has attracted great attention for biomedical applications, in particular for three-dimensional cell cultures. However, the mechanisms of cell-CNF interactions and factors that affect these interactions are not yet fully understood. In this work, multi-parametric surface plasmon resonance (SPR) was used to study how the adsorption of human hepatocellular carcinoma (HepG2) cells on CNF films is affected by the different proteins and components of the cell medium. Both human recombinant laminin-521 (LN-521, a natural protein of the extracellular matrix) and poly-l-lysine (PLL) adsorbed on CNF films and enhanced the attachment of HepG2 cells. Cell medium components (glucose and amino acids) and serum proteins (fetal bovine serum, FBS) also adsorbed on both bare CNF and on protein-coated CNF substrates. However, the adsorption of FBS hindered the attachment of HepG2 cells to LN-521- and PLL-coated CNF substrates, suggesting that serum proteins blocked the formation of laminin-integrin bonds and decreased favorable PLL-cell electrostatic interactions. This work sheds light on the effect of different factors on cell attachment to CNF, paving the way for the utilization and optimization of CNF-based materials for different tissue engineering applications.

Well-Defined Lignin Model Films from Colloidal Lignin Particles.

The transformation of a molecularly complex and irregularly shaped lignin into a nanoscale spherical architecture is anticipated to play a pivotal role in the promotion of lignin valorization. From the standpoint of using colloidal lignin particles (CLPs) as building blocks for a diverse range of applications, it has become essential to study their interactions with soluble compounds of varied origin. However, the lack of model films with well-defined surface properties similar to those of CLPs has hindered fundamental studies using surface-sensitive techniques. Here, we report well-defined and stable thin films prepared from CLPs and demonstrate their suitability for investigation of surface phenomena. Direct adsorption on substrates coated with a cationic anchoring polymer resulted in uniform distribution of CLPs as shown with atomic force microscopy (AFM). Quartz crystal microbalance with dissipation monitoring (QCM-D) experiments revealed higher adsorbed mass of cationic lignin onto the CLP-coated substrate in comparison to the film prepared from dissolved lignin, suggesting preferential adsorption via the carboxylic acid enriched surfaces of CLPs. QCM-D further enabled detection of small changes such as particle swelling or partial dissolution not detectable via bulk methods such as light scattering. The CLP thin films remained stable until pH 8 and displayed only a low degree of swelling. Increasing the pH to 10 led to some instability, but their spherical geometry remained intact until complete dissolution was observed at pH 12. Particles prepared from aqueous acetone or aqueous tetrahydrofuran solution followed similar trends regarding adsorption, pH stability, and wetting, although the particle size affected the magnitude of adsorption. Overall, our results present a practical way to prepare well-defined CLP thin films that will be useful not only for fundamental studies but also as a platform for testing stability and interactions of lignin nanoparticles with materials of technical and biomedical relevance.

Author Correction: Quantified forces between HepG2 hepatocarcinoma and WA07 pluripotent stem cells with natural biomaterials correlate with in vitro cell behavior.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Three-Dimensional Printed Cell Culture Model Based on Spherical Colloidal Lignin Particles and Cellulose Nanofibril-Alginate Hydrogel.

Three-dimensional (3D) printing has been an emerging technique to fabricate precise scaffolds for biomedical applications. Cellulose nanofibril (CNF) hydrogels have attracted considerable attention as a material for 3D printing because of their shear-thinning properties. Combining cellulose nanofibril hydrogels with alginate is an effective method to enable cross-linking of the printed scaffolds in the presence of Ca2+ ions. In this work, spherical colloidal lignin particles (CLPs, also known as spherical lignin nanoparticles) were used to prepare CNF-alginate-CLP nanocomposite scaffolds. High-resolution images obtained by atomic force microscopy (AFM) showed that CLPs were homogeneously mixed with the CNF hydrogel. CLPs brought antioxidant properties to the CNF-alginate-CLP scaffolds in a concentration-dependent manner and increased the viscosity of the hydrogels at a low shear rate, which correspondingly provide better shape fidelity and printing resolution to the scaffolds. Interestingly, the CLPs did not affect the viscosity at high shear rates, showing that the shear thinning behavior typical for CNF hydrogels was retained, enabling easy printing. The CNF-alginate-CLP scaffolds demonstrated shape stability after printing, cross-linking, and storage in Dulbecco's phosphate buffer solution (DPBS +) containing Ca2+ and Mg2+ ions, up to 7 days. The 3D-printed scaffolds showed relative rehydration ratio values above 80% after freeze-drying, demonstrating a high water-retaining capability. Cell viability tests using hepatocellular carcinoma cell line HepG2 showed no negative effect of CLPs on cell proliferation. Fluorescence microscopy indicated that HepG2 cells grew not only on the surfaces but also inside the porous scaffolds. Overall, our results demonstrate that nanocomposite CNF-alginate-CLP scaffolds have high potential in soft-tissue engineering and regenerative-medicine applications.

AFM Force Spectroscopy Reveals the Role of Integrins and Their Activation in Cell-Biomaterial Interactions.

Transmembrane protein integrins play a key role in cell adhesion. Cell-biomaterial interactions are affected by integrin expression and conformation, which are actively controlled by cells. Although integrin structure and function have been studied in detail, quantitative analyses of integrin-mediated cell-biomaterial interactions are still scarce. Here, we have used atomic force spectroscopy to study how integrin distribution and activation (via intracellular mechanisms in living cells or by divalent cations) affect the interaction of human pluripotent stem cells (WA07) and human hepatocarcinoma cells (HepG2) with promising biomaterials -human recombinant laminin-521 (LN-521) and cellulose nanofibrils (CNF). Cell adhesion to LN-521-coated probes was remarkably influenced by cell viability, divalent cations, and integrin density in WA07 colonies, indicating that specific bonds between LN-521 and activated integrins play a significant role in the interactions between LN-521 and HepG2 and WA07 cells. In contrast, the interactions between CNF and cells were nonspecific and not influenced by cell viability or the presence of divalent cations. These results shed light on the underlying mechanisms of cell adhesion, with direct impact on cell culture and tissue engineering applications.

Understanding hemicellulose-cellulose interactions in cellulose nanofibril-based composites.

Plant-based polysaccharides (cellulose and hemicellulose) are a very interesting option for the preparation of sustainable composite materials to replace fossil plastics, but the optimum bonding mechanism between the hard and soft components is still not well known. In this work, composite films made of cellulose nanofibrils (CNF) and various modified and unmodified polysaccharides (galactoglucomannan, GGM; hydrolyzed and oxidized guar gum, GGhydHox; and guar gum grafted with polyethylene glycol, GG-g-PEG) were characterized from the nano- to macroscopic level to better understand how the interactions between the composite components at nano/microscale affect macroscopic mechanical properties, like toughness and strength. All the polysaccharides studied adsorbed well on CNF, although with different adsorption rates, as measured by quartz crystal microbalance with dissipation monitoring (QCM-D). Direct surface and friction force experiments using the colloidal probe technique revealed that the adsorbed polysaccharides provided repulsive forces-well described by a polyelectrolyte brush model - and a moderate reduction in friction between cellulose surfaces, which may prevent CNF aggregates during composite formation and, consequently, enhance the strength of dry films. High affinity for cellulose and moderate hydration were found to be important requirements for polysaccharides to improve the mechanical properties of CNF-based composites in wet conditions. The results of this work provide fundamental information on hemicellulose-cellulose interactions and can support the development of polysaccharide-based materials for different packaging and medical applications.

Quantified forces between HepG2 hepatocarcinoma and WA07 pluripotent stem cells with natural biomaterials correlate with in vitro cell behavior.

In vitro cell culture or tissue models that mimic in vivo cellular response have potential in tissue engineering and regenerative medicine, and are a more economical and accurate option for drug toxicity tests than animal experimentation. The design of in vivo-like cell culture models should take into account how the cells interact with the surrounding materials and how these interactions affect the cell behavior. Cell-material interactions are furthermore important in cancer metastasis and tumor progression, so deeper understanding of them can support the development of new cancer treatments. Herein, the colloidal probe microscopy technique was used to quantify the interactions of two cell lines (human pluripotent stem cell line WA07 and human hepatocellular carcinoma cell line HepG2) with natural, xeno-free biomaterials of different chemistry, morphology, and origin. Key components of extracellular matrices -human collagens I and IV, and human recombinant laminin-521-, as well as wood-derived, cellulose nanofibrils -with evidenced potential for 3D cell culture and tissue engineering- were analysed. Both strength of adhesion and force curve profiles depended on biomaterial nature and cell characteristics. The successful growth of the cells on a particular biomaterial required cell-biomaterial adhesion energies above 0.23 nJ/m. The information obtained in this work supports the development of new materials or hybrid scaffolds with tuned cell adhesion properties for tissue engineering, and provides a better understanding of the interactions of normal and cancerous cells with biomaterials in the human body.

Micelle carriers based on dendritic macromolecules containing bis-MPA and glycine for antimalarial drug delivery.

Biomaterials for antimalarial drug transport still need to be investigated in order to attain nanocarriers that can tackle essential issues related to malaria treatment, e.g. complying with size requirements and targeting specificity for their entry into Plasmodium-infected red blood cells (pRBCs), and limiting premature drug elimination or drug resistance evolution. Two types of dendritic macromolecule that can form vehicles suitable for antimalarial drug transport are herein explored. A new hybrid dendritic-linear-dendritic block copolymer based on Pluronic® F127 and amino terminated 2,2'-bis(glycyloxymethyl)propionic acid dendrons with a poly(ester amide) skeleton (HDLDBC-bGMPA) and an amino terminated dendronized hyperbranched polymer with a polyester skeleton derived from 2,2'-bis(hydroxymethyl)propionic acid (DHP-bMPA) have provided self-assembled and unimolecular micelles. Both types of micelle carrier are biocompatible and exhibit appropriate sizes to enter into pRBCs. Targeting studies have revealed different behaviors for each nanocarrier that may open new perspectives for antimalarial therapeutic approaches. Whereas DHP-bMPA exhibits a clear targeting specificity for pRBCs, HDLDBC-bGMPA is incorporated by all erythrocytes. It has also been observed that DHP-bMPA and HDLDBC-bGMPA incorporate into human umbilical vein endothelial cells with different subcellular localization, i.e. cytosolic and nuclear, respectively. Drug loading capacity and encapsulation efficiencies for the antimalarial compounds chloroquine, primaquine and quinacrine ranging from 30% to 60% have been determined for both carriers. The resulting drug-loaded nanocarriers have been tested for their capacity to inhibit Plasmodium growth in in vitro and in vivo assays.

Quantifying the interactions between biomimetic biomaterials - collagen I, collagen IV, laminin 521 and cellulose nanofibrils - by colloidal probe microscopy.

Biomaterials of different nature have been and are widely studied for various biomedical applications. In many cases, biomaterial assemblies are designed to mimic biological systems. Although biomaterials have been thoroughly characterized in many aspects, not much quantitative information on the molecular level interactions between different biomaterials is available. That information is very important, on the one hand, to understand the properties of biological systems and, on the other hand, to develop new composite biomaterials for special applications. This work presents a systematic, quantitative analysis of self- and cross-interactions between films of collagen I (Col I), collagen IV (Col IV), laminin (LN-521), and cellulose nanofibrils (CNF), that is, biomaterials of different nature and structure that either exist in biological systems (e.g., extracellular matrices) or have shown potential for 3D cell culture and tissue engineering. Direct surface forces and adhesion between biomaterials-coated spherical microparticles and flat substrates were measured in phosphate-buffered saline using an atomic force microscope and the colloidal probe technique. Different methods (Langmuir-Schaefer deposition, spin-coating, or adsorption) were applied to completely coat the flat substrates and the spherical microparticles with homogeneous biomaterial films. The adhesion between biomaterials films increased with the time that the films were kept in contact. The strongest adhesion was observed between Col IV films, and between Col IV and LN-521 films after 30 s contact time. In contrast, low adhesion was measured between CNF films, as well as between CNF and LN-521 films. Nevertheless, a good adhesion between CNF and collagen films (especially Col I) was observed. These results increase our understanding of the structure of biological systems and can support the design of new matrices or scaffolds where different biomaterials are combined for diverse biological or medical applications.

Colloidal Lignin Particles as Adhesives for Soft Materials.

Lignin has interesting functionalities to be exploited in adhesives for medicine, foods and textiles. Nanoparticles (NPs) < 100 nm coated with poly (L-lysine), PL and poly(L-glutamic acid) PGA were prepared from the laccase treated lignin to coat nanocellulose fibrils (CNF) with heat. NPs ca. 300 nm were prepared, ß-casein coated and cross-linked with transglutaminase (Tgase) to agglutinate chamois. Size exclusion chromatography (SEC) and Fourier-transform infrared (FTIR) spectroscopy were used to characterize polymerized lignin, while zeta potential and dynamic light scattering (DLS) to ensure coating of colloidal lignin particles (CLPs). Protein adsorption on lignin was studied by quartz crystal microbalance (QCM). Atomic force microscopy (AFM) was exploited to examine interactions between different polymers and to image NPs with transmission electron microscopy (TEM). Tensile testing showed, when using CLPs for the adhesion, the stress improved ca. 10 and strain ca. 6 times compared to unmodified Kraft. For the ß-casein NPs, the values were 20 and 8, respectively, and for the ß-casein coated CLPs between these two cases. When NPs were dispersed in adhesive formulation, the increased Young's moduli confirmed significant improvement in the stiffness of the joints over the adhesive alone. Exploitation of lignin in nanoparticulate morphology is a potential method to prepare bionanomaterials for advanced applications.

Polyamidoamine Nanoparticles for the Oral Administration of Antimalarial Drugs.

Current strategies for the mass administration of antimalarial drugs demand oral formulations to target the asexual Plasmodium stages in the peripheral bloodstream, whereas recommendations for future interventions stress the importance of also targeting the transmission stages of the parasite as it passes between humans and mosquitoes. Orally administered polyamidoamine (PAA) nanoparticles conjugated to chloroquine reached the blood circulation and cured Plasmodium yoelii-infected mice, slightly improving the activity of the free drug and inducing in the animals immunity against malaria. Liquid chromatography with tandem mass spectrometry analysis of affinity chromatography-purified PAA ligands suggested a high adhesiveness of PAAs to Plasmodium falciparum proteins, which might be the mechanism responsible for the preferential binding of PAAs to Plasmodium-infected erythrocytes vs. non-infected red blood cells. The weak antimalarial activity of some PAAs was found to operate through inhibition of parasite invasion, whereas the observed polymer intake by macrophages indicated a potential of PAAs for the treatment of certain coinfections such as Plasmodium and Leishmania. When fluorescein-labeled PAAs were fed to females of the malaria mosquito vectors Anopheles atroparvus and Anopheles gambiae, persistent fluorescence was observed in the midgut and in other insect's tissues. These results present PAAs as a versatile platform for the encapsulation of orally administered antimalarial drugs and for direct administration of antimalarials to mosquitoes, targeting mosquito stages of Plasmodium.