3.5.3. Investigations from Cellular Uptake Using Fluorescent Nanoparticles

The human non-small-cell lung cancer cell line A549 was obtained from the National Centre for Cell Sciences (NCCS, Pune, Maharashtra, India). The cells were grown in DMEM medium supplemented with 10% FBS at 37 ◦C with 5% CO<sup>2</sup> and 95% humidity. The media was replaced every 2–3 days, and the cells were detached from the culture flask using a 0.25% trypsin–0.02% EDTA solution after reaching a confluence level of 80–90%. For visualization of the cellular internalization by confocal laser scanning microscopy (CLSM, LSM 780, Carl Zeiss MicroImaging GmbH, Jena, Germany), A549 cells were seeded in a 24-well plate at a density of 1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells per well along with a coverslip, allowed to adhere and grow for 24 h. Before the experiment, the cells were washed thrice with Dulbecco's buffer solution. Then, the cells were incubated with rhodamine 6G (Rho) loaded Rho-PLGA nanoparticles and ADN-RhoPLGA nanoparticles dispersed in the cell culture medium at 37 ◦C. After 2 h treatments, the cells were washed three times with cold phosphate buffer saline and treated with Hoechst blue 33342 (100 ng/mL) for 30 min. The media was removed, and cells were washed with phosphate buffer saline, fixed with 4% formaldehyde, and mounted on a coverslip. To preserve the samples, coverslips were placed on microscope slides. The CLSM apparatus was used to capture microscopy pictures. While recording the images, the microscopy gain and offset settings were kept constant throughout the study. Fluorescence in the cells was observed in CLSM with excitation wavelengths at 525 and 548 nm and emission wavelengths at 504 and 461 nm for rhodamine 6G and Hoechst blue 33342, respectively [27]. The mean fluorescence intensities were calculated from CLSM images using Image J software and plotted graphically.

#### 3.5.4. In Vitro Hemocompatibility Assay

Biocompatibility was assessed using hemolysis testing. Biocompatibility of DPLGA and ADN-DPLGA was confirmed by incubating the formulations with red blood cells. Fresh blood was obtained from rats and centrifuged at 2500 rpm for 10 min at 4 ◦C in heparinized tubes. The pellet obtained after centrifugation was washed thrice with phosphate buffer saline, and cells were finally resuspended in phosphate buffer saline. In a 96-well plate, an equal volume of 100 µL of erythrocyte suspension and the nanoparticles' dispersion were combined. The plate was incubated at 37 ◦C for 1 h. After 1 h, the plate was centrifuged, and the supernatant was transferred to another 96-well plate. The absorbance was measured at 540 nm using a microplate reader (Erba LisaScan EM, Transasia, Mumbai, India). The supernatant generated from the centrifuged blood sample was used as a blank, and the supernatant derived from the blood sample treated with 1% Triton *w*/*v* was utilized as a positive control. Cells treated with 0.1% *v*/*v* DMSO in phosphate buffer saline were considered a negative control. All measurements were repeated (n = 6), and the percent hemolysis was calculated [26,27].

## *3.6. In Vivo Pharmacokinetics, Biodistribution, and Acute Toxicity Studies*

The experimental protocol was approved by the Institutional Animals Ethics Committee (Vidya Siri College of Pharmacy, Bangalore, Karnataka, India). The experiment was carried out in accordance with the rules for experimental animal care established by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA). The protocol approval number is VSCP/EC/1405/2021/2, with a date of approval 14 May 2021. Female Sprague Dawley rats (150–200 g) were used for pharmacokinetic and biodistribution evaluations. Female Swiss albino mice (20–25 g) were utilized for toxicity evaluation. The animals were housed in normal wire mesh plastic cages in a room kept at 22 ± 0.5 ◦C with a 12 h light and 12 h dark cycle, and they were fed a standard pellet diet and provided water ad libitum. Experiments were carried out between 09:00 and 17:00 h.

## 3.6.1. Pharmacokinetic Studies

The rats were assigned to one of three treatment groups: Docepar® (commercially available product), DPLGA, or ADN-DPLGA nanoparticles. Each group had six animals (n = 6). The dose equivalent to 6 mg/kg of DTX was administered intravenously (IV) [22]. At different time intervals, blood samples were withdrawn and centrifuged at a fixed speed of 10,000 rpm for 5 min at 4 ◦C. The plasma samples were kept at −80 ◦C until they were processed. DTX concentrations were measured using the previously indicated verified and calibrated HPLC technique. Mean plasma concentration vs. time profile was represented graphically, and the area under the curve (AUC) was calculated [39] for comparison purposes.

#### 3.6.2. Tissue Distribution Analysis

Animals were randomly divided into three treatment groups, namely, Docepar® (A commercially available product), DPLGA, and ADN-DPLGA nanoparticles. Individual groups had an equal number of animals. Each group received a single fixed dose of respective formulations equivalent to 5 mg/kg of DTX by IV route of administration. Mice (n = 6) were sacrificed at 1, 2, 4, and 8 h of post-dose and were dissected to isolate the heart, liver, spleen, kidney, and lungs. Tissues were weighed, homogenized in phosphate buffer saline, and were stored at −80 ◦C until further processing [40]. DTX concentration in each tissue was assessed after extracting in the organic phase and analyzing by validated RP-HPLC as described earlier. The lung targeting ability of the DTX nanoparticles was calculated using plasma concentration data. The tissue targeting ability of the delivery system was measured based on the drug targeting efficiency (*Te*) calculated using the equation below.

$$T\_{\varepsilon} = \frac{(AUC\_0^{\infty})Target\,\,tissue}{\left(AUC\_0^{\infty}\right)\,\,Non\,target\,\,tissue} \tag{1}$$

3.6.3. In Vivo Toxicity—Biochemical Analysis and Histopathology

To estimate drug-induced toxicities, mice were randomly divided into four different formulation groups, namely, untreated normal control (phosphate buffer saline, PBS), Docepar®, DPLGA, and ADN-DPLGA nanoparticles containing nanoparticles in an equal number of animals (n = 6). Formulations with a dose equivalent to 5 mg/kg of DTX were administered intravenously. The untreated normal group similarly received only normal saline [41]. Animals were humanly sacrificed after 7 days, followed by the collection of blood samples using cardiac puncture. Serum was separated, and several biochemical markers such as blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine levels were analyzed according to the instructions of the commercial kits (Sigma Aldrich, Bangalore, Karnataka, India). The vital organs, namely the liver, spleen, and kidney, were preserved in 10% formaldehyde in phosphate buffer saline, embedded in paraffin wax, and sliced into layers using a microtome (Leica, Wetzlar, Germany). The sections were observed in a light microscope (Olympus, Tokyo, Japan) after staining with Hematoxylin and Eosin (H&E).

#### *3.7. Statistical Analysis*

The statistical significance of the data was determined using a one-way analysis of variance (ANOVA) at a 95% confidence level. The Newman–Keul's test for statistical significance at the 95% confidence level was used to examine any significant differences between groups.

#### **4. Conclusions**

Lung cancer continues to be the most lethal form of cancer today. The present investigation is a proof-of-concept for developing targeted nanoparticulate interventions for non-small lung cancer (NSCLC) overexpressing adenosine (ADN) receptors (ARs). In the current investigation, an ADN ligand having a high affinity for ARs was postulated to develop ADN-conjugated PLGA nanoparticulate formulations containing docetaxel (DTX) as a chemotherapeutic agent. A series of investigations were conducted, and inferences were drawn in favor of the developed formulation. Planned comparisons between conventional clinically used formulations, i.e., Docepar® and tested formulations established the supremacy of the developed formulation over Docepar®.

Ligand-conjugated nanoparticulate systems offer a flexible and versatile technology that can be adapted to various drugs by modulating the process parameters to achieve the desired therapeutic response. When administered systemically, such ADN-conjugated nanoparticles can also serve as a platform technology for the active targeting of drugs to the cells with overexpressed ADN receptors with minimal non-target side effects.

**Author Contributions:** Conceptualization, H.M.A. and N.S.; data curation, S.S., N.A.A., R.B.B., A.A.H., N.S. and S.M.B.-E.; formal analysis, H.M.A., S.S., N.A.A., R.B.B., A.A.H., N.S. and S.M.B.-E.; funding acquisition, H.M.A., S.S., N.A.A., R.B.B., A.A.H., N.S. and S.M.B.-E.; investigation, H.M.A., S.S., N.A.A., R.B.B., A.A.H., N.S. and S.M.B.-E.; methodology, H.M.A., S.S., N.A.A., R.B.B., A.A.H., N.S. and S.M.B.-E.; project administration, H.M.A. and S.M.B.-E.; resources, A.A.H.; software, N.A.A.; supervision, S.S., R.B.B. and S.M.B.-E.; validation, S.M.B.-E.; visualization, H.M.A.; writing—original draft, H.M.A., S.S., N.A.A., R.B.B., A.A.H. and N.S.; writing—review and editing, H.M.A., N.A.A., R.B.B., A.A.H., N.S. and S.M.B.-E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project was funded by Institutional Fund Projects under grant no. (IFPRC-070-249-2020).

**Institutional Review Board Statement:** The experimental protocol was approved by the Institutional Animals Ethics Committee (Vidya Siri College of Pharmacy, Bangalore, Karnataka, India). The experiment was carried out in accordance with the rules for experimental animal care established by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA). The protocol approval number is VSCP/EC/1405/2021/2, with a date of approval 14 May 2021.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

**Acknowledgments:** The authors extend their appreciation to the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia for funding this research work through the project number IFPRC-070-249-2020 and King Abdulaziz University, DSR, Jeddah, Saudi Arabia.

**Conflicts of Interest:** The authors declare no conflict of interest.
