A layer of mucus covers the surface of all wet epithelia throughout the human body. Mucus is a hydrogel mainly composed of water, mucins (glycoproteins), DNA, proteins, lipids, and cell debris. This ...complex composition yields a tenacious viscoelastic hydrogel that lubricates and protects the exposed epithelia from external threats and enzymatic degradation. The natural protective role of mucus is nowadays acknowledged as a major barrier to be overcome in non-invasive drug delivery. The heterogeneity of mucus components offers a wide range of potential chemical interaction sites for macromolecules, while the mesh-like architecture given to mucus by the intermolecular cross-linking of mucin molecules results in a dense network that physically, and in a size-dependent manner, hinders the diffusion of nanoparticles through mucus. Consequently, drug diffusion, epithelial absorption, drug bioavailability, and ultimately therapeutic outcomes of mucosal drug delivery can be attenuated.
Display omitted
Calcium phosphate system has been used widely in in vitro gene delivery for almost four decades. Excellent biocompatibility and simple application have motivated the researchers to always consider ...this system in their transfection experiments. However, there was a major drawback regarding the low transfection efficiency of calcium phosphate. Hence, there have been many efforts in order to increase the gene delivery potential of this system. In this paper, the application of calcium phosphate in gene delivery is introduced. Moreover, the recent progresses in the application of calcium phosphate in the delivery of (oligo)nucleotides and different approaches to improve the properties of this system are reviewed.
Messenger RNA (mRNA) has gained remarkable attention as an alternative to DNA-based therapies in biomedical research. A variety of biodegradable nanoparticles (NPs) has been developed including ...lipid-based and polymer-based systems for mRNA delivery. However, both systems still lack in achieving an efficient transfection rate and a detailed understanding of the mRNA transgene expression kinetics. Therefore, quantitative analysis of the time-dependent translation behavior would provide a better understanding of mRNA's transient nature and further aid the enhancement of appropriate carriers with the perspective to generate future precision nanomedicines with quick response to treat various diseases.
A lipid-polymer hybrid system complexed with mRNA was evaluated regarding its efficiency to transfect dendritic cells (DCs) by simultaneous live cell video imaging of both particle uptake and reporter gene expression. We prepared and optimized NPs consisting of poly (lactid-co-glycolid) (PLGA) coated with the cationic lipid 1, 2-di-O-octadecenyl-3-trimethylammonium propane abbreviated as LPNs. An earlier developed polymer-based delivery system (chitosan-PLGA NPs) served for comparison. Both NPs types were complexed with mRNA-mCherry at various ratios. While cellular uptake and toxicity of either NPs was comparable, LPNs showed a significantly higher transfection efficiency of ~ 80% while chitosan-PLGA NPs revealed only ~ 5%. Further kinetic analysis elicited a start of protein translation after 1 h, with a maximum after 4 h and drop of transgene expression after 48 h post-transfection, in agreement with the transient nature of mRNA.
Charge-mediated complexation of mRNA to NPs enables efficient and fast cellular delivery and subsequent protein translation. While cellular uptake of both NP types was comparable, mRNA transgene expression was superior to polymer-based NPs when delivered by lipid-polymer NPs.
Bacterial invasion into eukaryotic cells and the establishment of intracellular infection has proven to be an effective means of resisting antibiotic action, as anti‐infective agents commonly exhibit ...a poor permeability across the host cell membrane. Encapsulation of anti‐infectives into nanoscaled delivery systems, such as liposomes, is shown to result in an enhancement of intracellular delivery. The aim of the current work is, therefore, to formulate colistin, a poorly permeable anti‐infective, into liposomes suitable for oral delivery, and to functionalize these carriers with a bacteria‐derived invasive moiety to enhance their intracellular delivery. Different combinations of phospholipids and cholesterol are explored to optimize liposomal drug encapsulation and stability in biorelevant media. These liposomes are then surface‐functionalized with extracellular adherence protein (Eap), derived from Staphylococcus aureus. Treatment of HEp‐2 and Caco‐2 cells infected with Salmonella enterica using colistin‐containing, Eap‐functionalized liposomes resulted in a significant reduction of intracellular bacteria, in comparison to treatment with nonfunctionalized liposomes as well as colistin alone. This indicates that such bio‐invasive carriers are able to facilitate intracellular delivery of colistin, as necessary for intracellular anti‐infective activity. The developed Eap‐functionalized liposomes, therefore, present a promising strategy for improving the therapy of intracellular infections.
A bioinvasive delivery system consisting of colistin‐loaded liposomes surface functionalized with the bacterial invasion protein Eap is developed and characterized, for the purposes of facilitating intracellular delivery of anti‐infectives via the oral route. Cell invasion studies show the considerable promise of Eap‐functionalized nanocarriers as a strategy to facilitate the intracellular delivery of anti‐infective therapies.
Purpose
The aim of this work was to formulate and characterize surfactant-free glibenclamide nanoparticles using Eudragit RLPO and polyethylene glycol as sole stabilizer.
Methods
Glibenclamide ...nanoparticles were obtained by nanoprecipitation and evaluated in terms of drug content, encapsulation efficiency, apparent saturation solubility, drug release profile, solid state and storage stability. The influence of different stirring speed on the particle size, size distribution and zeta potential of the nanoparticles was investigated. The nanoparticle biocompatibility and permeability were analyzed in vitro on Caco-2 cell line (clone HTB-37) and its interaction with mucin was also investigated.
Results
It was found that increasing the molecular weight of polyethylene glycol from 400 to 6000 decreased drug encapsulation, whereas the aqueous solubility and dissolution rate of the drug increased. Particle size of the nanoformulations, with and without polyethylene glycol, were between 140 and 460 nm. Stability studies confirmed that glibenclamide nanoparticles were stable, in terms of particle size, after 120 days at 4°C. In vitro studies indicated minimal interactions of glibenclamide nanoparticles and mucin glycoproteins suggesting favorable properties to address the intestinal mucus barrier. Cell viability studies confirmed the safety profile of these nanoparticles and showed an increased permeation through epithelial cells.
Conclusion
Taking into consideration these findings, polyethylene glycol is a useful polymer for stabilizing these surfactant-free glibenclamide nanoparticles and represent a promising alternative to improve the treatment of non-insulin dependent diabetes.
Gene therapy has always been a promising therapeutic approach for Cystic Fibrosis (CF). However, numerous trials using DNA or viral vectors encoding the correct protein resulted in a general low ...efficacy. In the last years, chemically modified messenger RNA (cmRNA) has been proven to be a highly potent, pulmonary drug. Consequently, we first explored the expression, function and immunogenicity of human (h)CFTR encoded by cmRNA
in vitro and ex vivo, quantified the expression by flow cytometry, determined its function using a YFP based assay and checked the immune response in human whole blood. Similarly, we examined the function of cmRNA
in vivo after intratracheal (i.t.) or intravenous (i.v.) injection of the assembled cmRNA
together with Chitosan-coated PLGA (poly-D, L-lactide-co-glycolide 75:25 (Resomer RG 752 H)) nanoparticles (NPs) by FlexiVent. The amount of expression of human hCFTR encoded by cmRNA
was quantified by hCFTR ELISA, and cmRNA
values were assessed by RT-qPCR. Thereby, we observed a significant improvement of lung function, especially in regards to FEV
, suggesting NP-cmRNA
as promising therapeutic option for CF patients independent of their CFTR genotype.
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 N-(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.
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 (Chem. Mater. 2013, 25 (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.
Co-delivery of different species of protein-encoding polynucleotides, e.g., messenger RNA (mRNA) and plasmid DNA (pDNA), using the same nanocarrier is an interesting topic that remains scarcely ...researched in the field of nucleic acid delivery. The current study hence aims to explore the possibility of the simultaneous delivery of mRNA (mCherry) and pDNA (pAmCyan) using a single nanocarrier. The latter is based on gelatin type A, a biocompatible, and biodegradable biopolymer of broad pharmaceutical application. A core-shell nanostructure is designed with a thermally stabilized gelatin–pDNA coacervate in its center. Thermal stabilization enhances the core’s colloidal stability and pDNA shielding effect against nucleases as confirmed by nanoparticle tracking analysis and gel electrophoresis, respectively. The stabilized, pDNA-loaded core is coated with the cationic peptide protamine sulfate to enable additional surface-loading with mRNA. The dual-loaded core-shell system transfects murine dendritic cell line DC2.4 with both fluorescent reporter mRNA and pDNA simultaneously, showing a transfection efficiency of 61.4 ± 21.6% for mRNA and 37.6 ± 19.45% for pDNA, 48 h post-treatment, whereas established commercial, experimental, and clinical transfection reagents fail. Hence, the unique co-transfectional capacity and the negligible cytotoxicity of the reported system may hold prospects for vaccination among other downstream applications.
Despite the enormous potential of nanomedicine, the search for materials from renewable resources that balance bio-medical requirements and engineering aspects is still challenging. This study ...proposes an easy method to make nanoparticles composed of oxidized starch and chitosan, both isolated from natural biopolymers. The careful adjustment of C/N ratio, polymer concentration and molecular weight allowed for tuning of particle characteristics. The system's carrier capability was assessed both for anti-infectives and for nucleic acid. Higher starch content polyplexes were found to be suitable for high encapsulation efficiency of cationic anti-infectives and preserving their bactericidal function. A cationic carrier was obtained by coating the anionic polyplex with chitosan. Coating allowed for a minimal amount of cationic polymer to be employed and facilitated plasmid DNA loading both within the particle core and on the surface. Transfection studies showed encouraging result, approximately 5% of A549 cells with reporter gene expression. In summary, starch-chitosan complexes are suitable carriers with promising perspectives for pharmaceutical use.
PDF
