One strategy to consider is the route by which nanoparticle drones with radiosensitizers or drugs can navigate physiological barriers to reach the tumor with minimal systemic toxicities. This will depend on the tumor site. One tumor site that could significantly benefit from new strategies is lung cancer, the leading cause of cancer deaths. Two key routes for targeting nanoparticle drones to lung tumors are inhalation (INH or “air”) route and intravenous (IV or “sea”) route, i.e., through the blood stream.

Previous studies show that administering nanoparticles via IV route may result in up to 5% nanoparticles reaching the lung, compared to 3.5–14.6 times higher nanoparticle concentrations when administered via INH (13, 14). Taking this into account, we have previously investigated the potential for enhancing external beam radiotherapy for lung cancer using high-Z nanoparticles (made of gold or platinum-based chemotherapy drugs) administered via INH (15). The results of this work indicate that administering nanoparticles via the INH route could enable clinically significant damage enhancement to lung tumors compared to using IV routes of administration during external beam radiotherapy for lung cancer. Building on this work, we conducted additional experiments using nanoparticle drones based on GNP using transgenic mouse models.

The design of such nanoparticle drones described in our previous work (16) particularly takes size and nanoparticle functionalization into account. The size is optimized to ensure increased circulation and tumor uptake. Meanwhile, PEGylation confers stealth to the nanoparticle drones, thereby, also enabling longer circulation time for nanoparticles administered intravenously. Such longer circulation allows for higher amounts of nanoparticles to concentrate in the tumor. The hetero-bifunctional-polyethylene glycol with amine, carboxyl, and methoxy ligands, also allows for conjugating various moieties such as imaging or targeting ligands, as well as radiosensitizers or drugs. The imaging capability of these nanoplatforms has already been demonstrated in vitro and in vivo (16). A number of recent efforts have also developed nanoparticle drone platforms, including biogenic GNPs (17, 18) and drug-loaded gold plasmonic nanoparticles (19, 20) for different applications. For lung tumors, the nanoparticle drones are functionalized with RGD designed to target the integrin receptors on the lung tumor.

In experiments, transgenic mouse models bearing single-nodule lung adenocarcinoma were employed, with features that closely resemble human lung tumors, as described in recent work (21). Nanoparticle drones were administered to Cohort A mice (n = 5) via INH (instillation), while the same concentration of nanoparticle drones was administered to Cohort B (n = 5) mice via IV (Figure 1). The biodistribution of drones was measured via fluorescence imaging and ex vivo electron microcopy methods. Mice were dissected, and entire lungs were imaged 24 h post administration. The images were acquired with the same acquisition time of 88 ms for all the experiments. With spectral imaging software, small but meaningful spectral differences could be rapidly detected and analyzed. Spectral unmixing algorithms were employed to generate unmixed images of “pure” autofluorescence and “pure” fluorescence signal. A quantitative estimation of the fluorescence intensity was done using the Maestro software and Image J.

Meanwhile, transmission electron microscopy was carried out using a JEOL model JEM-1000 microscope at an acceleration voltage of 80 kV. The samples were prepared by drop casting method on the formvar coated copper grids. 1 mm³ of lung tissues were fixed in 2.5% formaldehyde and 2.5% glutaraldehyde solution for few hours. The post fixation was carried out using 1% osmium tetraoxide for 1 h followed by dehydration in varying alcohol concentrations and overnight infiltration using Squetol resin. The polymerized resin with tissue was sectioned using ultramicrotome. The sections were placed on copper grids, and silver enhancement was done following the manufacturer’s recommendations. The grids were dried and imaged.

After each study, animals were euthanized by CO₂ INH followed by cervical dislocation. Death was assured by harvesting tumor-bearing and other vital organs, including the cecum, liver, and lungs. All studies followed Dana-Farber Cancer Institute IACUC approved protocol.

The schematic (Figure 1A) illustrates the nanoparticle drone and the two different routes of administration in the transgenic lung tumor mouse model. The size of the nanoparticles as estimated by TEM (Figure 1B) showed fairly monodispersed nanoparticles of around 2–3 nm in diameter with spherical morphology. The overall hydrodynamic diameter of the drones was approximately 12 nm as estimated by the dynamic light scattering measurements (data not shown). Meanwhile, Figure 1C shows the photophysical characteristics of the nanoparticle drones. As the figure shows, drones conjugated with AF647 showed a characteristic fluorescence peak at λ_max 650 nm with a slight bathochromic shift of 5 nm. This can be attributed to slight aggregation or steric hindrance of the dye molecules after conjugation to PEG. The absorption studies showed a slight hump in the nanoparticle drone spectra at around 647 nm. However, a distinct absorption peak is masked due to higher degree of scattering by the GNP drones.

Figure 2 shows the comparative imaging studies with the nanoparticle drones administered via INH or IV routes at similar concentrations. The transgenic lung tumor model, which we used for this study, develops tumor only in the left lung (21). Figures 2A,B show white light optical images of sample lung tumoral masses with drones reaching their target after administration via INH and IV, respectively. The corresponding fluorescent images are shown in Figures 2C,D, respectively. From these images, significantly higher fluorescence due to the drones was consistently observed for the left lung in case of INH administration compared to IV. This is further corroborated by the TEM images in Figures 3A,B for IV and Figures 3C,D for INH. Consistent with the findings from fluorescence imaging, the TEM images show nanoparticle drones abundantly distributed throughout the target tumor tissue in mice with INH route of delivery compared to sparse distribution in sample tissue sections of mice with IV route.

For more perspective, the pixel intensity for randomly selected regions of interests was quantified and compared. From the quantification, the drones reaching their target tumors via the INH (air) route were averagely about 4.8 ± 0.4 times higher, compared to IV (“sea”) route. This finding for nanoparticle drones is consistent with the previous studies showing that INH route provides 3.5–14.6 times higher nanoparticle concentrations compared to IV (13, 14). In our study, the nanoparticle drones can also be visualized penetrating into the tumor tissue. This can be attributed to the small size of drones (<100 nm), which facilitates enhanced diffusion and adsorption and also targeting by RGD peptide. Larger nanoparticles are known to deposit in the extrathoracic and bronchial region airways during inspiration. Altogether, the results support the perspective that nanoparticle drones administration via INH could lead to significantly higher targeting to lung tumors compared to IV routes. Our previous work (15) has showed that this could lead to a clinical significant radiotherapy boosting to lung tumor cells compared to IV routes.