Neutral Surface Charge Research Articles

Bioinspired orthogonal-shaped protein-biometal nanocrystals enable oral protein absorption.

With the growing number of marketed biological drugs, the development of technological strategies for their oral systemic absorption, becomes increasingly important. The harsh gastrointestinal environment and low permeability of the intestinal epithelium, represent a huge challenge for their systemic delivery. Herein, bioinspired in the physiological insulin-Zn interaction, the design of orthogonal-shaped protein-biometal hybrid nanocrystals, further enveloped by a bilayer of functional biomaterials, is reported. The nanocrystals exhibited a size of 80 nm, a neutral surface charge and a high insulin loading. In vitro studies showed the capacity of the nanocomplexes to control the release of the associated insulin, while preserving its stability. In vivo evaluation showed sustained blood glucose reductions in both healthy and diabetic rats (up to 40 % and 80 %, respectively), while chronic immunotoxicity studies in mice indicated no toxicity effect. Preliminary efficacy studies in healthy awake pigs following oral capsule administration showed over 20 % absolute bioavailability.

Glycopolymeric Nanoparticles Enrich Less Immunogenic Protein Coronas, Reduce Mononuclear Phagocyte Clearance, and Improve Tumor Delivery Compared to PEGylated Nanoparticles.

Nanoparticles (NPs) offer significant promise as drug delivery vehicles; however, their in vivo efficacy is often hindered by the formation of a protein corona (PC), which influences key physiological responses such as blood circulation time, biodistribution, cellular uptake, and intracellular localization. Understanding NP-PC interactions is crucial for optimizing NP design for biomedical applications. Traditional approaches have utilized hydrophilic polymer coatings like polyethylene glycol (PEG) to resist protein adsorption, but glycopolymer-coated nanoparticles have emerged as potential alternatives due to their biocompatibility and ability to reduce the adsorption of highly immunogenic proteins. In this study, we synthesized and characterized glycopolymer-based poly[2-(diisopropylamino)ethyl methacrylate-b-poly(methacrylamidoglucopyranose) (PDPA-b-PMAG) NPs as an alternative to PEGylated NPs. We characterized the polymers using a range of techniques to establish their molecular weight and chemical composition. PMAG and PEG-based NPs showed equivalent physicochemical properties with sizes of ∼100 nm, spherical morphology, and neutral surface charges. We next assessed the magnitude of protein adsorption on both NPs and catalogued the identity of the adsorbed proteins using mass spectrometry-based techniques. The PMAG NPs were found to adsorb fewer proteins in vitro as well as fewer immunogenic proteins such as Immunoglobulins and Complement proteins. Flow cytometry and confocal microscopy were employed to examine cellular uptake in RAW 264.7 macrophages and MDA-MB-231 tumor cells, where PMAG NPs showed higher uptake into tumor cells over macrophages. In vivo studies in BALB/c mice with orthotopic 4T1 breast cancer xenografts showed that PMAG NPs exhibited prolonged circulation times and enhanced tumor accumulation compared to PEGylated NPs. The biodistribution analysis also revealed greater selectivity for tumor tissue over the liver for PMAG NPs. These findings highlight the potential of glycopolymeric NPs to improve tumor targeting and reduce macrophage uptake compared to PEGylated NPs, offering significant advancements in cancer nanomedicine and immunotherapy.

Anti-oxidative mesoporous polydopamine-based hypotensive nano-eyedrop for improved glaucoma management

Conventional hypotensive eye drops remain suboptimal for glaucoma management, primarily due to their limited intraocular bioavailability and the growing concern regarding ocular surface side effects. Therefore, there is an urgent need to develop innovative intraocular pressure (IOP)-lowering formulations that not only possess enhanced corneal penetration ability but also provide ocular surface protection. Herein, anti-oxidative mesoporous polydopamine nanoparticles (MPDA NPs) were explored as a nano-carrier for Brimonidine to address the above issues. Nearly monodisperse MPDA NPs with obvious nanopores were successfully prepared by template-removal method and used for encapsulation of Brimonidine benefiting from their high specific surface area. Interestingly, the PEGylated and drug loaded MPDA-PEG@Brim NPs showed a near neutral surface charge, which is expected to enhance intraocular drug delivery. Consequently, much higher concentration of Brimonidine in the aqueous humor was found after topical administration of MPDA-PEG@Brim nano-dispersion as compared to free Brimonidine solution. Accordingly, superior IOP reduction effect was achieved for the nano-formulation in both hypertensive and normotensive rat eyes. Moreover, MPDA-PEG NPs showed good capability in scavenging diverse free radicals, alleviating intracellular oxidative stress, and mitigating ocular surface oxidative level in a mouse model of preservative-induced dry eye. In addition, the excellent biosafety of this novel Brimonidine nanodrug was confirmed both in vitro and in vivo. Therefore, the present work may shed light on the development of next generation hypotensive formulations for extended ocular surface protection and glaucoma management.

Biocompatible PAMAM-PLGA-PCL Nanocarrier for Efficient Curcumin Delivery to Lung Cancer Cells: In Vitro Studies.

Lung cancer, as the leading cause of death among other types of cancer, has a high rate of incidence throughout the world. Although conventional modalities, like chemotherapy, have been applied for the inhibition of this cancer, they have not led to the suppression of lung cancer owing to their deficiencies. Thus, we developed a novel polylactic-co-glycolic acid (PLGA)-polyamidoamine G4 (PAMAM G4)-polycaprolactone (PCL) nanocarrier for efficient delivery of curcumin (Cur) to A549 lung cancer cells. The synthesized nanocarrier was characterized by applying analytical techniques, FT-IR, DLS, TEM, and TGA. Successful synthesis, nano-size diameter (40-80 nm), near neutral surface charge (8.0 mV), and high drug entrapment (11.5 %) were measured for the nanocarrier. Controlled (about 5 folds within first 2 h) and pH-sensitive (2-3 folds faster within first hours) Cur release observed for PLGA-PAMAM-PCL-Cur. Cell viability test (MTT assay) indicated the high capability of nanocarrier in suppression of A549 cancer cells (21 % viability after 24 h of treatment with 200 nM) while did not result in toxicity on MSC normal cells. The IC50 observed for 50 nM at 24 h of post-treatment in A549 cells. The qRT-PCR technique indicated inducing the expression of apoptotic genes (Caspase9 and Bax) by 6-8 folds and suppressing anti-apoptotic gene (Bcl2) by 7 folds. ROS considerably increased in cancer cells as well. This nanocarrier would be a promising drug delivery system against lung cancer.

Godanti bhasma (anhydrous CaSO4) induces massive cytoplasmic vacuolation in mammalian cells: A model for phagocytosis assay

Phagocytosis is an essential physiological mechanism; its impairment is associated with many diseases. A highly smart particle is required for understanding detailed sequential cellular events in phagocytosis. Recently, we identified an Indian traditional medicine named Godanti Bhasma (GB), a bioactive calcium sulfate particle prepared by thermo-transformation ofgypsum. Thermal processing of the gypsum transforms its native physicochemical properties by removing water molecules into the anhydrous GB, which was confirmed by Raman and FT-IR spectroscopy. GB particle showed a 0.5–5 µm size range and a neutral surface charge. Exposure of mammalian cells to GB particles showed a rapid cellular uptake through phagocytosis and induced massive cytoplasmic vacuolation in cells. Interestingly, no cellular uptake and cytoplasmic vacuolation were observed with the parent gypsum particle. The presence of the GB particles in intra-vacuolar space was confirmed using FESEM coupled with EDX. Flow cytometry analysis and live tracking of GB-treated cells showed particle internalization, vacuole formation, particle dissolution, and later vacuolar turnover. Quantification of GB-induced vacuolation was done using neutral red uptake assay in cells. Treatment of lysosomal inhibitors (BFA1 or CQ) with GB could not induce vacuolation, suggesting the requirement of an acidic environment for the vacuolation. In the mimicking experiment, GB particle dissolution in acidic cell-free solution suggested that degradation of GB occurs by acidic pH inside the cell vacuole. Vacuole formation generally accompanies with cell death, whereas GB-induced massive vacuolation does not cause cell death. Moreover, the cell divides and proliferates with the vacuolar process, intra-vacuolar cargo degradation, and eventually vacuolar turnover. Taken together, the sequential cellular events in this study suggest that GB can be used as a smart particle for phagocytosis assay development in animal cells.

Internalization of therapeutic antibodies into dendritic cells as a risk factor for immunogenicity.

Immunogenicity, the unwanted immune response triggered by therapeutic antibodies, poses significant challenges in biotherapeutic development. This response can lead to the production of anti-drug antibodies, potentially compromising the efficacy and safety of treatments. The internalization of therapeutic antibodies into dendritic cells (DCs) is a critical factor influencing immunogenicity. Using monoclonal antibodies, with differences in non-specific cellular uptake, as tools to explore the impact on the overall risk of immunogenicity, this study explores how internalization influences peptide presentation and subsequently T cell activation. To investigate the impact of antibody internalization on immunogenicity, untargeted toolantibodies with engineered positive or negative charge patches were utilized. Immature monocyte-derived DCs (moDCs), known for their physiologically relevant high endocytic activity, were employed for internalization assays, while mature moDCs were used for MHC-II associated peptide proteomics (MAPPs) assays. In addition to the lysosomal accumulation and peptide presentation, subsequent CD4+ T cell activation has been assessed. Consequently, a known CD4+ T cell epitope from ovalbumin was inserted into the tool antibodies to evaluate T cell activation on a single, shared epitope. Antibodies with positive charge patches exhibited higher rates of lysosomal accumulation and epitope presentation compared to those with negative charge patches or neutral surface charge. Furthermore, a direct correlation between internalization rate and presentation on MHC-II molecules could be established. To explore the link between internalization, peptide presentation and CD4+ T cell activation, tool antibodies containing the same OVA epitope were used. Previous observations were not altered by the insertion of the OVA epitope and ultimately, an enhanced CD4+ T cell response correlated with increased internalization in DCs and peptide presentation. These findings demonstrate that the biophysical properties of therapeutic antibodies, particularly surface charge, play a crucial role in their internalization into DCs. Antibodies internalized faster and processed by DCs, are also more prone to be presented on their surface leading to a higher risk of triggering an immune response. These insights underscore the importance of considering antibody surface charge and other properties that enhance cellular accumulation during the preclinical development of biotherapeutics to mitigate immunogenicity risks.

Engineering hyaluronic acid-based nanoassemblies for monoclonal antibody delivery – design, characterization, and biological insights

AbstractThe current spotlight of cancer therapeutics is shifting towards personalized medicine with the widespread use of monoclonal antibodies (mAbs). Despite their increasing potential, mAbs have an intrinsic limitation related to their inability to cross cell membranes and reach intracellular targets. Nanotechnology offers promising solutions to overcome this limitation, however, formulation challenges remain. These challenges are the limited loading capacity (often insufficient to achieve clinical dosing), the complex formulation methods, and the insufficient characterization of mAb-loaded nanocarriers. Here, we present a new nanocarrier consisting of hyaluronic acid-based nanoassemblies (HANAs) specifically designed to entrap mAbs with a high efficiency and an outstanding loading capacity (50%, w/w). HANAs composed by an mAb, modified HA and phosphatidylcholine (PC) resulted in sizes of ~ 100 nm and neutral surface charge. Computational modeling identified the principal factors governing the high affinity of mAbs with the amphiphilic HA and PC. HANAs composition and structural configuration were analyzed using the orthogonal techniques cryogenic transmission electron microscopy (cryo-TEM), asymmetrical flow field-flow fractionation (AF4), and small-angle X-ray scattering (SAXS). These techniques provided evidence of the formation of core-shell nanostructures comprising an aqueous core surrounded by a bilayer consisting of phospholipids and amphiphilic HA. In vitro experiments in cancer cell lines and macrophages confirmed HANAs’ low toxicity and ability to transport mAbs to the intracellular space. The reproducibility of this assembling process at industrial-scale batch sizes and the long-term stability was assessed. In conclusion, these results underscore the suitability of HANAs technology to load and deliver biologicals, which holds promise for future clinical translation.

Ionizable nanoemulsions for RNA delivery into the central nervous system – importance of diffusivity

Lipid nanoparticles (LNPs) currently dominate the RNA delivery landscape; however their limited diffusivity hampers targeted tissue dissemination, and, hence, their capacity for intracellular drug delivery. This is especially relevant for tissues such as the central nervous system (CNS), where overcoming proactive brain barriers is crucial for the efficacy of genetic therapeutics. This research aimed to create ionizable nanoemulsions (iNEs), a new generation of RNA delivery systems with enhanced diffusivity. The developed iNEs (consisting of the combination of C12–200, DOPE, Vitamin E, and DMG-PEG) with a size below 100 nm, neutral surface charge, and high RNA loading capacity, showed excellent cell viability and transfection efficiency in various cellular models, including neurons, astrocytes, and microglia. Subsequently, iNEs containing mRNA GFP were tested for CNS transfection, highlighting their exceptional diffusivity and selective transfection of neurons following intra-parenchymal administration.

“Click” amphotericin B in prodrug nanoformulations for enhanced systemic fungemia treatment

Severe nephrotoxicity and infusion-related side effects pose significant obstacles to the clinical application of Amphotericin B (AmB) in life-threatening systemic fungal infections. In pursuit of a cost-effective and safe formulation, we have introduced multiple phenylboronic acid (PBA) moieties onto a linear dendritic telodendrimer (TD) scaffold, enabling effective AmB conjugation via boronate chemistry through a rapid, high yield, catalysis-free and dialysis-free “Click” drug loading process. Optimized AmB-TD prodrugs self-assemble into monodispersed micelles characterized by small particle sizes and neutral surface charges. AmB prodrugs sustain drug release in circulation, which is accelerated in response to the acidic pH and Reactive Oxygen Species (ROS) in the infection and inflammation. Prodrugs mitigate the AmB aggregation status, reduce cytotoxicity and hemolytic activity compared to Fungizone®, and demonstrate superior antifungal activity to AmBisome®. AmB-PEG5kBA4 has a comparable maximum tolerated dose (MTD) to AmBisome®, while over 20-fold increase than Fungizone®. A single dose of AmB-PEG5kBA4 demonstrates superior efficacy to Fungizone® and AmBisome® in treating systemic fungal infections in both immunocompetent and immunocompromised mice.

Biocompatible nanoparticles for metals removal from fresh water with potential for rare earth extraction applications

Freshwater contamination by metals can come from a variety of sources and be damaging to wildlife, alter landscapes, and impact human health. Metals removal is desirable not only for improving water quality and preventing adverse effects but also for metals collection and recycling. Nanoadsorption of metals is economically feasible and nanoscale materials exhibit a high surface-area-to-volume ratio that is promising for high adsorption and reactivity. However, the extraordinarily small dimensions of these materials allow them to maneuver biological systems, and combined with high reactivity, this translocation can result in toxicity. In this work, nanoparticles (NPs) composed of a magnetite core coated in hydroxyapatite (HA) and functionalized for adsorption with titanium dioxide (TiHAMNPs) were synthesized. The magnetic core enabled NP retrieval, while HA enhanced adsorption and minimized toxicity. Here, synthesis and characterization are presented, revealing a stable NP structure exhibiting a near neutral surface charge. Results of adsorption studies showed that as compared to silica-coated magnetite nanoparticles (SiMNPs), traditionally used for this application, TiHAMNPs exhibited significantly higher adsorption (43.28% more Cu removal) after 24 h. The equilibrium rate constant for the adsorption of Cu by TiHAMNPs was 0.0003 g/(min*mg) and TiHAMNP adsorption data indicated that TiHAMNPs adsorb metals in a monolayer at the particle surface with a maximum capacity of 2.8 mmol/g. Metabolic and toxicity assays showed TiHAMNPs were highly biocompatible as compared to SiMNPs. This work also explores rare earth element (REE) separation applications of TiHAMNPs, finding that TiHAMNPs may provide a promising alternative for REE retrieval and/or separation.

Surface charge-dependent cytokine production using near-infrared emitting silicon quantum dots

Toll-like receptor 9 (TLR-9) is a protein that helps our immune system identify specific DNA types. Upon detection, CpG oligodeoxynucleotides signal the immune system to generate cytokines, essential proteins that contribute to the body’s defence against infectious diseases. Native phosphodiester type B CpG ODNs induce only Interleukin-6 with no effect on interferon-α. We prepared silicon quantum dots containing different surface charges, such as positive, negative, and neutral, using amine, acrylate-modified Plouronic F-127, and Plouronic F-127. Then, class B CpG ODNs are loaded on the surface of the prepared SiQDs. The uptake of ODNs varies based on the surface charge; positively charged SiQDs demonstrate higher adsorption compared to SiQDs with negative and neutral surface charges. The level of cytokine production in peripheral blood mononuclear cells was found to be associated with the surface charge of SiQDs prior to the binding of the CpG ODNs. Significantly higher levels of IL-6 and IFN-α induction were observed compared to neutral and negatively charged SiQDs loaded with CpG ODNs. This observation strongly supports the notion that the surface charge of SiQDs effectively regulates cytokine induction.

Evaluating a targeted Palbociclib-Trastuzumab loaded smart niosome platform for treating HER2 positive breast cancer cells

In this study, we present a targeted and pH-sensitive niosomal (pHSN) formulation, incorporating quantum dot (QD)-labeled Trastuzumab (Trz) molecules for the specific delivery of Palbociclib (Pal) to cells overexpressing human epidermal growth factor receptor 2 (HER2). FTIR analyses confirmed the successful preparation of the pHSNs and their bioconjugation. The labeled Trz-conjugated Pal-pHSNs (Trz-Pal-pHSNs) exhibited a size of approximately 170 nm, displaying a spherical shape with a neutral surface charge of −1.2 mV. Pal encapsulation reached ∼86%, and the release pattern followed a two-phase pH-dependent mechanism. MTT assessments demonstrated enhanced apoptosis induction, particularly in HER2-positive cells, by Trz-Pal-pHSNs. Fluorescence imaging further validated the internalization of particles into cells. In conclusion, Trz-Pal-pHSNs emerge as a promising platform for personalized medicine in the treatment of HER2-positive breast cancer.

Nanoparticles targeting the intestinal Fc receptor enhance intestinal cellular trafficking of semaglutide

Semaglutide is the first oral glucagon-like peptide-1 (GLP-1) analog commercially available for the treatment of type 2 diabetes. In this work, semaglutide was incorporated into poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticles (NPs) to improve its delivery across the intestinal barrier. The nanocarriers were surface-decorated with either a peptide or an affibody that target the human neonatal Fc receptor (hFcRn), located on the luminal cell surface of the enterocytes. Both ligands were successfully conjugated with the PLGA-PEG via maleimide-thiol chemistry and thereafter, the functionalized polymers were used to produce semaglutide-loaded NPs. Monodisperse NPs with an average size of 170 nm, neutral surface charge and 3% of semaglutide loading were obtained. Both FcRn-targeted NPs exhibited improved interaction and association with Caco-2 cells (cells that endogenously express the hFcRn), compared to non-targeted NPs. Additionally, the uptake of FcRn-targeted NPs was also observed to occur in human intestinal organoids (HIOs) expressing hFcRn through microinjection into the lumen of HIOs, resulting in potential increase of semaglutide permeability for both ligand-functionalized nanocarriers. Herein, our study demonstrates valuable data and insights that the FcRn-targeted NPs has the capacity to promote intestinal absorption of therapeutic peptides.

In silico and in vitro assessment of an optimized QbD-guided myoinositol and metformin-loaded mucus-penetrating particle-based gel for the amelioration of PCOS.

Polycystic ovarian syndrome (PCOS) is a multi-factorial endocrine disorder affecting women of reproductive age. However, its high prevalence and the unsuccessful translation of conventional modalities have made PCOS a pharmaco-therapeutic challenge. In the present study, we explored bi-formulations (comprising metformin-loaded mucus-penetrating nanoparticles, MTF-MPPs, and myoinositol-loaded mucus-penetrating particles, MI-MPPs) incorporated in a carbomer gel tailored for intravaginal administration. For the development and optimization of the MPPs-gel, a QbD (quality by design) approach was employed, including the initial and final risk assessment, central composite design of experts, and method validation. The optimized MTF-MPPs and MI-MPPs possessed an optimum nanometric particle size (195.0 nm and 178.8 nm, respectively) and a PDI of 0.150 and 0.123, respectively, together with a negligible negative zeta potential (-5.19 mV and -6.19 mV, respectively) through the vaginal mucus. It was observed that the MPPs are small and monodisperse with a neutral surface charge. It was observed that the MPPs-gel formulations released approximately 69.86 ± 4.65% of MTF and 67.14 ± 5.74% of MI within 120 h (5 days), which was observed to be sustained unlike MFT-MI-gel with approximately 94.89 ± 4.17% of MTF and 90.91 ± 15% of MI drugs released within 12 h. The confocal microscopy study of rhodamine-loaded MPPs indicated that they possessed a high fluorescence intensity at a depth of 15 μm, while as the penetration trajectory in the vaginal tissue increased to 35 μm, their intensity was reduced, appearing to be more prominent in the blood vessels. The analyzed data of MPPs-gel suggest that the optimized MPPs-gel formulation has potential to reach the targeted area via the uterovaginal mucosa, which has a wide network of blood vessels. Subsequently, in vivo studies were conducted and the results revealed that the proposed MPPs-gel formulation could regulate the estrous cycle of the reproductive system compared to the conventional formulation. Moreover, the formulation significantly reduced the weight of the ovaries compared to the control and conventional vaginal gel. Biochemical estimation showed improved insulin and sex hormone levels. Thus, the obtained data revealed that the deep penetration and deposition of MTF and MI on the targeted area through intravaginal delivery resulted in better therapeutic effects than the conventional vaginal gel. The obtained results confirmed the amelioration of PCOS upon treatment using the prepared MPPs-gel formulation. According to the relevant evaluation studies, it was concluded that MPPs-gel was retained in the vaginal cavity for systemic effects. Also, the sustained and non-irritating therapeutic effect meets the safety aspects. This work serves as a promising strategy for intravaginal drug delivery.

Intracellular trafficking kinetics of nucleic acid escape from lipid nanoparticles via fluorescence imaging.

Lipid nanoparticles (LNPs) are one of the most clinically advanced candidates for delivering nucleic acids to target cell populations, such as hepatocytes. Once LNPs are endocytosed, they must release their nucleic acid cargo into the cell cytoplasm. For delivering messenger RNA (mRNA), delivery into the cytosol is sufficient; however, for delivering DNA, there is an added diffusional barrier needed to facilitate nuclear uptake for transcription and therapeutic effect. Here, we use fluorescence microscopy to investigate the intracellular fate of different LNP formulations to determine the kinetics of localization to endosomes and lysosomes. LNPs used in the studies were prepared via self-assembly using a NanoAssemblr for microfluidic mixing. As the content of polyethylene glycol (PEG) within the LNP formulation influences cellular uptake by hepatocyte cells, the content and hydrocarbon chain length within the formulation were assessed for their impact on intracellular trafficking. Standard LNPs were then formed using three commercially available ionizable lipids, Dlin-MC3-DMA (MC3), Dlin-KC2-DMA (KC2), and SS-OP. Plasmid DNA (pDNA) and mRNA were used, more specifically with a mixture of Cyanine 3 (Cy3)-labeled and green fluorescence protein (GFP) producing plasmid DNA (pDNA) as well as Cy5-labeled GFP producing mRNA. After formulation, LNPs were characterized for the encapsulation efficiency of the nucleic acid, hydrodynamic diameter, polydispersity, and zeta potential. All standard LNPs were ~100 nm in diameter and had neutral surface charge. All LNPs resulted in encapsulation efficiency greater than 70%. Confocal fluorescence microscopy was used for the intracellular trafficking studies, where LNPs were incubated with HuH-7 hepatocyte cells at times ranging from 0-48 h. The cells were antibody-stained for subcellular components, including nuclei, endosomes, and lysosomes. Analysis was performed to quantify localization of pDNA to the endosomes and lysosomes. LNPs with 1.5 mol% PEG and a hydrocarbon chain C14 resulted in optimal endosomal escape and GFP production. Results from this study demonstrate that a higher percentage of C14 PEG leads to smaller LNPs with limited available phospholipid binding area for ApoE, resulting in decreased cellular uptake. We observed differences in the localization kinetics depending on the LNP formulation type for SS-OP, KC2, and MC3 ionizable lipids. The results also demonstrate the technique across different nucleic acid types, where mRNA resulted in more rapid and uniform GFP production compared to pDNA delivery. Here, we demonstrated the ability to track uptake and the sub-cellular fate of LNPs containing pDNA and mRNA, enabling improved screening prior to in vivo studies which would aid in formulation optimization.

TPGS/soluplus® blended micelles: an effective strategy for improving loading capacity of ferroptosis inducer erastin

Erastin is the most classical small molecular ferroptosis inducer but its clinical application is hindered due to its poor solubility. Nanotechnology has been used to improve ferroptosis inducer erastin’s solubility. The recently reported erastin-incorporated nanosystems (e.g., nanoparticulate ferritin, poly(ethylene glycol) methyl ether-blockpoly(lactide-co-glycolide) (mPEG-PLGA) nanoparticle, exosome, and liposome) could to some degree elevate its solubility, but were still poor in loading capacity. To be specific, the maximum entrapment efficiency and drug loading in previous studies were approximately 36% and 3.7%, respectively. In order to improve drug loading capacity, we constructed a novel carrier based on Vitamin E d-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) and polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus®) blended micelles. The erastin@TPGS/Soluplus® micelles were prepared by thin film hydration method, and formulation details including dosage of Soluplus®, the weight ratio of TPGS to Soluplus®, the weight ratio of erastin to TPGS/Soluplus® were optimized. The physicochemical properties of the erastin micelles were measured by DLS, TEM, FTIR, and TG-DSC analysis. The products exhibited a nanoscale and uniform particle size (78.11 ± 0.54 nm) and neutral surface charge (-0.43 ± 0.32 mV). Significantly improved entrapment efficiency (89.63 ± 1.81%) and drug loading rate (10.03 ± 0.14%) were obtained, which was over 2-fold of current reports. The in vitro release study demonstrated that erastin could release in a sustained yet complete manner, and the drug release might be governed by multiple mechanisms including anomalous diffusion of non-Fickian diffusion, matrix erosion and sustained release. In summary, this proposed erastin nanosystem provides a new strategy for improving loading capacity, and may probably augment ferroptosis-inducing therapy for cancer treatment.

Enhancement of antimicrobial and antibiofilm activities of liposomal fatty acids

Microbial biofilms are protected surface-attached communities of bacteria or fungi with high drug tolerance that typically cause persistent infections. Smart drug carriers are being explored as a promising platform of antimicrobials to address their recalcitrance to antibiotic agents and minimize the side effects of current therapies. In this study, soy lecithin liposomes loaded with lauric acid (LA) and myristoleic acid (MA) were formulated using an emulsification method, and their antibiofilm properties were evaluated. The physio-chemical properties of the most potent liposome were characterized using a zeta sizer, transmission electron microscopy (TEM), fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. TEM and zeta sizer analysis of the liposome revealed a homogeneous spherical structure with an average size of 159.2 nm and zeta potential of − 5.4 mV. The unilamellar liposomes loaded with LA at 0.1–0.5 µg/mL achieved obvious antibiofilm efficiency against Staphylococcus aureus and Candida albicans and their dual biofilms. Also, LA-loaded liposome formulation efficiently disrupted preformed biofilms of S. aureus and C. albicans. Furthermore, formulated liposomal LA (0.1 µg/mL) exhibited 100-fold increased dual biofilm inhibition compared to LA alone. The single biofilms and dual biofilm formation on polystyrene were reduced as determined by 3D-bright field and scanning electron microscopy. Zeta potential measurements exhibited neutralized surface charge of S. aureus, and the liposomes inhibited hyphae formation in C. albicans. These findings demonstrated that the LA-incorporated liposomes have great potential to become a new, effective, and good antibiofilm agent for treating S. aureus and C. albicans infections.

Drug repurposing for the treatment of COVID-19: Targeting nafamostat to the lungs by a liposomal delivery system

Despite tremendous global efforts since the beginning of the COVID-19 pandemic, still only a limited number of prophylactic and therapeutic options are available. Although vaccination is the most effective measure in preventing morbidity and mortality, there is a need for safe and effective post-infection treatment medication.In this study, we explored a pipeline of 21 potential candidates, examined in the Calu-3 cell line for their antiviral efficacy, for drug repurposing. Ralimetinib and nafamostat, clinically used drugs, have emerged as attractive candidates. Due to the inherent limitations of the selected drugs, we formulated targeted liposomes suitable for both systemic and intranasal administration. Non-targeted and targeted nafamostat liposomes (LipNaf) decorated with an Apolipoprotein B peptide (ApoB-P) as a specific lung-targeting ligand were successfully developed. The developed liposomal formulations of nafamostat were found to possess favorable physicochemical properties including nano size (119–147 nm), long-term stability of the normally rapidly degrading compound in aqueous solution, negligible leakage from the liposomes upon storage, and a neutral surface charge with low polydispersity index (PDI). Both nafamostat and ralimetinib liposomes showed good cellular uptake and lack of cytotoxicity, and non-targeted LipNaf demonstrated enhanced accumulation in the lungs following intranasal (IN) administration in non-infected mice. LipNaf retained its anti-SARS-CoV 2 activity in Calu 3 cells with only a modest decrease, exhibiting complete inhibition at concentrations >100 nM. IN, but not intraperitoneal (IP) treatment with targeted LipNaf resulted in a trend to reduced viral load in the lungs of K18-hACE2 mice compared to targeted empty Lip. Nevertheless, upon removal of outlier data, a statistically significant 1.9-fold reduction in viral load was achieved. This observation further highlights the importance of a targeted delivery into the respiratory tract. In summary, we were able to demonstrate a proof-of-concept of drug repurposing by liposomal formulations with anti-SARS-CoV-2 activity. The biodistribution and bioactivity studies with LipNaf suggest an IN or inhalation route of administration for optimal therapeutic efficacy.

Cell uptake and intracellular trafficking of bioreducible poly(amidoamine) nanoparticles for efficient mRNA translation in chondrocytes.

Disulfide-containing poly(amidoamine) (PAA) is a cationic and bioreducible polymer, with potential use as a nanocarrier for mRNA delivery in the treatment of several diseases including osteoarthritis (OA). Successful transfection of joint cells with PAA-based nanoparticles (NPs) was shown previously, but cell uptake, endosomal escape and nanoparticle biodegradation were not studied in detail. In this study, C28/I2 human chondrocytes were transfected with NPs co-formulated with a PEG-polymer coating and loaded with EGFP mRNA for confocal imaging of intracellular trafficking and evaluation of transfection efficiency. Compared with uncoated NPs, PEG-coated NPs showed smaller particle size, neutral surface charge, higher colloidal stability and superior transfection efficiency. Furthermore, endosomal entrapment of these PEG-coated NPs decreased over time and mRNA release could be visualized both in vitro and in live cells. Importantly, cell treatment with modulators of the intracellular reducing environment showed that glutathione (GSH) concentrations affect translation of the mRNA payload. Finally, we applied a D-optimal experimental design to test different polymer-to-RNA loading ratios and dosages, thus obtaining an optimal formulation with up to ≈80% of GFP-positive cells and without toxic effects. Together, the biocompatibility and high transfection efficiency of this system may be a promising tool for intra-articular delivery of therapeutical mRNA in OA treatment.

Statistical modelling and kinetics of copper loss to printed circuit board (PCB) fibreglass during PCB ammine leaching

ABSTRACT PCB dust leach feed contains fibreglass particles responsible for incomplete copper recovery observed during PCB dust leaching. Interaction of fibreglass particles with ammine leach solution was studied by varying contact time, operating temperature and pulp density at different initial concentrations. Box Behnken RSM design was utilised to design the experimental tests. The 3D plots, ANOVA, Pearson correlation and multiple regression analysis show the initial copper concentration impacts the response QCu most. Of the five non-linear adsorption kinetic models employed, Elovich and Avrami's kinetic model best fitted the experimental data with R2 of 0.9632 and 0.9435, respectively. The pHpzc analysis of fibreglass particles shows the material possesses a neutral surface charge at pH = 7.1. Evaluation of the kinetic models and pHpzc analysis of copper losses to PCB fibreglass particles supports dual mechanisms of physisorption and ion exchange co-occurring in the solid–liquid systems. At 1 g fibreglass particles in 100 mL of 50 mg/L copper ammine leach solution, maximum loss of about 1.5 mg/L copper was obtained in 12 h. This clearly indicates limitation to maximum target metal recovery. Copper loss to fibreglass particles occurs during PCB leaching up to an amount which depends on the mass fraction of the glass fiber particles in the pulp, leach solution concentration and time.