We present an innovative bioanalytical hybrid platform designed for the preclinical evaluation of cellular characteristics. The system combines a three-dimensional (3D) cell culture grown on an artificial extracellular matrix with a chromatography-inspired array configuration. Sponges, made from the structural protein silk fibroin, serve both as a biomimetic extracellular matrix and as a stationary phase. Silk fibroin sponges were produced in-house using a multistep process involving removal of inherent sericin proteins from raw silk fibers, followed by dissolution and dialysis to purify the fibroin solution, dissolution in organic solvent, and subsequent salt-bed casting to generate silk-based sponges with controlled porosity/pore sizes of 500-800 µm. Genetically modified breast cancer cell lines 4T1-iRFP720 and 4T1-wt (non-fluorescent control) were cultured within silk scaffolds using a continuous media flow via a pump, and their cellular growth and characteristics were analyzed non-invasively using optical imaging techniques (in vivo optical imaging instrument). By merging key advantages of chromatographic systems (automatization, reproducibility) with the biological relevance of advanced 3D cell cultures, the platform enables in vitro modeling of tissue-like architecture and morphology while facilitating the monitoring of dynamic cellular behavior. In parallel, the application of medical imaging technology enables real-time and prolonged monitoring of cellular migration and growth, among other factors. This approach offers substantial potential for investigating cellular behaviors at a macroscopic scale in a laminar-like flow system. By improving the physiological relevance of in vitro models, this method may help bridge the translational gap to in vivo studies and is consistent with the reduce, replace, refine (3R) framework for animal experimentation.

Background and Aims Aging significantly increases the risk of cardiovascular disease, characterized by progressive cardiac dysfunction. The vascular niche is crucial for maintaining cardiac homeostasis, yet endothelial cell (EC) impairment during aging remains poorly understood. This study investigates epigenetically regulated mechanisms underlying EC-dependent cardiac aging and identifies a critical role for zinc finger and BTB domain-containing protein 16 (ZBTB16).Methods Chromatin accessibility (snATAC-seq) and transcriptomic (snRNA-seq) analyses of aged hearts were performed to define age-related regulatory changes. Functional studies using genetic models were performed to assess cardiac aging phenotypes. In vitro assays examined EC senescence, secretory profiles, and effects of ZBTB16-deficient EC supernatants on fibroblasts, cardiomyocytes, and neurons. Overexpression experiments in vitro and in vivo tested whether ZBTB16 mitigates aging-associated dysfunction.Results Aged hearts exhibited decreased chromatin accessibility and reduced ZBTB16 expression in both humans and mice. Zbtb16 deletion in young mice, including Zbtb16-haploinsufficient and endothelial-specific knockout mice, led to premature aging, diastolic dysfunction, and increased secretion of pro-fibrotic and inflammatory factors. ZBTB16-deficient EC supernatants activated fibroblasts, induced cardiomyocyte hypertrophy, and impaired neuronal sprouting. Overexpression of ZBTB16 reversed these effects in senescent ECs and aged mice and reduced diastolic dysfunction. Mechanistic studies identified nuclear receptor-interacting protein 1 as a downstream target suppressed by ZBTB16, thereby limiting fibroblast activation and pro-fibrotic signalling.Conclusions ZBTB16 preserves endothelial integrity and vascular niche homeostasis, protecting against aging-associated cardiac dysfunction. Its loss promotes EC senescence and fibrosis, whereas restoring its expression may represent a therapeutic strategy to improve cardiac function and reduce cardiovascular disease risk during aging.

Receptor engagement plays a key role in the cellular uptake, intracellular trafficking, and overall transduction efficiency of adeno-associated virus (AAV) vectors. Although heparan sulfate proteoglycan (HSPG) and α5ß1 integrin-binding motifs of the AAV serotype 2 (AAV2) capsid were mapped, it has remained incompletely understood how loss of these interactions affects AAV vector performance. Hence, we generated capsid variants harboring mutations at capsid residues responsible for HSPG binding (AAV2ΔHSPG), α5ß1 integrin binding (AAV2ΔIntegrin), or both (AAV2ΔHSPGΔIntegrin), and investigated the mutants systematically ex vivo and in vivo. While neither production nor packaging efficiency was affected, variants revealed distinct physicochemical alterations, including altered electrophoretic mobility and thermal stability. Ex vivo, loss of HSPG binding completely abolished transgene expression across various cell lines, whereas ablation of α5ß1 integrin binding lowered transduction efficiency. While cellular uptake was reduced for AAV2ΔIntegrin and almost eliminated for AAV2ΔHSPG and AAV2ΔHSPGΔIntegrin, mutants nevertheless reached the nuclear compartment, albeit with lower efficiency compared with AAV2. Strikingly, in vivo performance diverged sharply from ex vivo findings as we observed a substantially enhanced transduction in multiple non-hepatic tissues for AAV2ΔHSPG and AAV2ΔHSPGΔIntegrin in C57BL/6N albino and BALB/c mice, as well as a strong liver detargeting, while AAV2ΔIntegrin was non-infectious in vivo. These data uncover a fundamental dichotomy between ex vivo and in vivo determinants of AAV2 transduction and identify receptor-binding ablation, especially integrin binding, as a potential alternative to detarget AAV2 vectors for next-generation capsid engineering and tissue-specific retargeting.

Silk fibroin scaffolds are a versatile platform for biomedical applications due to their biocompatibility and tunable properties. Successful clinical translation requires standardized production and characterization methods to ensure high reproducibility in cell seeding, growth profiling and recovery for downstream analysis. The intrinsic autofluorescence of silk and the limited diffusion of reagents through its porous structure present significant challenges for conventional assays, such as cell viability tests, DNA quantification, and optical imaging-based approaches. These assays are a requirement for validation procedures and characterization. In this study, we introduce a standardized protocol for efficiently assessing cell seeding and growth behavior. By analyzing the physicochemical properties of the silk sponge, we determined the optimal volumes required for silk swelling and cell seeding. Additionally, we developed a spin-down system that enables the application of endpoint assays while ensuring gentle cell recovery. We established and experimentally validated the relationship between silk sponge volume and the growth limitations of embedded cells. Overall, this study underscores the importance of a standardized procedure for efficient cell seeding and recovery, ultimately facilitating clinical translation.

BACKGROUND:Endothelial cells (ECs) play pivotal roles in maintaining cardiac blood supply and regulating inflammation by acting as gatekeepers for immune cell activity. This study unveils a novel immunomodulatory function of cardiac ECs following myocardial infarction. METHODS:We used single-cell RNA sequencing and spatial transcriptomics to identify EC states after acute myocardial infarction in mice. Subsequently, we mimicked the cytokine environment that was predicted to induce EC activation in cell culture studies and confirmed the results in an endothelial-specific deletion mouse model. RESULTS:Single-cell RNA sequencing analysis identified a transient myeloid CD45+CD11b+Cdh5+ immunomodulatory EC phenotype (IMEC) emerging between days 1 and 3 after myocardial infarction. IMECs derived from Cdh5+ tissue resident cells as shown by bone marrow transplantation and lineage tracing experiment. Ligand-receptor interaction predictions indicated a cytokine-mediated activation of IMECs, which we validated through in vitro experiments in cultured ECs. Notably, while cytokine treatment with IL-1β and TGF-β (transforming growth factor β) induced mesenchymal gene expression, the addition of IFN-γ (interferon γ) facilitated the transition into the immunomodulatory phenotype. IMECs exhibited an upregulation of MHC-II (major histocompatibility complex class II) genes, along with the expression of RUNX1 (runt-related transcription factor-1) and proinflammatory cytokines, such as IL-6 and IL-12. IMECs induced T-cell activation through paracrine signaling and were colocalized with T cells in vivo. Inhibition of endothelial-specific IFN-γ-signaling in mice by IFN-γ receptor 1 deletion improved the recovery after myocardial infarction. CONCLUSIONS:These findings provide insight into the role of ECs regulating adaptive immune responses following myocardial infarction, offering potential insights into therapeutic interventions for postinfarction immunomodulation.

Splice-switching oligonucleotides (SSOs) can restore protein functionality in pathologies and are promising tools for manipulating the RNA-splicing machinery. Delivery vectors can considerably improve SSO functionality in vivo and allow dose reduction, thereby addressing the challenges of RNA-targeted therapeutics. Here, we report a biocompatible SSO nanocarrier, based on redox-responsive disulfide cross-linked low-molecular-weight linear polyethylenimine (cLPEI), for overcoming multiple biological barriers from subcellular compartments to en-route serum stability and finally in vivo delivery challenges. Intracellularly responsive cross-links of cLPEI significantly accelerated the endosomal escape and offered efficient SSO release to the cell's nucleus, thereby leading to high splice correction in vitro. In vivo performance of cLPEI-SSOs was investigated in a novel transgenic mouse model for splice correction, spatiotemporal tracking of SSO delivery in wild-type mice, and biodistribution in a colorectal cancer peritoneal metastasis model. A single intravenous application of 5 mg kg-1 cLPEI-SSOs induced splice correction in liver, lung, kidney, and bladder, giving functional protein, which was validated by RT-PCR. Near-infrared (NIR) fluorescence imaging and X-ray computed tomography revealed improved organ retention and reduced renal excretion of SSOs. NIR microscopy demonstrated the accumulation of SSOs in angiogenic tumors within the pancreas. Successful nuclear delivery of SSOs was observed in the hepatocytes. Thus, cLPEI nanocarriers resulted in highly efficient splice correction in vivo, highlighting the critical role of the enhanced SSO bioavailability.

BACKGROUND:Compounding and storage of intravitreal anti-vascular endothelial growth factor (anti-VEGF) agents in syringes is commonly performed in an off-label manner. However, the preservation of compound integrity and microbiological safety must be guaranteed. The aim of this study was to compare the chemical and physical stability, sterility and binding affinity to vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) of faricimab, a novel bispecific anti-VEGF/Ang-2 biologic, after compounding and storage in two different polypropylene syringe types for up to 28 days. METHODS:Faricimab was compounded into silicone oil-free and silicone oil-containing polypropylene syringes under controlled aseptic clean room conditions and stored under light protection at 2-8 °C for up to 28 days. Compound integrity was analysed by size exclusion chromatography, nano differential scanning fluorimetry, UV-vis and dynamic light scattering. The analysis of the simultaneous binding of VEGF and Ang-2 was performed by grating-coupled interferometry. Additionally, samples were tested for sterility and presence of bacterial endotoxins. One-way ANOVA test was used to analyse statistical significance (p ≤ 0.05). RESULTS:No significant differences in VEGF and ANG-2 binding affinity were found in faricimab samples stored in either syringe type after 28 days compared to control. Chemical and physical stability testing revealed no statistically significant variation. Furthermore, sterility and the absence of bacterial endotoxins could be maintained. CONCLUSION:Our findings confirm the pharmaceutical safety of compounded faricimab after storage for up to 28 days. This may facilitate a cost-effective off-label use of faricimab in clinical practice while maintaining safety in the treatment of patients.

Although cationic liposomes are efficient carriers for nucleic acid delivery, their toxicity often hampers the clinical translation. Polyethylene glycol (PEG) coating has been largely used to improve their stability and reduce toxicity. Nevertheless, it has been found to decrease the transfection process. In order to exploit the advantages of cationic liposomes and PEG decoration for nucleic acid delivery, liposomes decorated with tetraArg-[G-1]distearoyl glycerol (Arg4-DAG) dendronic oligo-cationic lipid enhancer (OCE) and PEG-lipid have been investigated. Non decorated or OCE-decorated lipoplexes (OCEfree-LPX and OCE-LPX, respectively) were obtained by lipid film hydration using oligonucleotide (ON) solutions. PEG and OCE/PEG decorated lipoplexes (PEG-OCEfreeLPX and PEG-OCE-LPX, respectively) were obtained by post-insertion of 2 or 5 kDa PEG-DSPE on preformed lipoplexes. The OCE decoration yielded lipoplexes with size of about 240 nm, 84% loading efficiency at 10 N/P ratio, ten times higher than OCEfree-LPX, and prevented the ON release when incubated with physiological heparin concentration or with plasma. The PEG decoration reduced the zeta potential, enhanced the lipoplex stability in serum and decreased both hemolysis and cytotoxicity, while it did not affect the lipoplex size and ON loading. With respect to OCEfree-LPX, the OCE-LPX remarkably associated with cells and were taken up by different cancer cell lines (HeLa and MDA-MB-231). Interestingly, 2 or 5 kDa PEG decoration did not reduce either the cell interaction or the cell up-take of the cationic lipoplexes. With siRNA as a payload, OCE enabled efficient internalization, but endosomal release was hampered. Post-transfection treatment with the lysosomotropic drug chloroquine allowed to identify the optimal time point for endosomal escape. Chloroquine treatment after 12 to 20 h of LPX pre-incubation enabled siRNA mediated target knockdown indicating that this is the time window of endo-lysosomal processing. This indicates that OCE can protect siRNA from lysosomal degradation for up to 20 h, as shown by these rescue experiments.

Background: Most frequently the functionalization of nanoparticles is hampered by time-consuming, sometimes harsh conjugation and purification procedures causing premature drug release and/or degradation. A strategy to circumvent multi-step protocols is to synthesize building blocks with different functionalities and to use mixtures thereof for nanoparticle preparation in one step. Methods: BrijS20 was converted into an amine derivative via a carbamate linkage. The Brij-amine readily reacts with pre-activated carboxyl-containing ligands such as folic acid. The structures of the building blocks were confirmed by different spectroscopic methods and their utility was assessed by one-step preparation and characterization of nanoparticles applying PLGA as a matrix polymer. Results: Nanoparticles were about 200 nm in diameter independent of the composition. Experiments with human folate expressing single cells and monolayer revealed that the nanoparticle building block Brij mediates a “stealth” effect and the Brij-amine-folate a “targeting” effect. As compared to plain nanoparticles, the stealth effect decreased the cell interaction by 13%, but the targeting effect increased the cell interaction by 45% in the monolayer. Moreover, the targeting ligand density and thus the cell association of the nanoparticles is easily fine-tuned by selection of the initial ratio of the building blocks. Conclusions: This strategy might be a first step towards the one-step preparation of nanoparticles with tailored functionalities. Relying on a non-ionic surfactant is a versatile approach as it might be extended to other hydrophobic matrix polymers and promising targeting ligands from the biotech pipeline.

AbstractBottlebrush polymers are highly promising as unimolecular nanomedicines due to their unique control over the critical parameters of size, shape and chemical function. However, since they are prepared from biopersistent carbon backbones, most known bottlebrush polymers are non‐degradable and thus unsuitable for systemic therapeutic administration. Herein, we report the design and synthesis of novel poly(organo)phosphazene‐g‐poly(α‐glutamate) (PPz‐g‐PGA) bottlebrush polymers with exceptional control over their structure and molecular dimensions (Dh ≈ 15–50 nm). These single macromolecules show outstanding aqueous solubility, ultra‐high multivalency and biodegradability, making them ideal as nanomedicines. While well‐established in polymer therapeutics, it has hitherto not been possible to prepare defined single macromolecules of PGA in these nanosized dimensions. A direct correlation was observed between the macromolecular dimensions of the bottlebrush polymers and their intracellular uptake in CT26 colon cancer cells. Furthermore, the bottlebrush macromolecular structure visibly enhanced the pharmacokinetics by reducing renal clearance and extending plasma half‐lives. Real‐time analysis of the biodistribution dynamics showed architecture‐driven organ distribution and enhanced tumor accumulation. This work, therefore, introduces a robust, controlled synthesis route to bottlebrush polypeptides, overcoming limitations of current polymer‐based nanomedicines and, in doing so, offers valuable insights into the influence of architecture on the in vivo performance of nanomedicines.

The epidermal growth factor receptor EGFR allows targeted delivery of macromolecular drugs to tumors. Its ligand, epidermal growth factor, binds EGFR with high affinity but acts mitogenic. Non-mitogenic peptides are utilized as targeting ligands, like the dodecapeptide GE11, although its low binding affinity warrants improvement. We applied a two-step computational approach with database search and molecular docking to design GE11 variants with improved binding. Synthesized peptides underwent binding studies on immobilized EGFR using surface plasmon resonance. Conjugates of peptides coupled via heterobifunctional PEG linker to linear polyethylenimine (LPEI) were used for transfection studies on EGFR-overexpressing cells using reporter gene encoding plasmid DNA. Docking studies unraveled similarities between GE11 and the EGFR dimerization arm. By skipping non-overlapping amino acids, a less hydrophobic segment (YTPQNVI) was identified to be directly involved in EGFR binding. By replacing valine by tyrosine, a full-length version with proposed enhanced binding (GE11m3) was developed. While hydrophobic or hydrophilic segments and variations thereof exhibited low binding, GE11m3 exhibited 3-fold increase in binding compared to GE11, validating in silico predictions. In transfection studies, polyplexes with GE11m3 induced a significantly higher reporter gene expression when compared to GE11 polyplexes both on murine and human cancer cells overexpressing EGFR.

Adeno-associated viruses (AAVs) are frequently used for gene transfer and gene editing in vivo, except for endothelial cells, which are remarkably resistant to unmodified AAV-transduction. AAVs are retargeted here toward endothelial cells by coating with second-generation polyamidoamine dendrimers (G2) linked to endothelial-affine peptides (CNN). G2(CNN) AAV9-Cre (encoding Cre recombinase) are injected into mTmG-mice or mTmG-pigs, cell-specifically converting red to green fluorescence upon Cre-activity. Three endothelial-specific functions are assessed: in vivo quantification of adherent leukocytes after systemic injection of - G2(CNN) AAV9 encoding 1) an artificial adhesion molecule (S1FG) in wildtype mice (day 10) or 2) anti-inflammatory Annexin A1 (Anxa1) in ApoE(-/-) mice (day 28). Moreover, 3) in Cas9-transgenic mice, blood pressure is monitored till day 56 after systemic application of G2(CNN) AAV9-gRNAs, targeting exons 6-10 of endothelial nitric oxide synthase (eNOS), a vasodilatory enzyme. G2(CNN) AAV9-Cre transduces microvascular endothelial cells in mTmG-mice or mTmG-pigs. Functionally, G2(CNN) AAV9-S1FG mediates S1FG-leukocyte adhesion, whereas G2(CNN) AAV9-Anxa1-application reduces long-term leukocyte recruitment. Moreover, blood pressure increases in Cas9-expressing mice subjected to G2(CNN) AAV9-gRNA(eNOS). Therefore, G2(CNN) AAV9 may enable gene transfer in vascular and atherosclerosis models.

Formulations based on ionizable amino-lipids have been put into focus as nucleic acid delivery systems. Recently, the in vitro efficacy of the lipid formulation OH4:DOPE has been explored. However, in vitro performance of nanomedicines cannot correctly predict in vivo efficacy, thereby considerably limiting pre-clinical translation. This is further exacerbated by limited access to mammalian models. The present work proposes to close this gap by investigating in vivo nucleic acid delivery within simpler models, but which still offers physiologically complex environments and also adheres to the 3R guidelines (replace/reduce/refine) to improve animal experiments. The efficacy of OH4:DOPE as a delivery system for nucleic acids is demonstrated using in vivo approaches. It is shown that the formulation is able to transfect complex tissues using the chicken chorioallantoic membrane model. The efficacy of DNA and mRNA lipoplexes is tested extensively in the zebra fish (Danio rerio) embryo which allows the screening of biodistribution and transfection efficiency. Effective transfection of blood vessel endothelial cells is seen, especially in the endocardium. Both model systems allow an efficacy screening according to the 3R guidelines bypassing the in vitro-in vivo gap. Pilot studies in mice are performed to correlate the efficacy of in vivo transfection.

CD47 protects healthy cells from macrophage attack by binding to signal regulatory protein α (SIRPα), while its upregulation in cancer prevents immune clearance. Systemic treatment with CD47 antibodies requires a weakened Fc-mediated effector function or lower CD47-binding affinity to prevent side effects. Our approach combines "the best of both worlds," i.e., maximized CD47 binding and full Fc-mediated immune activity, by exploiting gene therapy for paracrine release. We developed a plasmid vector encoding for the secreted fusion protein sCV1-hIgG1, comprising highly efficient CD47-blocking moiety CV1 and Fc domain of human immunoglobulin G1 (IgG1) with maximized immune activation. sCV1-hIgG1 exhibited a potent bystander effect, blocking CD47 on all cells via fusion protein secreted from only a fraction of cells or when transferring transfection supernatant to untransfected cells. The CpG-free plasmid ensured sustained secretion of sCV1-hIgG1. In orthotopic human triple-negative breast cancer in CB17-severe combined immunodeficiency (SCID) mice, ex vivo transfection significantly delayed tumor growth and eradicated one-third of tumors. In intratumoral transfection experiments, CD47 blockage and increased migration of macrophages into the tumor were observed within 17 h of a single injection. Natural killer (NK) cell-mediated lysis of sCV1-hIgG1-expressing cells was demonstrated in vitro. Taken together, this approach also opens the opportunity to block, in principle, any immune checkpoints.

Extracellular vesicles produced by different types of cells have recently attracted great attention, not only for their role in physiology and pathology, but also because of the emerging applications in gene therapy, vaccine production and diagnostics. Less well known than their eukaryotic counterpart, also bacteria produce extracellular vesicles, in the case of the Gram-negative E. coli the main species is termed outer membrane vesicles (OMVs). In this study, we show for the first time the functional surface modification of E. coli OMVs with glycosylphosphatidylinositol (GPI)-anchored protein, exploiting a process variably described as molecular painting or protein engineering in eukaryotic membranes, whereby the lipid part of the GPI anchor inserts in cell membranes. By transferring the process to bacterial vesicles, we can generate a hybrid of perfectly eukaryotic proteins (in terms of folding and post-translational modifications) on a prokaryotic platform. We could demonstrate that two different GPI proteins can be displayed on the same OMV. In addition to fluorescent marker proteins, cytokines, growth factors and antigens canb be potentially transferred, generating a versatile modular platform for a novel vaccine strategy.

Current nucleic acid (NA) nanotherapeutic approaches face challenges because of shortcomings such as limited control on loading efficiency, complex formulation procedure involving purification steps, low load of NA cargo per nanoparticle, endosomal trapping, and hampered release inside the cell. When combined, these factors significantly limit the amount of biologically active NA delivered per cell in vitro, delivered dosages in vivo for a prolonged biological effect, and the upscalability potential, thereby warranting early consideration in the design and developmental phase. Here, we report a versatile nanotherapeutic platform, termed auropolyplexes, for improved and efficient delivery of small interfering RNA (siRNA). Semitelechelic, thiolated linear polyethylenimine (PEI) was chemisorbed onto gold nanoparticles to endow them with positive charge. A simple two-step complexation method offers tunable loading of siRNA at concentrations relevant for in vivo studies and the flexibility for inclusion of multiple functionalities without any purification steps. SiRNA was electrostatically complexed with these cationic gold nanoparticles and further condensed with polycation or polyethyleneglycol-polycation conjugates. The resulting auropolyplexes ensured complete complexation of siRNA into nanoparticles with a high load of ∼15,500 siRNA molecules/nanoparticle. After efficient internalization into the tumor cell, an 80% knockdown of the luciferase reporter gene was achieved. Auropolyplexes were applied intratracheally in Balb/c mice for pulmonary delivery, and their biodistribution were studied spatio-temporally and quantitatively by optical tomography. Auropolyplexes were well tolerated with ∼25% of the siRNA dose remaining in the lungs after 24 h. Importantly, siRNA was released from auropolyplexes in vivo and a fraction also crossed the air-blood barrier, which was then excreted via kidneys, whereas >97% of gold nanoparticles were retained in the lung. Linear PEI-based auropolyplexes offer a combination of successful endosomal escape and better biocompatibility profile in vivo. Taken together, combined chemisorption and complexation endow auropolyplexes with crucial biophysical attributes, enabling a versatile and upscalable nanogold-based platform for siRNA delivery in vitro and in vivo.

Peptide ligands can enhance delivery of nucleic acid-loaded nanoparticles to tumors by promoting their cell binding and internalization. Lung tumor lesions accessible from the alveolar side can be transfected, in principle, using gene vectors delivered as an aerosol. The cell surface marker CD49f (Integrin alpha 6) is frequently upregulated in metastasizing, highly aggressive tumors. In this study, we utilize a CD49f binding peptide coupled to linear polyethylenimine (LPEI) promoting gene delivery into CD49f-overexpressing tumor cells in vitro and into lung lesions in vivo. We have synthesized a molecular conjugate based on LPEI covalently attached to the CD49f binding peptide CYESIKVAVS via a polyethylene glycol (PEG) spacer. Particles formed with plasmid DNA were small (<200 nm) and could be aerosolized without causing major aggregation or particle loss. In vitro, CD49f targeting significantly improved plasmid uptake and reporter gene expression on both human and murine tumor cell lines. For evaluation in vivo, localization and morphology of 4T1 murine triple-negative breast cancer tumor lesions in the lung of syngeneic BALB/c mice were identified by MRI. Polyplexes applied via intratracheal aerosolization were well tolerated and resulted in measurable transgene activity of the reporter gene firefly luciferase in tumor areas by bioluminescence imaging (BLI). Transfectability of tumors correlated with their accessibility for the aerosol. With CD49f-targeted polyplexes, luciferase activity was considerably increased and was restricted to the tumor area.