ABSTRACT
Antibiotic resistance is a cause of serious illness and death, originating often from insufficient permeability into gram-negative bacteria. Nanoparticles (NP) can increase antibiotic delivery in bacterial cells, however, may as well increase internalization in mammalian cells and toxicity. In this work, NP in liposome (NP-Lip) formulations were used to enhance the selectivity of the antibiotics (3C and tobramycin) and quorum sensing inhibitor (HIPS-1635) towards Pseudomonas aeruginosa by fusing with bacterial outer membranes and reducing uptake in mammalian cells due to their larger size. Poly (lactic-co-glycolic) acid NPs were prepared using emulsion solvent evaporation and incorporated in larger liposomes. Cytotoxicity and uptake studies were conducted on two lung cell lines, Calu-3 and H460. NP-Lip showed lower toxicity and uptake in both cell lines. Then formulations were investigated for suitability for oral inhalation. The deposition of NP and NP-Lip in the lungs was assessed by next generation impactor and corresponded to 75% and 45% deposition in the terminal bronchi and the alveoli respectively. Colloidal stability and mucus-interaction studies were conducted. NP-Lip showed higher diffusion through mucus compared to NPs with the use of nanoparticle tracking analyzer. Moreover, the permeation of delivery systems across a liquid-liquid interface epithelial barrier model of Calu-3 cells indicated that NP-Lip could cause less systemic toxicity upon in-vivo like administration by aerosol deposition. Monoculture and Pseudomonas aeruginosa biofilm with Calu-3 cells co-culture experiments were conducted, NP-Lip achieved highest toxicity towards bacterial biofilms and least toxicity % of the Calu-3 cells. Therefore, the NP- liposomal platform offers a promising approach for enhancing antibiotic selectivity and treating pulmonary infections.
ABSTRACT
Hair follicle-penetrating nanoparticles offer a promising avenue for targeted antibiotic delivery, especially in challenging infections like acne inversa or folliculitis decalvans. However, demonstrating their efficacy with existing preclinical models remains difficult. This study presents an innovative approach using a 3D in vitro organ culture system with human hair follicles to investigate the hypothesis that antibiotic nanocarriers may reach bacteria within the follicular cleft more effectively than free drugs. Living human hair follicles were transplanted into a collagen matrix within a 3D printed polymer scaffold to replicate the follicle's microenvironment. Hair growth kinetics over 7 days resembled those of simple floating cultures. In the 3D model, fluorescent nanoparticles exhibited some penetration into the follicle, not observed in floating cultures. Staphylococcus aureus bacteria displayed similar distribution profiles postinfection of follicles. While rifampicin-loaded lipid nanocapsules were as effective as free rifampicin in floating cultures, only nanoencapsulated rifampicin achieved the same reduction of CFU/mL in the 3D model. This underscores the hair follicle microenvironment's critical role in limiting conventional antibiotic treatment efficacy. By mimicking this microenvironment, the 3D model demonstrates the advantage of topically administered nanocarriers for targeted antibiotic therapy against follicular infections.
Subject(s)
Anti-Bacterial Agents , Hair Follicle , Printing, Three-Dimensional , Rifampin , Staphylococcus aureus , Hair Follicle/microbiology , Hair Follicle/drug effects , Humans , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/therapeutic use , Staphylococcus aureus/drug effects , Rifampin/pharmacology , Rifampin/therapeutic use , Rifampin/administration & dosage , Drug Delivery Systems , Nanoparticles/chemistry , Nanocapsules/chemistry , Staphylococcal Infections/drug therapyABSTRACT
Co-delivery of different protein-encoding polynucleotide species with varying expression kinetics of their therapeutic product will become a prominent requirement in the realm of combined nucleic acid(NA)-based therapies in the upcoming years. The current study explores the capacity for time-staggered expression of encoded proteins by simultaneous delivery of plasmid DNA (pDNA) in the core and mRNA on the shell of the same nanocarrier. The core is based on a Gelatin Type A-pDNA coacervate, thermally stabilized to form an irreversible nanogel stable enough for the deposition of cationic coats namely, protamine sulfate or LNP-related lipid mixtures. Only the protamine-coated nanocarriers remained colloidally stable following mRNA loading and could successfully co-transfect murine dendritic cell line DC2.4 with fluorescent reporter mRNA(mCherry) and pDNA (pAmCyan1). Further investigation of the protamine-coated nanosystem only, the transfection efficiency (percentage of transfected cells) and level of protein expression (mean fluorescence intensity, MFI) of mRNA and pDNA, simultaneously delivered by the same nanocarrier, were compared and kinetically assessed over 48 h in DC2.4 using flow cytometry. The onset of transfection for both nucleotides was initially delayed, with levels < 5% at 6 h. Thereafter, mRNA transfection reached 90% after 24 h and continued to slightly increase until 48 h. In contrast, pDNA transfection was clearly slower, reaching approximately 40% after 24 h, but continuing to increase to reach 94% at 48 h. The time course of protein expression (represented by MFI) for both NAs essentially followed that of transfection. Model-independent as well as model-dependent kinetic parameters applied to the data further confirmed such time-staggered expression of the two NA's where mRNA's rate of transfection and protein expression initially exceeded those of pDNA in the first 24 h of the experiment whereas the opposite was true during the second 24 h of the experiment where pDNA displayed the higher response rates. We expect that innovative nanocarriers capable of time-staggered co-delivery of different nucleotides could open new perspectives for multi-dosing, pulsatile or sustained expression of nucleic acid-based therapeutics in protein replacement, vaccination, and CRISPR-mediated gene editing scenarios.
ABSTRACT
Pseudomonas aeruginosa (PA) is a major healthcare concern due to its tolerance to antibiotics when enclosed in biofilms. Tobramycin (Tob), an effective cationic aminoglycoside antibiotic against planktonic PA, loses potency within PA biofilms due to hindered diffusion caused by interactions with anionic biofilm components. Loading Tob into nano-carriers can enhance its biofilm efficacy by shielding its charge. Polyion complex vesicles (PIC-somes) are promising nano-carriers for charged drugs, allowing higher drug loadings than liposomes and polymersomes. In this study, a new class of nano-sized PIC-somes, formed by Tob-diblock copolymer complexation is presented. This approach replaces conventional linear PEG with brush-like poly[ethylene glycol (methyl ether methacrylate)] (PEGMA) in the shell-forming block, distinguishing it from past methods. Tob paired with a block copolymer containing hydrophilic PEGMA induces micelle formation (PIC-micelles), while incorporating hydrophobic pyridyldisulfide ethyl methacrylate (PDSMA) monomer into PEGMA chains reduces shell hydrophilicity, leads to the formation of vesicles (PIC-somes). PDSMA unit incorporation enables unprecedented dynamic disulfide bond-based shell cross-linking, significantly enhancing stability under saline conditions. Neither PIC-somes nor PIC-micelles show any relevant cytotoxicity on A549, Calu-3, and dTHP-1 cells. Tob's antimicrobial efficacy against planktonic PA remains unaffected after encapsulation into PIC-somes and PIC-micelles, but its potency within PA biofilms significantly increases.
ABSTRACT
Background: Therapeutic proteins and peptides offer great advantages compared to traditional synthetic molecular drugs. However, stable protein loading and precise control of protein release pose significant challenges due to the extensive range of physicochemical properties inherent to proteins. The development of a comprehensive protein delivery strategy becomes imperative accounting for the diverse nature of therapeutic proteins. Methods: Biodynamers are amphiphilic proteoid dynamic polymers consisting of amino acid derivatives connected through pH-responsive dynamic covalent chemistry. Taking advantage of the amphiphilic nature of the biodynamers, PNCs and DEs were possible to be prepared and investigated to compare the delivery efficiency in drug loading, stability, and cell uptake. Results: As a result, the optimized PNCs showed 3-fold encapsulation (<90%) and 5-fold loading capacity (30%) compared to DE-NPs. PNCs enhanced the delivery efficiency into the cells but aggregated easily on the cell membrane due to the limited stability. Although DE-NPs were limited in loading capacity compared to PNCs, they exhibit superior adaptability in stability and capacity for delivering a wider range of proteins compared to PNCs. Conclusion: Our study highlights the potential of formulating both PNCs and DE-NPs using the same biodynamers, providing a comparative view on protein delivery efficacy using formulation methods.
Subject(s)
Emulsions , Peptides , Peptides/chemistry , Peptides/administration & dosage , Peptides/pharmacokinetics , Emulsions/chemistry , Humans , Proteins/chemistry , Proteins/administration & dosage , Proteins/pharmacokinetics , Drug Delivery Systems/methods , Polymers/chemistry , Nanoparticles/chemistry , Hydrogen-Ion Concentration , Amino Acids/chemistry , Drug Carriers/chemistry , Drug Carriers/pharmacokinetics , Drug Liberation , Cell Survival/drug effectsABSTRACT
Responsive and adaptive soft-matter systems represent an advanced category of materials with potential applications in drug delivery. Among these, liquid crystals (LCs) emerge as multifunctional anisotropic scaffolds capable of reacting to temperature, light, electric or magnetic fields. Specifically, the ordering and physical characteristics of thermotropic LCs are primarily contingent on temperature as an external stimulus. This comprehensive review aims to bridge a notable gap in the biomedical application of thermotropic mesogens by exclusively focusing on drug delivery. Anticipated to inspire diverse ideas, the review intends to facilitate the elegant exploitation of controllable and temperature-induced characteristics of LCs to enhance drug permeation. Here, we delineate recent advancements in thermally-driven LCs with a substantial emphasis on LC monomer mixtures, elastomers, polymers, microcapsules and membranes. Moreover, special emphasis is placed on the biocompatibility and toxicity of LCs as the foremost prerequisite for their application in healthcare. Given the promising prospect of thermotropic LC formulations in a clinical context, a special section is devoted to skin drug delivery. The review covers content from multiple disciplines, primarily targeting researchers interested in innovative strategies in drug delivery. It also appeals to those enthusiastic about firsthand exploration of the feasible biomedical applications of thermotropic LCs. To the best of our knowledge, this marks the first review addressing thermotropic LCs as tunable soft-matter systems for drug delivery.
Subject(s)
Delayed-Action Preparations , Drug Carriers , Drug Delivery Systems , Liquid Crystals , Temperature , Liquid Crystals/chemistry , Humans , Drug Delivery Systems/methods , Drug Carriers/chemistry , Animals , Drug Liberation , Polymers/chemistry , Skin/metabolism , Skin/drug effects , Administration, CutaneousABSTRACT
Antimicrobial resistance is becoming more prominent day after day due to a number of mechanisms by microbes, especially the sophisticated biological barriers of bacteria, especially in Gram-negatives. There, the lipopolysaccharides (LPS) layer is a unique component of the outer leaflet of the outer membrane which is highly impermeable and prevents antibiotics from passing passively into the intracellular compartments. Biodynamers, a novel class of dynamically bio-responsive polymers, may open new perspectives to overcome this particular barrier by accommodating various secondary structures and form supramolecular structures in such bacterial microenvironments. Generally, bio-responsive polymers are not only candidates as bio-active molecules against bacteria but also carriers via their interactions with the cargo. Based on their dynamicity, design flexibility, biodegradability, biocompatibility, and pH-responsiveness, we investigated the potential of two peptide-based biodynamers for improving antimicrobial drug delivery. By a range of experimental methods, we discovered a greater affinity of Arg-biodynamers for bacterial membranes than for mammalian membranes as well as an enhanced LPS targeting on the bacterial membrane, opening perspectives for enhancing the delivery of antimicrobials across the Gram-negative bacterial cell envelope. This could be explained by the change of the secondary structure of Arg-biodynamers into a predominant ß-sheet character in the LPS microenvironment, by contrast to the α-helical structure typically observed for most lipid membrane-permeabilizing peptides. In comparison to poly-L-arginine, the intrinsic antibacterial activity of Arg-biodynamers was nearly unchanged, but its toxicity against mammalian cells was >128-fold reduced. When used in bacterio as an antibiotic potentiator, however, Arg-biodynamers improved the minimum inhibitory concentration (MIC) against Escherichia coli by 32 times compared to colistin alone. Similar effect has also been observed in two stains of Pseudomonas aeruginosa. Arg-biodynamers may therefore represent an interesting option as an adjuvant for antibiotics against Gram-negative bacteria and to overcome antimicrobial resistance.
Subject(s)
Anti-Bacterial Agents , Gram-Negative Bacteria , Lipopolysaccharides , Microbial Sensitivity Tests , Lipopolysaccharides/pharmacology , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Gram-Negative Bacteria/drug effects , Bacterial Outer Membrane/drug effects , Bacterial Outer Membrane/metabolism , Humans , Escherichia coli/drug effects , Polymers/chemistry , Arginine/chemistry , Drug Delivery Systems/methodsABSTRACT
Herein, we report the properties of nanostructured lipid carriers (NLCs) prepared with a gradient concentration of Bergenin (BGN) isolated from Pentaclethra macrophylla stem bark powder. A gradient concentration of BGN (BGN 0, 50, 100, 150, and 200 mg) was prepared in a 5 % lipid matrix consisting of Transcutol HP (75 %), Phospholipon 90H (15 %), and Gelucire 43/01 (10 %) to which a surfactant aqueous phase consisting of Tween 80, sorbitol, and sorbic acid was dissolved. The NLCs were evaluated by size, polydispersity index (PDI), zeta potential, Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), encapsulation efficiency, and in vitro drug release. The result shows polydispersed nanoparticles with high drug encapsulation (94.26-99.50 %). The nanoparticles were mostly spherical, but those from the 50 mg BGN batch were more cuboidal than spherical. The drug release was highest from the latter to the tune of 40 % compared to the pure BGN solution, which released about 15 % BGN. The anti-inflammatory activity of the BGN-NLC and total plant extract was studied on lipopolysaccharide (LPS)-inflamed macrophages. The cell study showed that BGN and plant extract had low cytotoxicity on macrophages and exhibited a dose-dependent anti-inflammatory effect on the LPS-induced inflammatory process in macrophages.
Subject(s)
Anti-Inflammatory Agents , Benzopyrans , Drug Carriers , Lipids , Lipopolysaccharides , Macrophages , Nanoparticles , Lipopolysaccharides/pharmacology , Animals , Mice , Macrophages/drug effects , Macrophages/metabolism , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/chemistry , Drug Carriers/chemistry , Benzopyrans/pharmacology , Benzopyrans/administration & dosage , Benzopyrans/chemistry , Nanoparticles/chemistry , Lipids/chemistry , RAW 264.7 Cells , Drug Liberation , Nanostructures/chemistry , Saxifragaceae/chemistry , Particle Size , Inflammation/drug therapy , Inflammation/chemically induced , Plant Extracts/pharmacology , Plant Extracts/administration & dosage , Plant Extracts/chemistryABSTRACT
The administration of insulins by subcutaneous injection is nowadays widely prevalent. The injection site is located below the dermis and composed of cells and the extracellular matrix formed of a network of macromolecules such as hyaluronic acid and collagen. Following an injection, the insulins from the formulated products are timely released as drug molecules from the injection site into systemic circulation. In this publication, we show the development of an in vitro setup utilizing a hydrogel composed of a special collagen-hyaluronic acid mixture that mimics the extracellular matrix. Another setup was used for differentiation of the commercially available and research insulin formulations by determining the in vitro permeation characteristics with the results that were correlated with the human in vivo data. Significant differentiation was achieved at 90 % confidence level between the permeation curves of insulin glulisine containing formulations (U100 and a concentrated research formulation), while in case of the insulin lispro containing formulations (U100 and U200) the permeation curves showed similarity. These results demonstrated that the in vitro setup may be used as a tool for formulation development and drug candidate profiling as it is able to differentiate or show similarities between the agglomeration states and concentration of the active pharmaceutical ingredients.
Subject(s)
Hyaluronic Acid , Insulins , Humans , Insulin , Insulin Lispro , Collagen , Hypoglycemic AgentsABSTRACT
Antimicrobial resistance and tolerance (AMR&T) are urgent global health concerns, with alarmingly increasing numbers of antimicrobial drugs failing and a corresponding rise in related deaths. Several reasons for this situation can be cited, such as the misuse of traditional antibiotics, the massive use of sanitizing measures, and the overuse of antibiotics in agriculture, fisheries, and cattle. AMR&T management requires a multifaceted approach involving various strategies at different levels, such as increasing the patient's awareness of the situation and measures to reduce new resistances, reduction of current misuse or abuse, and improvement of selectivity of treatments. Also, the identification of new antibiotics, including small molecules and more complex approaches, is a key factor. Among these, novel DNA- or RNA-based approaches, the use of phages, or CRISPR technologies are some potent strategies under development. In this perspective article, emerging and experienced leaders in drug delivery discuss the most important biological barriers for drugs to reach infectious bacteria (bacterial bioavailability). They explore how overcoming these barriers is crucial for producing the desired effects and discuss the ways in which drug delivery systems can facilitate this process.
Subject(s)
Anti-Bacterial Agents , Drug Delivery Systems , Humans , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/chemistry , Animals , Drug Resistance, Microbial , Drug Resistance, Bacterial , Bacteria/drug effects , Drug ToleranceABSTRACT
An inhalation-based Biopharmaceutics Classification System for pulmonary drugs (iBCS) holds the perspective to allow for scientifically sound prediction of differences in the in vivo performance of orally inhaled drug products (OIDPs). A set of nine drug substances were selected, that are administered via both the oral and pulmonary routes. Their solubility was determined in media representative for the oral (Fasted State Simulated Intestinal Fluid (FaSSIF)) and pulmonary (Alveofact medium and Simulated Lung Fluid (SLF)) routes of administration to confirm the need for a novel approach for inhaled drugs. The complexity of these media was then stepwise reduced with the purpose of understanding the contribution of their components to the solubilizing capacity of the media. A second reason for varying the complexity was to identify a medium that would allow robust but accurate dissolution testing. Hence, Hank's balanced salt solution (HBSS) as a medium used in many in vitro biological tests, non-buffered saline solution, and water were included. For some drug substances (salbutamol sulfate, tobramycin, isoniazid, and tiotropium bromide), no significant differences were observed between the solubility in the media used. For other drugs, however, we observed either just small (rifampicin, budesonide, salmeterol) or unexpectedly large differences (beclomethasone dipropionate). Based on the minimum theoretical solubility required for their common pulmonary dose in 10 ml of lung lining fluid, drug solubility was classified as either high or low. Two high solubility and two low solubility compounds were then selected for refined solubility testing in pulmonary relevant media by varying their content of phospholipids, surfactant proteins and other proteins. The solubility of drug substances in simulated lung lining fluids was found to be dependent on the physicochemical properties of the drug substance and the composition of the media. While a pulmonary dissolution medium that would fit all drugs could not be established, our approach may provide guidance for finding the most suitable dissolution medium for a given drug substance and better designing in vitro tests for predicting the in vivo performance of inhalable drug products.
Subject(s)
Biopharmaceutics , Intestines , Pharmaceutical Preparations/chemistry , Solubility , Administration, InhalationABSTRACT
Bacterial pathogens are constantly evolving to outsmart the host immune system and antibiotics developed to eradicate them. One key strategy involves the ability of bacteria to survive and replicate within host cells, thereby causing intracellular infections. To address this unmet clinical need, researchers are adopting new approaches, such as the development of novel molecules that can penetrate host cells, thus exerting their antimicrobial activity intracellularly, or repurposing existing antibiotics using nanocarriers (i.e., nanoantibiotics) for site-specific delivery. However, inconsistency in information reported across published studies makes it challenging for scientific comparison and judgment of experiments for future direction by researchers. Together with the lack of reproducibility of experiments, these inconsistencies limit the translation of experimental results beyond pre-clinical evaluation. Minimum information guidelines have been instrumental in addressing such challenges in other fields of biomedical research. Guidelines and recommendations provided herein have been designed for researchers as essential parameters to be disclosed when publishing their methodology and results, divided into four main categories: (i) experimental design, (ii) establishing an in vitro model, (iii) assessment of efficacy of novel therapeutics, and (iv) statistical assessment. These guidelines have been designed with the intention to improve the reproducibility and rigor of future studies while enabling quantitative comparisons of published studies, ultimately facilitating translation of emerging antimicrobial technologies into clinically viable therapies that safely and effectively treat intracellular infections.
Subject(s)
Anti-Infective Agents , Research Design , Reproducibility of Results , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , BacteriaABSTRACT
Stimuli-responsive and tunable soft-matter systems are an advanced class of materials applicable for drug delivery. Liquid crystals (LCs) are promising candidates as multifunctional materials that can respond to temperature, light or magnetic field. Particularly, ordering and physical properties of thermoresponsive LCs depend predominantly on temperature as external trigger. The current work addresses an elegant strategy to implement the anisotropic properties of thermoresponsive LCs with a view to extending their application for drug delivery. We firstly fabricated novel compositions with a thermotropic core based on natural products - cholesteryl esters and mono-/bicyclic terpenoids. The distinctive feature of aforementioned systems is their temperature-induced switchability of drug release by transition to the LC state, depending on the skin temperature. Their mesomorphic and optical behavior was characterized via differential scanning calorimetry and polarizing optical microscopy. Furthermore, we describe the dependence of helical pitch on LC formulation for various ternary cholesteric systems doped with terpenoids, suggesting that these stimuli-responsive chiral dopants are nominally untwisting. Data from fluorescence probe technique indicate that cholesteryl esters and terpenoids as essential components of those LC systems jointly disrupt the tight structure of phospholipid bilayer packing enabling the facilitated penetration of drugs. The potential of LC formulations was explored for several model drugs with diverse physicochemical properties by in vitro and ex vivo penetration tests using artificial membranes and full human skin. Our findings confirm the potential of LC systems for various applications in skin drug delivery.
ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) can establish latent lifelong infections in infected individuals. During viral latency, the latency-associated nuclear antigen (LANA) mediates the replication of the latent viral genome in dividing cells and tethers them to mitotic chromosomes, thus ensuring their partitioning into daughter cells during mitosis. This study aims to inhibit Kaposi's sarcoma-associated herpesvirus (KSHV) latent replication by targeting the LANA-DNA interaction using small molecular entities. Drawing from first-generation inhibitors and using growth vectors identified through STD-NMR, we expanded these compounds using Suzuki-Miyaura cross-coupling. This led to a deeper understanding of SAR achieved by microscale thermophoresis (MST) measurements and cell-free tests via electrophoretic mobility shift assays (EMSA). Our most potent compounds successfully inhibit LANA-mediated replication in cell-based assays and demonstrate favorable in vitro ADMET-profiles, including suitable metabolic stability, Caco-2 permeability, and cytotoxicity. These compounds could serve as qualified leads for the future refinement of small molecule inhibitors of KSHV latent replication.
Subject(s)
Herpesvirus 8, Human , Humans , Herpesvirus 8, Human/metabolism , Caco-2 Cells , Virus Replication , Virus LatencyABSTRACT
Polymicrobial infections involving various combinations of microorganisms, such as Escherichia, Pseudomonas, or Yersinia, can lead to acute and chronic diseases in for example the gastrointestinal and respiratory tracts. Our aim is to modulate microbial communities by targeting the posttranscriptional regulator system called carbon storage regulator A (CsrA) (or also repressor of secondary metabolites (RsmA)). In previous studies, we identified easily accessible CsrA binding scaffolds and macrocyclic CsrA binding peptides through biophysical screening and phage display technology. However, due to the lack of an appropriate in bacterio assay to evaluate the cellular effects of these inhibitor hits, the focus of the present study is to establish an in bacterio assay capable of probing and quantifying the impact on CsrA-regulated cellular mechanisms. We have successfully developed an assay based on a luciferase reporter gene assay, which in combination with a qPCR expression gene assay, allows for the monitoring of expression levels of different downstream targets of CsrA. The chaperone protein CesT was used as a suitable positive control for the assay, and in time-dependent experiments, we observed a CesT-mediated increase in bioluminescence over time. By this means, the cellular on-target effects of non-bactericidal/non-bacteriostatic virulence modulating compounds targeting CsrA/RsmA can be evaluated.
Subject(s)
Escherichia coli Proteins , Escherichia coli Proteins/metabolism , Carbon/metabolism , RNA-Binding Proteins/chemistry , Gene Expression , Genes, Reporter , Gene Expression Regulation, Bacterial , Bacterial Proteins/metabolismABSTRACT
Gene knockdown by siRNA offers an unrestricted choice of targets and specificity based on the principle of complementary Watson-Crick base pairing with mRNA. However, the negative charge, large molecular size, and susceptibility to enzymatic degradation of siRNA impede its successful transfection, hence limiting its potential for therapeutic use. The development of efficient and safe siRNA transfection agents is, therefore, critical for siRNA-based therapy. Herein, we developed a protein-based biodynamic polymer (biodynamer) that showed potential as a siRNA transfection vector, owing to its excellent biocompatibility, easy tunability, and dynamic polymerization under acidic environments. The positively charged biodynamers formed stable dynamic nanocomplexes (XL-DPs, hydrodynamic diameter of approximately 104 nm) with siRNA via electrostatic interactions and chemical cross-linking. As a proof of concept, the optimized XL-DPs were stable in physiological conditions with serum proteins and demonstrated significant pH-dependent size change and degradability, as well as siRNA release capability. The minimal cytotoxicity and excellent cellular uptake of XL-DPs effectively supported the intracellular delivery of siRNA. Our study demonstrated that the XL-DPs in survivin siRNA delivery enabled potent knockdown of survivin mRNA and induced notable apoptosis of carcinoma cells (2.2 times higher than a lipid-based transfection agent, Lipofectamine 2000). These findings suggested that our XL-DPs hold immense potential as a promising platform for siRNA delivery and can be considered strong candidates in the advancement of next-generation transfection agents.
Subject(s)
Apoptosis , Survivin/genetics , RNA, Small Interfering , Transfection , Hydrogen-Ion Concentration , RNA, Messenger , Cell Line, TumorABSTRACT
Addressing antimicrobial resistance requires new approaches in various disciplines of pharmaceutical sciences. The fluoroquinolone levofloxacin (LEV) plays an important role in the therapy of lung infections. However, its effectiveness is limited by its severe side effects involving tendinopathy, muscle weakness and psychiatric disturbance. Therefore, there is a need for the development of an effective formulation of LEV with reduced systemic drug concentrations, thereby also reducing the consumption and excretion of antibiotics or metabolites. This study aimed for the development of a pulmonary-applicable LEV formulation. Co-amorphous LEV-L-arginine (ARG) particles were prepared by spray drying and characterised by scanning electron microscopy, modulated differential scanning calorimetry, X-ray powder diffraction, Fourier-transform infrared spectroscopy and next generation impactor analysis. Co-amorphous LEV-ARG salts were produced independently of varying process parameters. The use of 30% (v/v) ethanol as a solvent led to better aerodynamic properties compared to an aqueous solution. With a mass median aerodynamic diameter of just over 2 µm, a fine particle fraction of over 50% and an emitted dose of over 95%, the product was deemed suitable for a pulmonary application. The created process was robust towards the influence of temperature and feed rate, as changing these parameters did not have a significant influence on the critical quality attributes, indicating the feasibility of producing pulmonary-applicable co-amorphous particles for sustainable antibiotic therapy.
ABSTRACT
Benznidazole, a poorly soluble in water drug, is the first-line medication for the treatment of Chagas disease, but long treatment periods at high dosages cause several adverse effects with insufficient activity in the chronic phase. According to these facts, there is a serious need for novel benznidazole formulations for improving the chemotherapy of Chagas disease. Thus, this work aimed to incorporate benznidazole into lipid nanocapsules for improving its solubility, dissolution rate in different media, and permeability. Lipid nanocapsules were prepared by the phase inversion technique and were fully characterized. Three formulations were obtained with a diameter of 30, 50, and 100 nm and monomodal size distribution with a low polydispersity index and almost neutral zeta potential. Drug encapsulation efficiency was between 83 and 92 % and the drug loading was between 0.66 and 1.04 %. Loaded formulations were stable under storage for one year at 4 °C. Lipid nanocapsules were found to protect benznidazole in simulated gastric fluid and provide a sustained release platform for the drug in a simulated intestinal fluid containing pancreatic enzymes. The small size and the almost neutral surface charge of these lipid nanocarriers improved their penetration through mucus and such formulations showed a reduced chemical interaction with gastric mucin glycoproteins. LNCs. The incorporation of benznidazole in lipid nanocapsules improved the drug permeability across intestinal epithelium by 10-fold compared with the non-encapsulated drug while the exposure of the cell monolayers to these nanoformulations did not affect the integrity of the epithelium.
Subject(s)
Nanocapsules , Nanocapsules/chemistry , Drug Liberation , Lipids/chemistry , Permeability , Drug StabilityABSTRACT
Nanopharmaceuticals represent a group of nanoparticles engineered for medical purposes. Nowadays, nanotechnology offers several possibilities to improve the safety and efficacy of medicines by designing advanced carrier systems which have been found to offer particular advantages when formulated in the nanoscale. Some of the initially marketed nano-formulations already demonstrate advantages over conventional formulations. Innovative delivery systems offer the possibility to not only control drug release but also to overcome biological barriers. For the translation of new drug products from bench to bedside, however, it is pivotal to test and prove their safety. This is of course also true for nanopharmaceuticals, where in particular the biocompatibility and also the clearance/biodegradation of the carrier material after drug delivery has to be demonstrated. The pulmonary route offers some great opportunities for noninvasive drug delivery but also implicates peculiar challenges. Advanced aerosol formulations with innovative drug carriers have already contributed to the significant progress of inhalation therapy. However, in spite of the large alveolar epithelial surface area, the respiratory tract still features diverse efficient biological barriers, primarily designed by nature to protect the human body against inhaled pollutants and pathogens. Only a thorough understanding of particle-lung interactions will allow the rational design of novel nanopharmaceuticals capable of overcoming these barriers, while of course always keeping in mind the strict demands for their safety. While the recent resurrection of inhaled insulin has already confirmed the potential of the pulmonary route for systemic delivery of biopharmaceuticals, inhaled nanopharmaceuticals, currently under investigation, promise to improve also local therapies like anti-infectives.
Subject(s)
Drug Carriers , Drug Delivery Systems , Humans , Administration, Inhalation , Drug Liberation , ExcipientsABSTRACT
Biofilm-associated infections are causing over half a million deaths each year, raising the requirement for innovative therapeutic approaches. For developing novel therapeutics against bacterial biofilm infections, complexin vitromodels that allow to study drug effects on both pathogens and host cells as well as their interaction under controlled, physiologically relevant conditions appear as highly desirable. Nonetheless, building such models is quite challenging because (1) rapid bacterial growth and release of virulence factors may lead to premature host cell death and (2) maintaining the biofilm status under suitable co-culture requires a highly controlled environment. To approach that problem, we chose 3D bioprinting. However, printing living bacterial biofilms in defined shapes on human cell models, requires bioinks with very specific properties. Hence, this work aims to develop a 3D bioprinting biofilm method to build robustin vitroinfection models. Based on rheology, printability and bacterial growth, a bioink containing 3% gelatin and 1% alginate in Luria-Bertani-medium was found optimal forEscherichia coliMG1655 biofilms. Biofilm properties were maintained after printing, as shown visually via microscopy techniques as well as in antibiotic susceptibility assays. Metabolic profile analysis of bioprinted biofilms showed high similarity to native biofilms. After printing on human bronchial epithelial cells (Calu-3), the shape of printed biofilms was maintained even after dissolution of non-crosslinked bioink, while no cytotoxicity was observed over 24 h. Therefore, the approach presented here may provide a platform for building complexin vitroinfection models comprising bacterial biofilms and human host cells.