Tendon injuries, common in both athletic and non-athletic populations, present significant challenges due to their slow healing and the formation of scar tissue, which impairs function and potentially increases the risk of (re-)rupture. Conventional treatments often yield suboptimal functional and structural repair. This study investigates the potential of mRNA-based therapeutics to enhance tendon healing by targeting 2 distinct pathways via the delivery of chemically modified ARCA-capped mRNAs (cmRNAs) encoding Interleukin-1 receptor antagonist (IL1RA) and Platelet-Derived Growth Factor-BB (PDGF-BB) using injectable nanoparticle (NP) carriers. In vitro experiments demonstrate successful cmRNA delivery and translation, resulting in increased tendon cell proliferation, migration, and anti-inflammatory responses. In vivo, cmRNA treatment notably enhances tendon repair in a rat patellar tendon defect model, by reducing pro-inflammatory cytokines and fibrotic markers while enhancing repair tissue structure. These findings suggest that NP-based cmRNA delivery represents a promising therapeutic strategy for improving tendon healing, offering better outcomes over existing treatments by targeting both inflammatory and regenerative pathways. Statement of significance: In this study, we investigate an mRNA-based therapeutic approach aimed at enhancing tendon healing in a small animal model. Utilizing bioreducible poly(amidoamine)-based polymeric nanoparticles (PAA PNPs) for the delivery of cmRNAs encoding Interleukin-1 receptor antagonist (IL1RA) and Platelet-Derived Growth Factor-BB (PDGF-BB), we demonstrate effective delivery and protein translation in vitro and ex vivo, resulting in enhanced tendon cell proliferation, migration, and robust anti-inflammatory responses. By combining these therapeutic cmRNAs, we show improved tendon repair in vivo, with accelerated tissue regeneration, better collagen fiber organization, and signs of reduced fibrotic scarring. These findings highlight the potential of nanoparticle-mediated cmRNA delivery targeting two distinct pathways to improve tendon healing, offering a promising alternative to current treatments that often yield suboptimal results.

Osteoarthritis (OA) is a progressive and degenerative disease of the joints, characterized by inflammation and loss of cartilage. Recently, mRNA therapies have emerged as promising disease-modifying treatments for cartilage repair and regeneration. Poly(amidoamine)-based polymeric nanoparticles (PAA-based NPs) were previously developed for intracellular mRNA delivery in chondrocytes, showing high biocompatibility and transfection efficiency. In this work, we aimed to evaluate this delivery system in models simulating the complex joint environment and in vivo in rat knee joints. For this purpose, cationic uncoated NPs and neutral PEG-coated NPs were formulated to test mRNA delivery in different models: (1) a 2D culture of chondrocytes supplemented with synthetic synovial fluid, (2) a cartilage-on-chip platform, (3) an ex vivo culture of mouse knee joints, and (4) an in vivo OA rat model. In the presence of synovial fluid, the PEG-coated NPs showed favorable physicochemical properties, higher cell uptake and equivalent GFP expression as uncoated NPs in the 2D cell culture. Similar observations were made using the cartilage-on-chip platform. In contrast, both NPs appeared to display cartilage penetration and uptake by tissue-resident chondrocytes in ex vivo joint culture. Upon intra-articular administration in vivo, the PAA-based NPs did not affect cartilage integrity in healthy nor OA rat knee joints, although enhanced synovial inflammation was observed. Uncoated NPs showed prolonged retention compared to PEG-coated NPs and higher luciferase expression in OA knee joints than in healthy joints of rats, whereas no difference was found for coated NPs. These results suggest that electrostatic interactions between cationic NPs and the anionic components of the extracellular matrix play a significant role in mRNA delivery to the articular cartilage, and that disease status may affect delivery of nucleic acids dependent on NP properties. In conclusion, PAA-based NPs are a promising platform for intra-articular mRNA delivery in the joints. STATEMENT OF SIGNIFICANCE: In this study, we investigate the application of poly(amidoamine)-based polymeric nanoparticles (PAA-based NPs) for mRNA delivery in the joints, aiming for use in osteoarthritis (OA) treatment. The formulations were tested in in vitro models mimicking the joint environment, and also following intra-articular injection ex vivo and in vivo (OA-induced rats). We demonstrate for positively charged uncoated NPs higher in vivo gene expression in OA knee joints than neutral PEG-coated NPs. However, PEG-coated NPs induced more consistent gene expression in both healthy and OA knee joints. These findings highlight the potential of PAA-based NPs for osteoarthritis research and how the interplay between the NP properties, joint biology and disease state can affect mRNA delivery.

New therapies and vaccines based on nucleic acids combined with an efficient nanoparticle delivery vehicle have a broad applicability for different disease indications. An alternative delivery technology for the successfully applied lipid nanoparticles in mRNA SARS-CoV-2 vaccines are nanoparticles composed of biodegradable poly(amido)amine-based polymers with mRNA payload. To show that these polymeric nanoparticles can efficiently deliver influenza hemagglutinin mRNA to target tissues and elicit protective immune responses, a relevant ferret influenza challenge model was used. In this model, our nanoparticle-based vaccine elicited strong humoral and cellular immune responses in the absence of local and systemic reactogenicity. Upon virus challenge, vaccinated animals exhibited reduced clinical signs and virus load relative to unvaccinated control animals. Based on these findings, further investigation of the polymeric nanoparticles in the context of prophylactic vaccination is warranted. Future studies will focus on optimizing the payload, the nanoparticle stability, the efficacy in the context of pre-existing immunity, and the applicability of the technology to prevent other infectious diseases.

Chronic lower back pain caused by intervertebral disc degeneration and osteoarthritis (OA) are highly prevalent chronic diseases. Although pain management and surgery can alleviate symptoms, no disease-modifying treatments are available. mRNA delivery could halt inflammation and degeneration and induce regeneration by overexpressing anti-inflammatory cytokines or growth factors involved in cartilage regeneration. Here, we investigated poly(amidoamine)-based polymeric nanoparticles to deliver mRNA to human joint and intervertebral disc cells. Human OA chondrocytes, human nucleus pulposus (NP) cells, human annulus fibrosus (AF) cells, fibroblast-like synoviocytes (FLS) and M1-like macrophages were cultured and transfected with uncoated or PGA-PEG-coated nanoparticles loaded with EGFP-encoding mRNA. Cell viability and transfection efficiency were analyzed for all cell types. Nanoparticle internalization was investigated in FLS and M1-like macrophages. No significant decrease in cell viability was observed in most conditions. Only macrophages showed a dose-dependent reduction of viability. Transfection with either nanoparticle version resulted in EGFP expression in NP cells, AF cells, OA chondrocytes and FLS. Macrophages showed internalization of nanoparticles by particle–cell co-localization, but no detectable expression of EGFP. Taken together, our data show that poly (amidoamine)-based nanoparticles can be used for mRNA delivery into cells of the human joint and intervertebral disc, indicating its potential future use as an mRNA delivery system in OA and IVDD, except for macrophages.

The successful use of mRNA vaccines enabled and accelerated the development of several new vaccine candidates and therapeutics based on the delivery of mRNA. In this study, we developed bioreducible poly (amidoamine)-based polymeric nanoparticles (PAA PNPs) for the delivery of mRNA with improved transfection efficiency. The polymers were functionalized with chloroquinoline (Q) moieties for improved endosomal escape and further stabilization of the mRNA-polymer construct. Moreover, these PAAQ polymers were cova-lently assembled around a core of multi-armed ethylenediamine (Mw 800, 2 % w/w) to form a pre-organized polymeric scaffolded PAAQ (ps-PAAQ) as a precursor for the formation of the mRNA-loaded nanoparticles. Transfection of mammalian cell lines with EGFP mRNA loaded into these PNPs showed a favorable effect of the Q incorporation on GFP protein expression. Additionally, these ps-PAAQ NPs were co-formulated with PEG-polymer coatings to shield the positive surface charge for increased stability and better in vivo applicability. The ps-PAAQ NPs coated with PEG-polymer displayed smaller particle size, electroneutral surface charge, and higher thermal stability. Importantly, these nanoparticles with both Q and PEG-polymer coating induced significantly higher luciferase activity in mice muscle than uncoated ps-PAAQ NPs, following intramuscular injection of PNPs loaded with luciferase mRNA. The developed technology is broadly applicable and holds promise for the development of new nucleotide-based vaccines and therapeutics in a range of infectious and chronic diseases.

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.

Brain and nervous system disorders represent a large, unmet medical need affecting two billion people worldwide; a number that is expected to grow with increasing life expectancy and the expanding global population. CNS drug development is hampered by the restricted transport of drug candidates across the blood-brain barrier (BBB). We will discuss blood-to-brain drug delivery strategies that make use of nanocarriers, like liposomes, albumin nanoparticles, and polymeric nanoparticles. The focus will be on the key pharmaceutical, pharmacological, and regulatory aspects towards the clinical development of nanocarriers. Clinical development of treatments employing nanocarriers is not as straightforward as for a single active moiety; therefore, we will highlight the issues that should be considered when translating basic research towards clinical development. Although it is still unrealistic to expect a magic bullet for exclusive CNS drug delivery, much progress has been made towards successful development of novel treatments for patients with devastating brain diseases.

Osteoarthritis (OA) is a degenerative musculoskeletal disorder affecting the whole synovial joint and globally impacts more than one in five individuals aged 40 and over, representing a huge socioeconomic burden. Drug penetration into and retention within the joints are major challenges in the development of regenerative therapies for OA. During the recent years, polymeric nanoparticles (PNPs) have emerged as promising drug carrier candidates due to their biodegradable properties, nanoscale structure, functional versatility, and reproducible manufacturing, which makes them particularly attractive for cartilage penetration and joint retention. In this review, we discuss the current development state of natural and synthetic PNPs for drug delivery and OA treatment. Evidence from in vitro and pre-clinical in vivo studies is used to show how disease pathology and key cellular pathways of joint inflammation are modulated by these nanoparticle-based therapies. Furthermore, we compare the biodegradability and surface modification of these nanocarriers in relation to the drug release profile and tissue targeting. Finally, the main challenges for nanoparticle delivery to the cartilage are discussed, as a function of disease state and physicochemical properties of PNPs such as size and surface charge.

model: a new approach for osteoarthritis treatment. Valentina Basoli1, Andreas Traweger2, Christian Plank3, Jaap Rip4, Mauro Alini1 and Sibylle Grad1. 1AO Research Institute Davos, Clavadelerstrasse 8, 7270 Davos Platz, Switzerland. 2Institute of Tendon and Bone Regeneration, Spinal Cord Injury & Tissue Regeneration Center Salzburg, Paracelsus Medical University, 5020 Salzburg, Austria 3ETHRIS GmbH, 82152 Planegg, Germany 420Med Therapeutics B.V., Galileiweg 8, 2333BD Leiden, The Netherlands

Biodegradable and bioresponsive polymer-based nanoparticles (NPs) can be used for oligonucleotide delivery, making them a promising candidate for mRNA-based therapeutics. In this study, we evaluated and optimized the efficiency of a cationic, hyperbranched poly(amidoamine)s-based nanoparticle system to deliver tdTomato mRNA to primary human bone marrow stromal cells (hBMSC), human synovial derived stem cells (hSDSC), bovine chondrocytes (bCH), and rat tendon derived stem/progenitor cells (rTDSPC). Transfection efficiencies varied among the cell types tested (bCH 28.4% ± 22.87, rTDSPC 18.13% ± 12.07, hBMSC 18.23% ± 14.80, hSDSC 26.63% ± 8.81) and while an increase of NPs with a constant amount of mRNA generally improved the transfection efficiency, an increase of the mRNA loading ratio (2:50, 4:50, or 6:50 w/w mRNA:NPs) had no impact. However, metabolic activity of bCHs and rTDSPCs was significantly reduced when using higher amounts of NPs, indicating a dose-dependent cytotoxic response. Finally, we demonstrate the feasibility of transfecting extracellular matrix-rich 3D cell culture constructs using the nanoparticle system, making it a promising transfection strategy for musculoskeletal tissues that exhibit a complex, dense extracellular matrix.

Despite the promising features of liposomes as brain drug delivery vehicles, it remains uncertain how they influence the brain uptake in vivo. In order to gain a better fundamental understanding of the interaction between liposomes and the blood-brain barrier (BBB), it is indispensable to test if liposomes affect drugs with different BBB transport properties (active influx or efflux) differently. The aim of this study was to quantitatively evaluate how PEGylated (PEG) liposomes influence brain delivery of diphenhydramine (DPH), a drug with active influx at the BBB, in rats. The brain uptake of DPH after 30 min intravenous infusion of free DPH, PEG liposomal DPH, or free DPH + empty PEG liposomes was compared by determining the unbound DPH concentrations in brain interstitial fluid and plasma with microdialysis. Regular blood samples were taken to measure total DPH concentrations in plasma. Free DPH was actively taken up into the brain time-dependently, with higher uptake at early time points followed by an unbound brain-to-plasma exposure ratio ( Kp,uu) of 3.0. The encapsulation in PEG liposomes significantly decreased brain uptake of DPH, with a reduction of Kp,uu to 1.5 ( p < 0.05). When empty PEG liposomes were coadministered with free drug, DPH brain uptake had a tendency to decrease ( Kp,uu 2.3), and DPH was found to bind to the liposomes. This study showed that PEG liposomes decreased the brain delivery of DPH in a complex manner, contributing to the understanding of the intricate interactions between drug, liposomes, and the BBB.

The impact of liposomal formulations on the in vivo release and brain delivery of methotrexate (MTX) was quantitatively assessed in rats. Two PEGylated liposomal MTX formulations based on hydrogenated soy phosphatidylcholine (HSPC) or egg-yolk phosphatidylcholine (EYPC) were prepared. The drug release and uptake into the brain after intravenous administration of both formulations were compared with unformulated MTX by determining the released, unbound MTX in brain and plasma using microdialysis. Total MTX concentrations in plasma were determined using regular blood sampling. The administration of both high- and low-dose EYPC liposomes resulted in 10 times higher extent of MTX release in plasma compared to that obtained from HSPC liposomes (p < 0.05). MTX itself possessed limited brain uptake with steady-state unbound brain-to-plasma concentration ratio (Kp,uu) of 0.10 ± 0.06. Encapsulation in HSPC liposomes did not affect MTX brain uptake (Kp,uu 0.11 ± 0.05). In contrast, EYPC liposomes significantly improved MTX brain delivery with a 3-fold increase of Kp,uu (0.28 ± 0.14 and 0.32 ± 0.13 for high- and low-dose EYPC liposomal MTX, respectively, p < 0.05). These results provide unique quantitative evidence that liposomal formulations based on different phospholipids can result in very different brain delivery of MTX.

The purpose of this study was to evaluate formulation factors causing improvement in brain delivery of a small peptide after encapsulation into a targeted nanocarrier in vivo.

The blood-brain barrier (BBB) represents a major obstacle for the delivery and development of drugs curing brain pathologies. However, this biological barrier presents numerous endogenous specialized transport systems that can be exploited by engineered nanoparticles to enable drug delivery to the brain. In particular, conjugation of glutathione (GSH) onto PEGylated liposomes (G-Technology®) showed to safely enhance delivery of encapsulated drugs to the brain. Yet, understanding of the mechanism of action remains limited and full mechanistic understanding will aid in the further optimization of the technology. In order to elucidate the mechanism of brain targeting by GSH-PEG liposomes, we here demonstrate that the in vivo delivery of liposomal ribavirin is increased in brain extracellular fluid according to the extent of GSH conjugation onto the liposomes. In vitro, using the hCMEC/D3 human cerebral microvascular endothelial (CMEC) cell line, as well as primary bovine and porcine CMEC (and in contrast to non-brain derived endothelial and epithelial cells), we show that liposomal uptake occurs through the process of endocytosis and that the brain-specific uptake is also glutathione conjugation-dependent. Interestingly, the uptake mechanism is an active process that is temperature-, time- and dose-dependent. Finally, early endocytosis events rely on cytoskeleton remodeling, as well as dynamin- and clathrin-dependent endocytosis pathways. Overall, our data demonstrate that the glutathione-dependent uptake mechanism of the G-Technology involves a specific endocytosis pathway indicative of a receptor-mediated mechanism, and supports the benefit of this drug delivery technology for the treatment of devastating brain diseases.

Brain and nervous system disorders represent a large unmet medical need. Central nervous system drug development is hampered by the restricted transport of drugs across the blood-brain barrier. Different strategies to deliver drugs to the brain have been developed. We discuss the current status of development of liposomal drug delivery to the brain. There is a growing interest in targeted delivery of liposomes to the brain and much progress has been made towards successful development of novel treatments for patients with devastating brain diseases.

Treatment of neurodegenerative disorders such as Alzheimer's disease is hampered by the blood-brain barrier (BBB). This tight cerebral vascular endothelium regulates selective diffusion and active transport of endogenous molecules and xenobiotics into and out of the brain parenchyma. In this study, glutathione targeted PEGylated (GSH-PEG) liposomes were designed to deliver amyloid-targeting antibody fragments across the BBB into the brain. Two different formulations of GSH-PEG liposomes based on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and egg-yolk phosphatidylcholine (EYPC) were produced. Both formulations encapsulate 15kDa amyloid beta binding llama single domain antibody fragments (VHH-pa2H). To follow the biodistribution of VHH-pa2H rather than the liposome, the antibody fragment was labeled with the radioisotope indium-111. To prolong the shelf life of the construct beyond the limit of radioactive decay, an active-loading method was developed to efficiently radiolabel the antibody fragments after encapsulation into the liposomes, with radiolabeling efficiencies of up to 68% after purification. The radiolabeled liposomes were administered via a single intravenous bolus injection to APPswe/PS1dE9 double transgenic mice, a mouse model of Alzheimer's disease, and their wildtype littermates. Both GSH-PEG DMPC and GSH-PEG EYPC liposomes significantly increased the standard uptake values (SUV) of VHH-pa2H in the blood of the animals compared to free VHH-pa2H. Encapsulation in GSH-PEG EYPC liposomes resulted in the highest increase in SUV in the brains of transgenic animals. Overall, these data provide evidence that GSH-PEG liposomes may be suitable for specific delivery of single domain antibody fragments over the BBB into the brain.

Partly due to poor blood-brain barrier drug penetration the treatment options for many brain diseases are limited. To safely enhance drug delivery to the brain, glutathione PEGylated liposomes (G-Technology (R)) were developed. In this study, in rats, we compared the pharmacokinetics and organ distribution of GSH-PEG liposomes using an autoquenched fluorescent tracer after intraperitoneal administration and intravenous administration. Although the appearance of liposomes in the circulation was much slower after intraperitoneal administration, comparable maximum levels of long circulating liposomes were found between 4 and 24 h after injection. Furthermore, 24 h after injection a similar tissue distribution was found. To investigate the effect of GSH coating on brain delivery in vitro uptake studies in rat brain endothelial cells (RBE4) and an in vivo brain microdialysis study in rats were used. Significantly more fluorescent tracer was found in RBE4 cell homogenates incubated with GSH-PEG liposomes compared to non-targeted PEG liposomes (1.8-fold, p<0.001). In the microdialysis study 4-fold higher (p<0.001) brain levels of fluorescent tracer were found after intravenous injection of GSH-PEG liposomes compared with PEG control liposomes. The results support further investigation into the versatility of GSH-PEG liposomes for enhanced drug delivery to the brain within a tolerable therapeutic window.

PURPOSE:Ocular inflammation is associated with the loss of visual acuity and subsequent blindness. Since their development, glucocorticoids have been the mainstay of therapy for ocular inflammatory diseases. However, the clinical benefit is limited by side effects due to the chronic use and generally high dosage that is required for effective treatment. We have developed the G-Technology to provide a means for sustained drug delivery, increased drug half-life, and reduced bodily drug exposure. Glutathione PEGylated liposomal methylprednisolone (2B3-201) has been developed as treatment for neuroinflammatory conditions and was evaluated in ocular inflammation.METHODS:The efficacy of 2B3-201 was investigated in rats with experimental autoimmune uveitis (EAU). Rats received 10 mg/kg of 2B3-201 intravenously at disease onset and at peak of the disease. The same dose of free methylprednisolone served as control treatment. Clinical signs of ocular inflammation were assessed by slit-lamp and immunohistochemistry.RESULTS:Whereas free methylprednisolone was ineffective, two doses of 2B3-201 almost completely abolished clinical signs of EAU. This was corroborated further by immunohistochemical analyses of isolated eyes. Treatment with 2B3-201 significantly reduced the infiltration of inflammatory cells and subsequent destruction of the retina cell layers.CONCLUSIONS:In this study, we show that systemic treatment with 2B3-201, a glutathione PEGylated liposomal methylprednisolone formulation, resulted in a superior efficacy in rats with EAU. Altogether, our findings hold promise for the development of a safe and more convenient systemic treatment for uveitis.