Aerosol technology to mimic dry powder inhalation in vitro using pulmonary cell models.

Inhaled therapy confers key advantages for the treatment of topical pulmonary diseases and offers potential for systemic delivery of medicines. Dry powder inhalers (DPIs) are generally the preferred devices for pulmonary delivery due to improved stability and satisfactory patient compliance. However, the mechanisms governing drug powder dissolution and availability in the lung and poorly understood. Here, we report a new in vitro system to study epithelial absorption of inhaled dry powders in lung barrier models of the upper and lower airway. The system is based on a CULTEX® RFS (Radial Flow System) cell exposure module joined to a Vilnius aerosol generator and allows the coupling of drug dissolution and permeability assessments. The cellular models recapitulate the barrier morphology and function of healthy and diseased pulmonary epithelium and incorporate the mucosal barrier to enable the investigation of drug powder dissolution in biorelevant conditions. With this system, we found differences in permeability across the airway tree and pinpointed the impact of diseased barriers in paracellular drug transport. Furthermore, we identified a different rank order of permeability for compounds tested in solution or powder form. These results highlight the value of this in vitro drug aerosolization setup for use in research and development of inhaled medicines.

Delivery of transcription factors as modulators of cell differentiation.

Fundamental studies performed during the last decades have shown that cell fate is much more plastic than previously considered, and technologies for its manipulation are a keystone for many new tissue regeneration therapies. Transcription factors (TFs) are DNA-binding proteins that control gene expression, and they have critical roles in the control of cell fate and other cellular behavior. TF-based therapies have much medical potential, but their use as drugs depends on the development of suitable delivery technologies that can help them reach their action site inside of the cells. TFs can be used either as proteins or encoded in polynucleotides. When used in protein form, many TFs require to be associated to a cell-penetrating peptide or another transduction domain. As polynucleotides, they can be delivered either by viral carriers or by non-viral systems such as polyplexes and lipoplexes. TF-based therapies have extensively shown their potential to solve many tissue-engineering problems, including bone, cartilage and cardiac regeneration. Yet, their use has expanded beyond regenerative medicine to other prominent disease areas such as cancer therapy and immunomodulation. This review summarizes some of the delivery options for effective TF-based therapies and their current main applications.

Extracellular matrix mechanics regulate transfection and SOX9-directed differentiation of mesenchymal stem cells.

Gene delivery within hydrogel matrices can potentially direct mesenchymal stem cells (MSCs) towards a chondrogenic fate to promote regeneration of cartilage. Here, we investigated whether the mechanical properties of the hydrogel containing the gene delivery systems could enhance transfection and chondrogenic programming of primary human bone marrow-derived MSCs. We developed collagen-I-alginate interpenetrating polymer network hydrogels with tunable stiffness and adhesion properties. The hydrogels were activated with nanocomplexed SOX9 polynucleotides to direct chondrogenic differentiation of MSCs. MSCs transfected within the hydrogels showed higher expression of chondrogenic markers compared to MSCs transfected in 2D prior to encapsulation. The nanocomplex uptake and resulting expression of transfected SOX9 were jointly enhanced by increased stiffness and cell-adhesion ligand density in the hydrogels. Further, transfection of SOX9 effectively induced MSCs chondrogenesis and reduced markers of hypertrophy compared to control matrices. These findings highlight the importance of matrix stiffness and adhesion as design parameters in gene-activated matrices for regenerative medicine. STATEMENT OF SIGNIFICANCE: Gene-activated matrices (GAMs) are biodegradable polymer networks integrating gene therapies, and they are promising technologies for supporting tissue regeneration. Despite this interest, there is still limited information on how to rationally design these systems. Here, we provide a systematic study of the effect of matrix stiffness and cell adhesion ligands on gene transfer efficiency. We show that high stiffness and the presence of cell-binding sites promote transfection efficiency and that this result is related to more efficient internalization and trafficking of the gene therapies. GAMs with optimized mechanical properties can induce cartilage formation and result in tissues with better characteristics for articular cartilage tissue engineering as compared to previously described standard methods.

mRNA-activated matrices encoding transcription factors as primers of cell differentiation in tissue engineering.

Gene-activated matrices (GAMs) encoding pivotal transcription factors (TFs) represent a powerful tool to direct stem cell specification for tissue engineering applications. However, current TF-based GAMs activated with pDNA, are challenged by their low transfection efficiency and delayed transgene expression. Here, we report a GAM technology activated with mRNAs encoding TFs SOX9 (cartilage) and MYOD (muscle). We find that these mRNA-GAMs induce a higher and faster TF expression compared to pDNA-GAMs, especially in the case of RNase resistant mRNA sequences. This potent TF expression was translated into a high synthesis of cartilage- and muscle-specific markers, and ultimately, into successful tissue specification in vitro. Additionally, we show that the expression of tissue-specific markers can be further modulated by altering the properties of the mRNA-GAM environment. These results highlight the value of this GAM technology for priming cell lineage specification, a key centerpiece for future tissue engineering devices.

Co-delivery of RNAi and chemokine by polyarginine nanocapsules enables the modulation of myeloid-derived suppressor cells.

Myeloid-Derived Suppressor Cells (MDSCs), immunosuppressive cells that promote tumor growth, represent an attractive target in cancer immunotherapy. However, the clinical success of this strategy is limited by the lack of efficient drug delivery vehicles targeting this cell compartment. The objective of this work was to develop a delivery carrier, multilayer polymer nanocapsules, with the capacity to co-encapsulate two types of immunomodulatory drugs, a chemokine and an RNAi sequence, aimed at reverting MDSC-mediated immunosuppression. The chemokine CCL2, intended to attract monocyte-macrophage MDSCs, was encapsulated within the L2 inverse micellar aqueous domains of the lipid core of these nanocapsules. On the other hand, two different RNAi sequences that modulate the CCAAT/enhancer-binding protein beta (C/EBPß) pathway, shC/EBPß and miR 142-3p, were successfully associated to their polymer shell. These RNAi sequences were covered by subsequent layers of polyarginine and hyaluronic acid, thereby creating multi-layered assemblies that protected them and facilitated their targeted delivery. The in vitro studies performed in primary MDSCs cultures showed the capacity of miR 142-3p-loaded nanocapsules to reduce the highly immunosuppressive monocyte-macrophage subset. Additionally, the encapsulation of CCL2 within the nanocapsules induced a potent monocyte-macrophage chemoattraction that could be used to direct the therapy to these cell subsets. Finally, in vitro and in vivo studies showed the capacity of shC/EBPß-loaded nanocapsules to downregulate C/EBPß levels in MDSCs and to reduce monocyte differentiation into tumor-associated macrophages in an MCA-203 fibrosarcoma mice model. In conclusion, the multilayer polymer nanocapsules described here are efficient vehicles for the co-delivery of proteins and RNA, and are potential candidates as nanomedicines for the modulation of MDSCs.