Efficient nuclear delivery of DNA remains a significant challenge for non-viral vectors, limiting their broader application in gene therapy. A key intracellular barrier is represented by the nuclear envelope, where the nuclear pore complex (NPC) restricts passive diffusion of macromolecules, and prevents more than 97% of delivered cytosolic DNA from entering the nucleus. Our study addresses this major limitation with a targeted delivery strategy leveraging nuclear localization signaling (NLS) peptides to enhance nuclear import of plasmid DNA (pDNA). NLS peptides were non-covalently complexed with pDNA and co-encapsulated in lipid nanoparticles (LNPs). Using fluorescently labeled LNPs and pDNA, the intracellular trafficking behavior from cellular uptake to endosomal escape and nuclear entry was characterized with and without the co-encapsulation of the NLS peptide. pDNA-NLS-functionalized LNPs achieved a 2.5-fold increase in transfection efficiency compared to non-complexed controls in vitro. Mechanistic studies indicate that cytosolic importins recognize the co-delivered NLS peptides, facilitating active transport of the released pDNA through the nuclear pore complex. In vivo studies in zebrafish larvae and wildtype mice are consistent with an NLS-dependent enhancement in gene expression, displaying a 10-fold increase after 72 h in mice. These findings demonstrate that LNPs can be combined with NLS peptides to promote non-viral DNA delivery to the nucleus by endogenous nuclear import pathways.

The water-soluble and biocompatible synthetic polypeptide poly(sarcosine) (pSar) can be used as an alternative to polyethylene glycol for the steric stabilization of lipid nanoparticles (LNPs). In the present study, a pSar lipid conjugate, mPep14-C14 (mPep-001), was synthesized by solid phase peptide synthesis, consisting of a monodisperse polysarcosine chain units (degree of polymerization: 14) linked via serine residue to two myristic acid chains. This lipopeptide was characterized and compare to clinically used DMG-PEG2000. pDNA-LNPs prepared with mPep14-C14 had a 2-fold increased transfection efficiency in vitro using human hepatoma HuH-7 cells. In vivo, using zebrafish larvae as a vertebrate screening model, mPep14-C14 pDNA-LNPs showed more than 6-fold higher systemic exposure, enhanced transgene expression, limited macrophage uptake, and no apparent signs of acute toxicity. Notably, mPep14-C14 induced distinct bleb-like protrusions on the LNP surface, possibly contributing to improved transfection efficiency. We conclude that mPep14-C14 combines efficient steric stabilization with superior transgene expression, demonstrating its potential as a promising PEG-free lipid alternative for pDNA-based gene delivery systems.​

Intravital lung imaging has been employed to study physiological and pathophysiological processes related to nanoparticle deposition in the alveolar lung, particularly in the context of air pollution and drug delivery. However, optical imaging depth is limited, often attributed to the refractive index (RI) mismatch at the alveolar air-tissue interface. To investigate this, we evaluated two complementary strategies. First, we demonstrated that eliminating the RI mismatch via partial liquid ventilation with oxygenated perfluorocarbon (PFC) did not enhance the imaging depth. A second approach, utilizing ex vivo optical tissue clearing (with RI matching), was only successful in improving imaging penetration depth if it included removal of scattering lipids such as pulmonary surfactant. Nevertheless, partial liquid ventilation with PFC in vivo enabled the homogeneous delivery of nanoparticles to the alveoli, allowing real-time observation of their interactions with lung epithelium. This finding opens new avenues for studying inhaled particulates and optimizing inhalation-based drug delivery.

The organic anion transporting polypeptide (OATP) 2B1 is considered an emerging drug transporter that is found expressed in pharmacokinetically relevant organs such as the liver, small intestine, and kidney. Despite its interaction with various substrate drugs, the understanding of its in vivo relevance is still limited. In this study, we first validated the interaction of atorvastatin with rat OATP2B1 using transiently transfected HeLa cells. Moreover, we characterized our rSlco2b1-knockout and SLCO2B1-knockin rats for mRNA, protein expression, and localization of OATP2B1 in the liver, small intestine, and kidney. The transporter showed the highest expression in the liver followed by the small intestine. In humanized rats, human OATP2B1 is localized on the sinusoidal membrane of hepatocytes. In enterocytes of wild-type and humanized rats, the transporter was detected in the luminal membrane with the vast majority being localized subapical. Subsequently, we assessed atorvastatin pharmacokinetics in male wild-type, rSlco2b1-knockout, and SLCO2B1-knockin rats after a single-dose administration (orally and intravenously). Investigating the contribution of rat OATP2B1 or human OATP2B1 to oral atorvastatin pharmacokinetics revealed no differences in concentration-time profiles or pharmacokinetic parameters. However, when comparing the pharmacokinetics of atorvastatin after intravenous administration in SLCO2B1-humanized rats and knockout animals, notable differences were observed. In particular, the systemic exposure (area under the curve) decreased by approximately 40% in humanized animals, whereas the clearance was 57% higher in animals expressing human OATP2B1. These findings indicate that human OATP2B1 influences pharmacokinetics of atorvastatin after intravenous administration, most likely by contributing to the hepatic uptake. SIGNIFICANCE STATEMENT Wild-type, rSlco2b1-knockout, and SLCO2B1-humanized Wistar rats were characterized for the expression of rat and human SLCO2B1 /OATP2B1. Pharmacokinetic studies of atorvastatin over 24 hours were conducted in male wild-type, rSlco2b1-knockout, and SLCO2B1-humanized rats. After a single-dose intravenous administration, a lower systemic exposure and an increase in clearance were observed in SLCO2B1-humanized rats compared with knockout animals indicating a contribution of OATP2B1 to the hepatic clearance.

Lipid nanoparticles (LNPs) are successfully used for RNA-based gene delivery. In the context of gene replacement therapies, however, delivery of DNA expression plasmids using LNPs as a non-viral vector could be a promising strategy for the induction of longer-lasting effects.Therefore, DNA expression plasmids (3 to 4 kbp) coding for fluorescent markers or luciferase were combined with LNPs. Different clinically used ionizable lipids (DLin-MC3-DMA, SM-102, and ALC-0315) were tested to compare their influence on DNA plasmid delivery.DNA-LNPs were characterized with respect to their colloidal properties (size, polydispersity, ζ-potential, morphology), in vitro performance (cellular uptake, DNA delivery, and gene expression), and in vivo characteristics (biodistribution and luciferase gene expression). At an optimized N/P ratio of 6, spherical, small and monodisperse particles with anionic ζ-potential were obtained. Efficient transgene expression was achieved with a minimum amount of 1 pg DNA per initially plated cells. Zebrafish studies allowed selection of DNA-LNPs, which demonstrated prolonged blood circulation, avoidance of macrophage clearance, and vascular extravasation.Our comparative study demonstrates a high impact of the ionizable lipid type on DNA-LNP performance. Superior transfection efficiency of DNA-LNPs containing the ionizable lipid ALC-0315 was confirmed in wildtype mice.

The mechanism governing pharmaceutical tablet disintegration is far from fully understood. Despite the importance of controlling a formulation's disintegration process to maximize the active pharmaceutical ingredient's bioavailability and ensure predictable and consistent release profiles, the current understanding of the process is based on indirect or superficial measurements. Formulation science could, therefore, additionally deepen the understanding of the fundamental physical principles governing disintegration based on direct observations of the process. We aim to help bridge the gap by generating a series of time-resolved X-ray micro-computed tomography (μCT) images capturing volumetric images of a broad range of mini-tablet formulations undergoing disintegration. Automated image segmentation was a prerequisite to overcoming the challenges of analyzing multiple time series of heterogeneous tomographic images at high magnification. We devised and trained a convolutional neural network (CNN) based on the U-Net architecture for autonomous, rapid, and consistent image segmentation. We created our own μCT data reconstruction pipeline and parameterized it to deliver image quality optimal for our CNN-based segmentation. Our approach enabled us to visualize the internal microstructures of the tablets during disintegration and to extract parameters of disintegration kinetics from the time-resolved data. We determine by factor analysis the influence of the different formulation components on the disintegration process in terms of both qualitative and quantitative experimental responses. We relate our findings to known formulation component properties and established experimental results. Our direct imaging approach, enabled by deep learning-based image processing, delivers new insights into the disintegration mechanism of pharmaceutical tablets.

Lipid nanoparticles (LNPs) have emerged as the leading nonviral nucleic acid (NA) delivery system, gaining widespread attention for their use in COVID-19 vaccines. They are recognized for their efficient NA encapsulation, modifiability, and scalable production. However, LNPs face efficacy and potency limitations due to suboptimal intracellular processing, with endosomal escape efficiencies (ESE) below 2.5%. Additionally, up to 70% of NPs undergo recycling and exocytosis after cellular uptake. In contrast, cell-derived vesicles offer biocompatibility and high-delivery efficacy but are challenging to load with exogenous NAs and to manufacture at large-scale. To leverage the strengths of both systems, a hybrid system is designed by combining cell-derived vesicles, such as nano plasma membrane vesicles (nPMVs), with LNPs through microfluidic mixing and subsequent dialysis. These hybrids demonstrate up to tenfold increase in ESE and an 18-fold rise in reporter gene expression in vitro and in vivo in zebrafish larvae (ZFL) and mice, compared to traditional LNPs. These improvements are linked to their unique physico-chemical properties, composition, and morphology. By incorporating cell-derived vesicles, this strategy streamlines the development process, significantly enhancing the efficacy and potency of gene delivery systems without the need for extensive screening.

Lipid conjugates have advanced the field of lipid-based nanomedicine by promoting active-targeting (ligand, peptide, antibody), stability (PEGylation), controlled release (lipoid prodrug), and probe-based tracking (fluorophore). Recent findings indicate lipid conjugates dissociating from nanomedicine upon encountering a biological environment. Yet, implications for (pre)clinical outcomes remain unclear. In this study, using the zebrafish model (Danio rerio), we investigated the fate of liposome-incorporated lipid fluorophore conjugates (LFCs) after intravenous (IV) administration. LFCs having a bilayer mismatch and relatively polar fluorophore revealed counter-predictive outcomes for Caelyx/Doxil (clearance vs. circulating) and AmBisome-like liposomes (scavenger endothelial cell vs. macrophage uptake). Findings on LFC (mis)match for Caelyx/Doxil-like liposomes were supported by translational intravital imaging studies in mice. Importantly, contradicting observations suggest to originate from LFC dissociation in vivo, which was investigated by Asymmetric Flow Field-Flow Fractionation (AF4) upon liposome-serum incubation in situ. Our data suggests that LFCs matching with the liposome bilayer composition - that did not dissociate upon serum incubation - revealed improved predictive outcomes for liposome biodistribution profiles. Altogether, this study highlights the critical importance of fatty acid tail length and headgroup moiety when selecting lipid conjugates for lipid-based nanomedicine.

Extracellular vesicles (EVs) are highly interesting for the design of next-generation therapeutics. However, their preparation methods face challenges in standardization, yield, and reproducibility. Here, we describe a highly efficient and reproducible EV preparation method for monodisperse nano plasma membrane vesicles (nPMVs), which yields 10 to 100 times more particles per cell and hour than conventional EV preparation methods. nPMVs are produced by homogenizing giant plasma membrane vesicles following cell membrane blebbing and apoptotic body secretion induced by chemical stressors. nPMVs showed no significant differences compared to native EVs from the same cell line in cryo-TEM analysis, in vitro cellular interactions, and in vivo biodistribution studies in zebrafish larvae. Proteomics and lipidomics, on the other hand, suggested substantial differences consistent with the divergent origin of these two EV types and indicated that nPMVs primarily derive from apoptotic extracellular vesicles. nPMVs may provide an attractive source for developing EV-based pharmaceutical therapeutics.

Polyethlyenimine (PEI) was introduced 1995 as a cationic polymer for nucleic acid delivery. PEI and its derivatives are extensively used in basic research and as reference formulations in the field of polymer-based gene delivery. Despite its widespread use, the number of clinical applications to date is limited. Thus, this review aims to consolidate the past applications of PEI in DNA delivery, elucidate the obstacles that hinder its transition to clinical use, and highlight potential prospects for novel iterations of PEI derivatives. The present review article is divided into three sections. The first section examines the mechanism of action employed by PEI, examining fundamental aspects of cellular delivery including uptake mechanisms, release from endosomes, and transport into the cell nucleus, along with potential strategies for enhancing these delivery phases. Moreover, an in-depth analysis is conducted concerning the mechanism underlying cellular toxicity, accompanied with approaches to overcome this major challenge. The second part is devoted to the in vivo performance of PEI and its application in various therapeutic indications. While systemic administration has proven to be challenging, alternative localized delivery routes hold promise, such as treatment of solid tumors, application as a vaccine, or serving as a therapeutic agent for pulmonary delivery. In the last section, the outcome of completed and ongoing clinical trials is summarized. Finally, an expert opinion is provided on the potential of PEI and its future applications. PEI-based formulations for nucleic acid delivery have a promising potential, it will be an important task for the years to come to introduce innovations that address PEI-associated shortcomings by introducing well-designed PEI formulations in combination with an appropriate route of administration.

Removal of particulate materials that would otherwise cumulate within the airspace and hinder the gas exchange is one of the central processes of maintaining lung homeostasis. While the importance of the particle uptake by alveolar macrophages and their expulsion via the airways mucociliary escalator is well established, very little is known about the alternative route for removing the particles via direct crossing the lung epithelium for transfer into the pulmonary lymph and bloodstream. This study dissected sequential mechanisms involved in nanoparticle transcytosis through the alveolar epithelial cell layer. By a combination of live cell, super resolution, and electron microscopy and RNA interference study, we have dissected temporal steps of nanoparticle transcytosis through alveolar epithelium. Our study revealed that caveolin is essential for the firm adhesion of the silica nanoparticle agglomerates to the apical membrane and their subsequent rapid internalization with the help of macropinocytic elements C-terminal-binding protein1 and Rabankyrin-5 but not dynamin. Actin, but not microtubules, played a major role in nanoparticle uptake and subsequent transportation. The compartments with nanoparticles were tethered to trans-Golgi network to be jointly transported along actin stress fibers across the cytoplasm, employing a myosin-dependent mechanism. The trans-Golgi nanoparticle transport machinery was positive to Rab6A, a marker linked to vesicle exocytosis. Exocytosis was primarily occurring at the basolateral plane of the alveolar epithelial cells. The high-proficiency novel caveolin and Rabankyrin-5 associated uptake and transcellular transport of nanoparticles across the AEC barrier supports its importance in clearance of amorphous silica and other types of non-inflammatory nanoparticles that are rapidly removed from the lungs following their inhalation.

The delivery of nucleic acids relies on vectors that condense and encapsulate their cargo. Especially nonviral gene delivery systems are of increasing interest. However, low transgene expression levels and limited tolerability of these systems remain a challenge. The improvement of nucleic acid delivery using depolymerized chitosan-polyethylenimine DNA complexes (dCS-PEI/DNA) is investigated. The secore complexes are further combined with chitosan-based shells and functionalized with polyethylene glycol (PEG) and cell penetrating peptides. This modular approach allows to evaluate the effect of functional shell components on physicochemical particle characteristics and biological effects. The optimized ternary complex combines a core-dCS-linear PEI/DNA complex with a shell consisting of dCS-PEG-COOH, which results in improved nucleic acid encapsulation, cellular uptake and transfection potency in human hepatoma HuH-7cells and murine primary hepatocytes. Effects on transgene expression are confirmed in wild-type mice following retrograde intrabiliary infusion. After administration of only 100 ng complexed DNA, ternary complexes induced a high reporter gene signal for three days. It is concluded that ternary coreshell structured nanoparticles comprising functionalized chitosan can be used for in vitro andin vivo gene delivery.

Hydroxychloroquine (HCQ) is a quinoline derivate used for the treatment of malaria and rheumatoid disorders. During early phases of the SARS-CoV2 (COVID-19) pandemic, preliminary and later not substantiated reports suggested that HCQ might benefit COVID-19 patients. This had sparked a worldwide and rapidly rising demand for HCQ drug products. Consequently, patients with pre-existing rheumatic diseases in Switzerland were confronted with an acute drug shortage.We have therefore designed, produced and characterized a generic HCQ drug formulation. The proposed HCQ formulation can be manufactured by using a minimal number of operation steps (mixing, wet granulation, sieving, blending, compression) and readily available pharmaceutical excipients.HCQ tablets were manufactured by granulation of the active pharmaceutical ingredient (API), blending with the external phase and compaction using a non instrumented single punch tablet press. Analytics and identification of the API was performed by a combination of NMR, ESI-MS, FTIR and HPLC. HCQ tablets met the quality criteria for an immediate release HCQ dosage form.We hope that free access to non-proprietary protocols covering analytical procedures, formulation design, and manufacturing instructions for HCQ tablets will help to bridge existing and future supply chain gaps.

Liposomal formulations are frequently used for oral, topical, or parenteral drug administration. However, liposome manufacturing and industrial scale-up remains a challenge, in particular if it comes to the preparation of liposome populations with a homogenous size distribution. Therefore, extrusion through filter membranes with defined pore size is traditionally used during the preparation of small unilamellar liposomes. Microfluidics is considered to be an alternative manufacturing method. Lipids, solvents and excipients are thereby passively mixed using a microfluidics device. While the microfluidic approach is highly scalable, most of the traditional liposome preparation protocols rely on extrusion. It was therefore the aim of the present study to compare liposomal formulations with identical composition, which were prepared using either extrusion or microfluidics protocols. Liposomal formulations produced by both methods were analyzed using dynamic light scattering (DLS) to compare size, polydispersity, and ζ-potential. Our results indicate significant differences between liposomal preparations obtained using the two manufacturing methods. We conclude that the two preparation methods should not be used interchangeably.

Buprenorphine is a short-acting analgesic drug. Its use in veterinary medicine requires several injections per day to alleviate pain in animals. We, therefore, designed a poly-lactic-co-glycolic acid (PLGA) based depot formulation of the opioid to prolong the analgesic effect. We characterized our novel microparticulate depot formulation with emphasis on a potential future product development in the present work. Microparticles showed low residual moisture levels and did meet bacterial endotoxin requirements defined by the European Pharmacopeia. Depot formulation showed physicochemical stability over 6 months at 4 degrees C, indicating sufficient shelf life. Reconstituted formulation stored at 4 degrees C showed unchanged release properties for 24 h. Terminal sterilization by x-rays with a dose of 30 kGy revealed the sensitivity of buprenorphine towards radiation, resulting in substantial drug degradation. However, microparticle properties were not affected. Our experiments indicate that sterilization of the final product is not possible, therefore requiring aseptic manufacturing protocols. In conclusion, our novel buprenorphine depot formulation based on PLGA microparticles can be stored for at least 6 month as a lyophilisate and can be used for at least 24 h after reconstitution. Pilot experiments suggest that scale-up and a future aseptic production should be feasible. We conclude that the novel depot formulation shows promising attributes, a prerequisite for future industrial production and commercialization.

There is an increasing interest in cationic polymers as important constituents of non-viral gene delivery vectors. In the present study, we developed a versatile synthetic route for the production of covalent polymeric conjugates consisting of water-soluble depolymerized chitosan (dCS; MW 6–9 kDa) and low molecular weight polyethylenimine (PEI; 2.5 kDa linear, 1.8 kDa branched). dCS-PEI derivatives were evaluated based on their physicochemical properties, including purity, covalent bonding, solubility in aqueous media, ability for DNA condensation, and colloidal stability of the resulting polyplexes. They were complexed with non-integrating DNA vectors coding for reporter genes by simple admixing and assessed in vitro using liver-derived HuH-7 cells for their transfection efficiency and cytotoxicity. Using a rational screening cascade, a lead compound was selected (dCS-Suc-LPEI-14) displaying the best balance of biocompatibility, cytotoxicity, and transfection efficiency. Scale-up and in vivo evaluation in wild-type mice allowed for a direct comparison with a commercially available non-viral delivery vector (in vivo-jetPEI). Hepatic expression of the reporter gene luciferase resulted in liver-specific bioluminescence, upon intrabiliary infusion of the chitosan-based polyplexes, which exceeded the signal of the in vivo jetPEI reference formulation by a factor of 10. We conclude that the novel chitosan-derivative dCS-Suc-LPEI-14 shows promise and potential as an efficient polymeric conjugate for non-viral in vivo gene therapy.

Metal-based nanoparticles are clinically used for diagnostic and therapeutic applications. After parenteral administration, they will distribute throughout different organs. Quantification of their distribution within tissues in the 3D space, however, remains a challenge owing to the small particle diameter. In this study, synchrotron radiation-based hard X-ray tomography (SRμCT) in absorption and phase contrast modes is evaluated for the localization of superparamagnetic iron oxide nanoparticles (SPIONs) in soft tissues based on their electron density and X-ray attenuation. Biodistribution of SPIONs is studied using zebrafish embryos as a vertebrate screening model. This label-free approach gives rise to an isotropic, 3D, direct space visualization of the entire 2.5 mm-long animal with a spatial resolution of around 2 µm. High resolution image stacks are available on a dedicated internet page (http://zebrafish.pharma-te.ch). X-ray tomography is combined with physico-chemical characterization and cellular uptake studies to confirm the safety and effectiveness of protective SPION coatings. It is demonstrated that SRμCT provides unprecedented insights into the zebrafish embryo anatomy and tissue distribution of label-free metal oxide nanoparticles.

In recent years, the stability of biotherapeutics in vivo has received increasing attention. Assessing the stability of biotherapeutics in serum may support the selection of adequate molecule candidates. In our study, we compared the physical stability of 8 different monoclonal antibodies (mAbs) in phosphate-buffered saline (PBS) and human serum. mAbs were Alexa Fluor 488-labeled and characterized with respect to fragmentation, aggregation, and proteinaceous particle formation. Samples were analyzed using size-exclusion chromatography, light obscuration, and flow imaging. In addition, novel methods such as flow cytometry and fluorescence microscopy were applied. mAbs were selected based on their hydrophobicity and isoelectric point. All mAbs studied were inherently less stable in human serum as compared to PBS. Particle size and particle counts increased in serum over time. Interestingly, certain mAbs showed significant levels of fragmentation in serum but not in PBS. We conclude that PBS cannot replicate the physical stability measured in serum. The stability of labeled mAbs in human serum did not correlate with their hydrophobicity and isoelectric point . Serum stability significantly differed amongst the tested mAbs.

In this study, we reported doxorubicin (DOX)-encapsulated nanoparticles (NPs) formulated with biocompatible and biodegradable poly (lactic-co-glycolic acid) (PLGA) and modified with a 13-amino acid peptide (S3) against sodium/potassium (Na+/K+)-ATPase pump alpha subunit to investigate its potential as antitumor agent. The morphological properties and size dispersity of the prepared nanoparticles were evaluated using scanning electron microscope (SEM) and dynamic light scattering (DLS). The encapsulation efficiency and in vitro release during 7 days were evaluated. Comparative in vitro cytotoxicity experiments demonstrated that the S3-conjugated nanoparticles (S3-PLGA-DOX NPs) had higher antiproliferative activity. Flow cytometry analysis confirmed the enhanced cellular uptake of S3-PLGA-DOX NPs in comparison with PLGA-DOX. In vivo study in 4T1 tumor-bearing BALB/C mice revealed that the S3-functionalized DOX-loaded NPs improved antitumor activity and survival rate of 4T1 tumor bearing mice. In this regard, conjugation of S3 peptide to the surface of DOX-loaded PLGA NPs provides site-specific delivery of DOX, inhibits 4T1 tumor growth in vivo and significantly decreases systemic toxicity. The obtained results suggested that the new (Na+/K+)-ATPase pump-targeted PLGA NPs as a target-selective delivery system for DOX has great potential for the treatment of breast cancer.