Design of Starch-<i>graft</i>-PEI Polymers: An Effective and Biodegradable Gene Delivery Platform

Starch and starch derivatives are widely utilized pharmaceutical excipients. The concept of this study was to make use of starch as a biodegradable backbone and to modify it with low-toxic, but poor transfecting low molecular weight polyethylenimine (PEI) in order to achieve better transfection efficacy while maintaining enzymatic degradability. A sufficiently controllable conjugation could be achieved via a water-soluble intermediate of oxidized starch and an optimized reaction protocol. Systematic variation of MW fraction of the starch backbone and the amount of cationic side chains (0.8 kDa bPEI) yielded a series of starch-<i>graft</i>-PEI copolymers. Following purification and chemical characterization, nanoscale complexes with plasmid DNA were generated and studied regarding cytotoxicity and transfection efficacy. The optimal starch-<i>graft</i>-PEI polymers consisted of >100 kDa MW starch and contained 30% (wt) of PEI, showing similar transfection levels as 25 kDa bPEI, and being less cytotoxic and enzymatically biodegradable

Self-Assembly and Shape Control of Hybrid Nanocarriers Based on Calcium Carbonate and Carbon Nanodots

We describe a platform for the synthesis of functional hybrid nanoparticles in the submicrometer range with tailorable anisotropic morphology. Fluorescent carbon dots (CDs) and poly­(acrylic acid) (PAA) are used to modify the crystallization and assembly of calcium carbonate (CaCO₃). Carboxylic groups on CDs sequester calcium ions and serve as templates for CaCO₃ precipitation when carbonate is added. This creates primary CaCO₃ nanoparticles, 7 nm in diameter, which self-assemble into spheres or rods depending on the PAA concentration. At increasing polymer concentration, oriented assembly becomes more prevalent yielding rod-like particles. The hybrid particles show colloidal stability in cell medium and absence of cytotoxicity as well as a loading efficiency of around 30% for Rhodamine B with pH-controlled release. Given the morphological control, simplicity of synthesis, and efficient loading capabilities the CD-CaCO₃ system could serve as a novel platform for advanced drug carrier systems

One-Step Synthesis of Nanosized and Stable Amino-Functionalized Calcium Phosphate Particles for DNA Transfection

Calcium phosphate (CaP) is used for in vitro transfection because of low toxicity and simple and low cost synthesis. The transfection results however vary because the precipitation lacks reproducibility and results in polydispersed, agglomerated particles. Here a reproducible, one-step procedure for the preparation of amino-modified CaP nanoparticles (NPs) is described using <i>N</i>-(2-aminoethyl)-3-aminopropyltrimethoxysilane as modifying and dispersing agent. The aim was to produce homogeneous, stable CaP NPs, which are loaded with DNA after particle formation. The refined wet-precipitation method yielded NPs with a narrow size distribution (∼140 nm) and positive zeta potential at physiological pH. FTIR and Raman spectroscopy analysis verified the aminosilane modification. Interestingly two types of CaP crystalline structures, Brushite and Hydroxyapatite, can be produced depending on the pH and without hydrothermal treatment. Both CaP crystalline phases were characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), and Brunauer–Emmett–Teller (BET) analysis. Both showed very low toxicity and enabled reproducible transfection of A549 cells. The higher surface functionalization density of Brushite NPs led to superior pDNA condensation capability and higher transfection in lower NP concentration. The advantages of the improved synthesis are the achievement of stable, crystalline CaP NPs in higher yield and narrow distributed size achieved by agglomeration reduction even without hazardous surfactant

Transfection System of Amino-Functionalized Calcium Phosphate Nanoparticles: In Vitro Efficacy, Biodegradability, and Immunogenicity Study

Many methods have been developed in order to use calcium phosphate (CaP) for delivering nucleotides into living cells. Surface functionalization of CaP nanoparticles (CaP NPs) with N-(2-aminoethyl)-3-aminopropyltrimethoxysilane was shown recently to achieve dispersed NPs with a positive surface charge, capable of transfection (<i>Chem. Mater.</i> <b>2013</b>, <i>25</i> (18), 3667). In this study, different crystal structures of amino-modified CaP NPs (brushite and hydroxyapatite) were investigated for their interaction in cell culture systems in more detail. Qualitative (confocal laser scanning microscopy) and quantitative (flow cytometry) transfection experiments with two cell lines showed the higher transfection efficacy of brushite versus hydroxyapatite. The transfection also revealed a cell type dependency. HEK293 cells were easier to transfect compared to A549 cells. This result was supported by the cytotoxicity results. A549 cells showed a higher degree of tolerance toward the CaP NPs. Further, the impact of the surface modification on the interaction with macrophages and complement as two important components of the innate immune system were considered. The amine surface functionalization had an effect of decreasing the release of proinflammatory cytokines. The complement interaction investigated by a C3a complement activation assay did show no significant differences between CaP NPs without or with amine modification and overall weak interaction. Finally, the degradation of CaP NPs in biological media was studied with respect to the two crystal structures and at acidic and neutral pH. Both amino-modified CaP NPs disintegrate within days at neutral pH, with a notable faster disintegration of brushite NPs at acidic pH. In summary, the fair transfection capability of this amino functionalized CaP NPs together with the excellent biocompatibility, biodegradability, and low immunogenicity make them interesting candidates for further evaluation

Size-Limited Penetration of Nanoparticles into Porcine Respiratory Mucus after Aerosol Deposition

We investigated the rheological properties and the penetration of differently sized carboxylated nanoparticles in pig pulmonary mucus, on different distance and time scales. Nanoparticles were either mechanically mixed into the mucus samples or deposited as an aerosol, the latter resembling a more physiologically relevant delivery scenario. After mechanical dispersion, 500 nm particles were locally trapped; a fraction of carboxylated tracer particles of 100 or 200 nm in diameter could however freely diffuse in these networks over distances of approximately 20 μm. In contrast, after aerosol deposition on top of the mucus layer only particles with a size of 100 nm were able to penetrate into mucus, suggesting the presence of smaller pores at the air-mucus interface compared to within mucus. These findings are relevant to an understanding of the fate of potentially harmful aerosol particles, such as pathogens, pollutants, and other nanomaterials after incidental inhalation, as well as for the design of pulmonary drug delivery systems

Size-Limited Penetration of Nanoparticles into Porcine Respiratory Mucus after Aerosol Deposition

We investigated the rheological properties and the penetration of differently sized carboxylated nanoparticles in pig pulmonary mucus, on different distance and time scales. Nanoparticles were either mechanically mixed into the mucus samples or deposited as an aerosol, the latter resembling a more physiologically relevant delivery scenario. After mechanical dispersion, 500 nm particles were locally trapped; a fraction of carboxylated tracer particles of 100 or 200 nm in diameter could however freely diffuse in these networks over distances of approximately 20 μm. In contrast, after aerosol deposition on top of the mucus layer only particles with a size of 100 nm were able to penetrate into mucus, suggesting the presence of smaller pores at the air-mucus interface compared to within mucus. These findings are relevant to an understanding of the fate of potentially harmful aerosol particles, such as pathogens, pollutants, and other nanomaterials after incidental inhalation, as well as for the design of pulmonary drug delivery systems

Nanoparticle Geometry and Surface Orientation Influence Mode of Cellular Uptake

In order to engineer safer nanomaterials, there is a need to understand, systematically evaluate, and develop constructs with appropriate cellular uptake and intracellular fates. The overall goal of this project is to determine the uptake patterns of silica nanoparticle geometries in model cells, in order to aid in the identification of the role of geometry on cellular uptake and transport. In our experiments we observed a significant difference in the viability of two phenotypes of primary macrophages; immortalized macrophages exhibited similar patterns. However, both primary and immortalized epithelial cells did not exhibit toxicity profiles. Interestingly uptake of these geometries in all cell lines exhibited very different time-dependent patterns. A screening of a series of chemical inhibitors of endocytosis was performed to isolate the uptake mechanisms of the different particles. The results show that all geometries exhibit very different uptake profiles and that this may be due to the orientation of the nanoparticles when they interact with the cell surface. Additionally, evidence suggests that these uptake patterns initialize different downstream cellular pathways, dependent on cell type and phenotype

Squalenoylation of Chitosan: A Platform for Drug Delivery?

The present study describes the synthesis of chitosan-squalene (chitosan-SQ), a unique amphiphilic chitosan derivative, which enables the efficient formation of nanoparticles in acetate buffer by self-assembly. The influence of different parameters on the nanoparticle size such as percentage of substitution, pH of the acetate buffer, concentration in chitosan-SQ, and time of stirring was studied. It could be demonstrated that this new polymer was nontoxic to cells, biodegradable, and preserved the anti-infective properties of the initial chitosan. Moreover, chitosan-SQ showed good carrier properties by allowing the encapsulation of both hydrophilic and hydrophobic model drug compounds

Atomic Force Microscopy and Analytical Ultracentrifugation for Probing Nanomaterial Protein Interactions

Upon contact with the human body, nanomaterials are known to interact with the physiological surroundings, especially with proteins. In this context, we explored analytical methods to provide biologically relevant information, in particular for manufactured nanomaterials as produced by the chemical industry. For this purpose, we selected two batches of SiO₂ nanoparticles as well as four batches of CeO₂ nanoparticles, each of comparably high chemical purity and similar physicochemical properties. Adsorption of serum proteins and bovine serum albumin (BSA) was quantified by SDS-PAGE in combination with densitometry and further investigated by atomic force microscopy (AFM) and analytical ultracentrifugation (AUC). The protein adsorption to SiO₂ nanoparticles was below the limit of detection, regardless of adjusting pH or osmolality to physiological conditions. In contrast, the four CeO₂ nanomaterials could be classified in two groups according to half-maximal protein adsorption. Measuring the work of adhesion and indention by AFM for the BSA-binding CeO₂ nanomaterials revealed the same classification, pointing to alterations in shape of the adsorbed protein. The same trend was also reflected in the agglomeration behavior/dispersibility of the four CeO₂ nanomaterials as revealed by AUC. We conclude that even small differences in physicochemical particle properties may nevertheless lead to differences in protein adsorption, possibly implicating a different disposition and other biological responses in the human body. Advanced analytical methods such as AFM and AUC may provide valuable additional information in this context

Proteomic and Lipidomic Analysis of Nanoparticle Corona upon Contact with Lung Surfactant Reveals Differences in Protein, but Not Lipid Composition

Pulmonary surfactant (PS) constitutes the first line of host defense in the deep lung. Because of its high content of phospholipids and surfactant specific proteins, the interaction of inhaled nanoparticles (NPs) with the pulmonary surfactant layer is likely to form a corona that is different to the one formed in plasma. Here we present a detailed lipidomic and proteomic analysis of NP corona formation using native porcine surfactant as a model. We analyzed the adsorbed biomolecules in the corona of three NP with different surface properties (PEG-, PLGA-, and Lipid-NP) after incubation with native porcine surfactant. Using label-free shotgun analysis for protein and LC–MS for lipid analysis, we quantitatively determined the corona composition. Our results show a conserved lipid composition in the coronas of all investigated NPs regardless of their surface properties, with only hydrophilic PEG-NPs adsorbing fewer lipids in total. In contrast, the analyzed NP displayed a marked difference in the protein corona, consisting of up to 417 different proteins. Among the proteins showing significant differences between the NP coronas, there was a striking prevalence of molecules with a notoriously high lipid and surface binding, such as, <i>e.g.</i>, SP-A, SP-D, DMBT1. Our data indicate that the selective adsorption of proteins mediates the relatively similar lipid pattern in the coronas of different NPs. On the basis of our lipidomic and proteomic analysis, we provide a detailed set of quantitative data on the composition of the surfactant corona formed upon NP inhalation, which is unique and markedly different to the plasma corona