Tailoring lipid nanoparticles for T-cell targeting in allergic asthma: Insights into efficacy and specificity.

Asthma impacts over 300 million patients globally, with significant health implications, especially in cases of its allergic subtype. The disease is characterized by a complex interplay of airway inflammation and immune responses, often mediated by Th2 cell-related cytokines. In this study, we engineered lipid nanoparticles (LNPs) to specifically deliver therapeutic siRNA via the transferrin receptor to T cells. Strain-promoted azide-alkyne cycloaddition (SPAAC) was employed for the conjugation of transferrin ligands to PEGylated lipids in the LNPs, with the goal of enhancing cellular uptake and gene knockdown. The obtained LNPs exhibited characteristics that make them suitable for pulmonary delivery. Using methods such as nanoparticle tracking analysis (NTA) and enzyme-linked immunosorbent assay (ELISA), we determined the average number of transferrin molecules bound to individual LNPs. Additionally, we found that cellular uptake was ligand-dependent, achieving a GATA3 knockdown of more than 50% in relevant in vitro and ex vivo models. Notably, our findings highlight the limitations inherent to modifying the surface of LNPs, particularly with regard to their targeting capabilities. This work paves the way for future research aimed at optimizing targeted LNPs for the treatment of immunologic diseases such as allergic asthma.

Efficient and Targeted siRNA Delivery to M2 Macrophages by Smart Polymer Blends for M1 Macrophage Repolarization as a Promising Strategy for Future Cancer Treatment.

Cancer remains an issue on a global scale. It is estimated that nearly 10 million people succumbed to cancer worldwide in 2020. New treatment options are urgently needed. A promising approach is a conversion of tumor-promoting M2 tumor-associated macrophages (TAMs) as part of the tumor microenvironment to tumor-suppressive M1 TAMs by small interfering RNA (siRNA). In this work, we present a well-characterized polymeric nanocarrier system capable of targeting M2 TAMs by a ligand-receptor interaction. Therefore, we developed a blended PEI-based polymeric nanoparticle system conjugated with mannose, which is internalized after interaction with macrophage mannose receptors (MMRs), showing low cytotoxicity and negligible IL-6 activation. The PEI-PCL-PEI (5 kDa-5 kDa-5 kDa) and Man-PEG-PCL (2 kDa-2 kDa) blended siRNA delivery system was optimized for maximum targeting capability and efficient endosomal escape by evaluation of different polymer and N/P ratios. The nanoparticles were formulated by surface acoustic wave-assisted microfluidics, achieving a size of ∼80 nm and a zeta potential of approximately +10 mV. Special attention was given to the endosomal escape as the so-called bottleneck of RNA drug delivery. To estimate the endosomal escape capability of the nanocarrier system, we developed a prediction method by evaluating the particle stability via the inflection temperature. Our predictions were then verified in an in vitro setting by applying confocal microscopy. For cellular experiments, however, human THP-1 cells were polarized to M2 macrophages by cytokine treatment and validated through MMR expression. To show the efficiency of the nanoparticle system, GAPDH and IκBα knockdown was performed in the presence or absence of an MMR blocking excess of mannan. Cellular uptake, GAPDH knockdown, and NF-κB western blot confirmed efficient mannose targeting. Herein, we presented a well-characterized nanoparticle delivery system and a promising approach for targeting M2 macrophages by a mannose-MMR interaction.

Pulmonary siRNA Delivery with Sophisticated Amphiphilic Poly(Spermine Acrylamides) for the Treatment of Lung Fibrosis.

RNA interference (RNAi) is an efficient strategy to post-transcriptionally silence gene expression. While all siRNA drugs on the market target the liver, the lung offers a variety of currently undruggable targets, which can potentially be treated with RNA therapeutics. To achieve this goal, the synthesis of poly(spermine acrylamides) (P(SpAA) is reported herein. Polymers are prepared via polymerization of N-acryloxysuccinimide (NAS) and afterward this active ester is converted into spermine-based pendant groups. Copolymerizations with decylacrylamide are employed to increase the hydrophobicity of the polymers. After deprotection, polymers show excellent siRNA encapsulation to obtain perfectly sized polyplexes at very low polymer/RNA ratios. In vitro 2D and 3D cell culture, ex vivo and in vivo experiments reveal superior properties of amphiphilic spermine-copolymers with respect to delivery of siRNA to lung cells in comparison to commonly used lipid-based transfection agents. In line with the in vitro results, siRNA delivery to human lung explants confirm more efficient gene silencing of protease-activated receptor 2 (PAR2), a G protein-coupled receptor involved in fibrosis. This study reveals the importance of the balance between efficient polyplex formation, cellular uptake, gene knockdown, and toxicity for efficient siRNA delivery in vitro, in vivo, and in fibrotic human lung tissue ex vivo.

Evaluation of the effects of storage conditions on spray-dried siRNA-LNPs before and after subsequent drying.

In an ideal world, pharmaceutical drugs would have infinite shelf life, no susceptibility to degradation, chemical reactions or loss of efficacy. In reality, these processes occur, however, making it desirable to extend a drugs' shelf life. Nucleic acid-based drugs are most commonly stored as aqueous suspension where they are vulnerable to microbial growth and degradation processes. Drying procedures, such as lyophilization and spray drying, help to reduce the products' residual moisture while increasing the products' shelf life and stability. The present study was designed to evaluate 90 days of storage of spray-dried siRNA-lipid nanoparticles (LNPs) at 4 °C and 25 °C. An updated Onpattro® composition modified with a positively charged helper lipid was used as the LNP carrier system. In an attempt to further reduce the residual moisture of our previously reported formulations, all LNP samples were subjected to a secondary drying step in the spray drying tower for 20 min. The measurement of physicochemical properties of spray-dried and subsequently dried LNPs resulted in sizes of 180 nm, PDI values of 0.1-0.15 and zeta potentials of + 3 mV. Spray drying resulted in residual moisture levels of 3.6-4 % and was reduced by subsequent drying to 2.8-3.1 %. Aerodynamic properties after storage showed discrepancies depending on the storage conditions. MMADs remained at 2.8 µm when stored at 4 °C, whereas an increase to 5 µm at 25 °C was observed. Subsequent drying led to sizes of 3.6-3.8 µm, independent of the storage conditions. Spray-dried LNPs maintained bioactivity resulting in > 95 % protein downregulation and confirming the lack of cytotoxic effects in a lung adenocarcinoma cell line. Furthermore, the spray-dried and subsequently dried LNPs stored for 3 months at 4 °C and 25 °C achieved up to 50 % gene silencing of the house-keeping gene GAPDH after deposition on the mucus layer of Calu-3 cells. This study confirms the stability of spray-dried and subsequently dried LNPs over at least 90 days at 4 °C and 25 °C emphasizing the potential of dry powder inhalation of RNA-loaded LNPs as a therapy option for pulmonary diseases.

Protein corona investigations of polyplexes with varying hydrophobicity - From method development to in vitro studies.

In the field of non-viral drug delivery, polyplexes (PXs) represent an advanced investigated and highly promising tool for the delivery of nucleic acids. Upon encountering physiological fluids, they adsorb biological molecules to form a protein corona (PC), that influence PXs biodistribution, transfection efficiencies and targeting abilities. In an effort to understand protein - PX interactions and the effect of PX material on corona composition, we utilized cationic branched 10 kDa polyethyleneimine (b-PEI) and a hydrophobically modified nylon-3 polymer (NM0.2/CP0.8) within this study to develop appropriate methods for PC investigations. A centrifugation procedure for isolating hard corona - PX complexes (PCPXs) from soft corona proteins after incubating the PXs in fetal bovine serum (FBS) for PC formation was successfully optimized and the identification of proteins by a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method clearly demonstrated that the PC composition is affected by the underlying PXs material. With regard to especially interesting functional proteins, which might be able to induce active targeting effects, several candidates could be detected on b-PEI and NM0.2/CP0.8 PXs. These results are of high interest to better understand how the design of PXs impacts the PC composition and subsequently PCPXs-cell interactions to enable precise adjustment of PXs for targeted drug delivery.

Targeted GATA3 knockdown in activated T cells via pulmonary siRNA delivery as novel therapy for allergic asthma.

GATA3 gene silencing in activated T cells displays a promising option to early-on undermine pathological pathways in the disease formation of allergic asthma. The central transcription factor of T helper 2 (Th2) cell cytokines IL-4, IL-5, and IL-13 plays a major role in immune and inflammatory cascades underlying asthmatic processes in the airways. Pulmonary delivery of small interfering RNAs (siRNA) to induce GATA3 knockdown within disease related T cells of asthmatic lungs via RNA interference (RNAi) presents an auspicious base to realize this strategy, however, still faces some major hurdles. Main obstacles for successful siRNA delivery in general comprise stability and targeting issues, while in addition the transfection of T cells presents a particularly challenging task itself. In previous studies, we have developed and advanced an eligible siRNA delivery system composed of polyethylenimine (PEI) as polycationic carrier, transferrin (Tf) as targeting ligand and melittin (Mel) as endosomolytic agent. Resulting Tf-Mel-PEI polyplexes exhibited ideal characteristics for targeted siRNA delivery to activated T cells and achieved efficient and sequence-specific gene knockdown in vitro. In this work, the therapeutic potential of this carrier system was evaluated in an optimized cellular model displaying the activated status of asthmatic T cells. Moreover, a suitable siRNA sequence combination was found for effective gene silencing of GATA3. To confirm the translatability of our findings, Tf-Mel-PEI polyplexes were additionally tested ex vivo in activated human precision-cut lung slices (PCLS). Here, the formulation showed a safe profile as well as successful delivery to the lung epithelium with 88% GATA3 silencing in lung explants. These findings support the feasibility of Tf-Mel-PEI as siRNA delivery system for targeted gene knockdown in activated T cells as a potential novel therapy for allergic asthma.