*2.5. In Vivo Pharmacokinetics, Biodistribution, and Acute Toxicity Testing* 2.5.1. Pharmacokinetic Studies

The mean plasma concentration vs. time profiles after single intravenous injection of DPLGA nanoparticles, ADN-DPLGA nanoparticles, and Docepar® (Parenteral Drugs India Ltd., Mumbai, India) in rats are shown in Figure 4. *Pharmaceuticals* **2022**, *15*, x FOR PEER REVIEW 8 of 17

**Figure 4.** The mean plasma docetaxel concentration vs. time profile after a single intravenous injection of three different formulations, namely Docepar®, DPLGA, ADN-DPLGA nanoparticles equivalent to DTX (5 mg/kg). More retention and slower excretion were seen for PLGA formulations (DPLGA and ADN-DPLGA), thus, causing a larger area under curve (AUC). **Figure 4.** The mean plasma docetaxel concentration vs. time profile after a single intravenous injection of three different formulations, namely Docepar®, DPLGA, ADN-DPLGA nanoparticles equivalent to DTX (5 mg/kg). More retention and slower excretion were seen for PLGA formulations (DPLGA and ADN-DPLGA), thus, causing a larger area under curve (AUC).

It is presumed that hydrophobic surfaces tend to face early clearance from systemic circulation. Accordingly, in the present study, we observed much earlier clearance of the DTX compared to the ADN-DPLGA nanoparticles. Since ADN is a hydrophilic molecule containing sugar, it decreases RES uptake leading to reduced clearance and higher area under the curve (AUC). However, higher AUC in the case of unconjugated DPLGA nanoparticles is debatable. We speculate that it might be due to the negative charge on the surface of PLGA nanoparticles. The negative charge of the particles allows them to circumvent the RES uptake resulting in significantly prolonged systemic circulation com-It is presumed that hydrophobic surfaces tend to face early clearance from systemic circulation. Accordingly, in the present study, we observed much earlier clearance of the DTX compared to the ADN-DPLGA nanoparticles. Since ADN is a hydrophilic molecule containing sugar, it decreases RES uptake leading to reduced clearance and higher area under the curve (AUC). However, higher AUC in the case of unconjugated DPLGA nanoparticles is debatable. We speculate that it might be due to the negative charge on the surface of PLGA nanoparticles. The negative charge of the particles allows them to circumvent the RES uptake resulting in significantly prolonged systemic circulation compared to naïve drugs [28].

pared to naïve drugs [28]. It can be seen that, compared with pure DTX, DTX nanoparticles, both DPLGA and ADN-DPLGA exhibited altered pharmacokinetic distribution of DTX in vivo and showed remarkably higher and prolonged plasma concentrations. The DPLGA nanoparticles exhibit almost ~3.38 times higher AUC (μg/mL.h) as compared to the pure drug ( <sup>ஶ</sup>DTX: It can be seen that, compared with pure DTX, DTX nanoparticles, both DPLGA and ADN-DPLGA exhibited altered pharmacokinetic distribution of DTX in vivo and showed remarkably higher and prolonged plasma concentrations. The DPLGA nanoparticles exhibit almost ~3.38 times higher AUC (µg/mL·h) as compared to the pure drug (*AUC* <sup>∞</sup> <sup>0</sup> DTX: 8.10

PLGA formulations. The findings were in agreement with the previous literature [7,29].

In vivo biodistribution behavior of DTX post intravenous administration of the DTX nanoparticles (both ADN-DPLGA and DPLGA) in rats was investigated and compared with that of DTX commercially available injection as a control. The amounts of the drug distributed in the heart, liver, spleen, lung, and kidney were measured at different pre-

New and novel nanoparticulate drug delivery systems overcome nonspecific distribution hurdles by targeting the drug to the related organ/cells. Biodistribution studies help predict the fate of nanoparticulate formulations; consequently, one can determine the exposure of different drug titers in various organs. This is an important finding in understanding the toxicity profile of the formulation. The current study findings indicate that exposure to different tissues is minimal compared to native clinical formulation, i.e., Docepar®. There is an insignificant difference among the PLGA formulations owing to similar basic characteristics of the formulation (Figure 5). Swami et al. explained that through biodistribution, organs are exposed to elevated levels of drug concentrations leading to

<sup>ஶ</sup> DPLGA: 27.41) while ADN-DPLGA nanoparticles show ~4.51 times higher

<sup>ஶ</sup> DTX: 8.10 vs.

<sup>ஶ</sup> DPLGA: 36.63). Though, the

AUC as compared to the pure drug (

2.5.2. Tissue Distribution Analysis

determined time points.

8.10 vs.

vs. *AUC* <sup>∞</sup> <sup>0</sup> DPLGA: 27.41) while ADN-DPLGA nanoparticles show ~4.51 times higher AUC as compared to the pure drug (*AUC* <sup>∞</sup> <sup>0</sup> DTX: 8.10 vs. *AUC* <sup>∞</sup> <sup>0</sup> DPLGA: 36.63). Though, the insignificant difference in mean plasma concentration was evident among the two tested PLGA formulations. The findings were in agreement with the previous literature [7,29].

#### 2.5.2. Tissue Distribution Analysis

In vivo biodistribution behavior of DTX post intravenous administration of the DTX nanoparticles (both ADN-DPLGA and DPLGA) in rats was investigated and compared with that of DTX commercially available injection as a control. The amounts of the drug distributed in the heart, liver, spleen, lung, and kidney were measured at different predetermined time points.

New and novel nanoparticulate drug delivery systems overcome nonspecific distribution hurdles by targeting the drug to the related organ/cells. Biodistribution studies help predict the fate of nanoparticulate formulations; consequently, one can determine the exposure of different drug titers in various organs. This is an important finding in understanding the toxicity profile of the formulation. The current study findings indicate that exposure to different tissues is minimal compared to native clinical formulation, i.e., Docepar®. There is an insignificant difference among the PLGA formulations owing to similar basic characteristics of the formulation (Figure 5). Swami et al. explained that through biodistribution, organs are exposed to elevated levels of drug concentrations leading to toxicity. Hence correlating the drug exposure with toxicity marker gives a holistic view of the targeting to toxicity potential of a formulation. *Pharmaceuticals* **2022**, *15*, x FOR PEER REVIEW 9 of 17 toxicity. Hence correlating the drug exposure with toxicity marker gives a holistic view of the targeting to toxicity potential of a formulation.

**Figure 5.** Tissue distribution studies. The concentration of DTX in various tissues after administration of three different formulations, namely, Docepar®, DPLGA, ADN-DPLGA nanoparticles equivalent to DTX (5 mg/kg) at four different time intervals, namely, 1, 2, 4, and 8 h. Data represents the mean of 6 determinations and error bars represent standard deviation. **Figure 5.** Tissue distribution studies. The concentration of DTX in various tissues after administration of three different formulations, namely, Docepar®, DPLGA, ADN-DPLGA nanoparticles equivalent to DTX (5 mg/kg) at four different time intervals, namely, 1, 2, 4, and 8 h. Data represents the mean of 6 determinations and error bars represent standard deviation.

To have a better understanding of the targeting efficiency (Te) of the DTX from both the nanoparticles, namely, DPLGA as well as ADN-DPLGA, parameter Te was calculated [30,31]. The *Te* demonstrates the ability of the delivery system to reach the target and nontarget tissues. The *Te* values indicate preferential accumulation of nanoparticles in the lung tissues compared to the pristine DTX (Table 2). To have a better understanding of the targeting efficiency (*Te*) of the DTX from both the nanoparticles, namely, DPLGA as well as ADN-DPLGA, parameter *Te* was calculated [30,31]. The *Te* demonstrates the ability of the delivery system to reach the target and non-target tissues. The *Te* values indicate preferential accumulation of nanoparticles in the lung tissues compared to the pristine DTX (Table 2).

**Table 2.** Comparative drug targeting efficiency of DTX form Docepar®, DPLGA, and ADN-DPLGA nanoparticles. **Organs** *Te* **DTX** *Te* **DPLGA** *Te* **ADN-DPLGA**  Lung 0.24 3.23 3.87 Liver 2.35 0.25 0.18 These results indicate the accumulation of DTX nanoparticles in the lungs. However, there is an insignificant difference between the accumulation of ADN-DPLGA and DPLGA nanoparticles in the lungs. Though results of cell uptake studies clearly show a preferential uptake of ADN-DPLGA nanoparticles by the cancerous lung cells. ADN-DPLGA nanoparticles are the adenosine-conjugated nanoparticles that are expected to preferentially accumulate in the cancerous lung cells that exhibit over-expressed ARs.

These results indicate the accumulation of DTX nanoparticles in the lungs. However, there is an insignificant difference between the accumulation of ADN-DPLGA and DPLGA nanoparticles in the lungs. Though results of cell uptake studies clearly show a preferential uptake of ADN-DPLGA nanoparticles by the cancerous lung cells. ADN-DPLGA nanoparticles are the adenosine-conjugated nanoparticles that are expected to preferentially accumulate in the cancerous lung cells that exhibit over-expressed ARs. However, the present study was carried out in non-cancerous, healthy animals with lung ARs. The absence of overexpressed adenosine receptors in healthy animals seems to be the reason for equivalent tissue accumulation of the ADN-DPLGA and DPLGA nanoparticles in the lungs. However, this study revealed a key component for designing future studies on disease/cancer models to understand adjoining effects of the ADN ligand (on nanoparticle surfaces) and overexpressed ARs (on lung cancer cells) on the migration of

Spleen 1.23 0.32 0.36 Kidney 2.08 0.92 0.98

ADN-DPLGA nanoparticles.

However, the present study was carried out in non-cancerous, healthy animals with lung ARs. The absence of overexpressed adenosine receptors in healthy animals seems to be the reason for equivalent tissue accumulation of the ADN-DPLGA and DPLGA nanoparticles in the lungs. However, this study revealed a key component for designing future studies on disease/cancer models to understand adjoining effects of the ADN ligand (on nanoparticle surfaces) and overexpressed ARs (on lung cancer cells) on the migration of ADN-DPLGA nanoparticles.

**Organs** *Te* **DTX** *Te* **DPLGA** *Te* **ADN-DPLGA** Lung 0.24 3.23 3.87 Liver 2.35 0.25 0.18 Spleen 1.23 0.32 0.36 Kidney 2.08 0.92 0.98 Heart 1.06 0.65 0.54

**Table 2.** Comparative drug targeting efficiency of DTX form Docepar®, DPLGA, and ADN-DPLGA nanoparticles.

#### 2.5.3. In Vivo Toxicity Evaluations

Docepar®, a clinically used formulation of DTX, utilizes a cocktail of surfactant and alcohol to solubilize the hydrophobic DTX to avoid drug precipitation in vitro and the systemic circulation after intravenous administration. However, the formulation is known for its toxic effects, such as hypersensitivity reactions, tissue toxicity, etc., which coincide with the native side effects of the DTX. Hence, assessing our developed formulations' toxicities and comparing them with the clinically used formulation is of utmost necessity. Variations in serum toxicity markers to analyze the abnormality in the blood hepatobiliary system (ALT, AST) and kidney (BUN, creatinine) exemplified significant improvement (*p* < 0.05) when compared with the developed nanoparticles, as presented in Figure 6. *Pharmaceuticals* **2022**, *15*, x FOR PEER REVIEW 10 of 17 2.5.3. In Vivo Toxicity Evaluations Docepar®, a clinically used formulation of DTX, utilizes a cocktail of surfactant and

Docepar® showed significantly higher toxicity (*p* < 0.001) as compared to PLGA formulations due to already stated reasons. Though insignificant, (*p* > 0.05) a difference between the DPLGA and ADN-DPLGA nanoparticles was evident. The histology evaluations also corroborated higher toxicity. The histological evaluations of organ (kidney, liver, and spleen) specimens revealed a normal pattern of morphology in the case of the control group, DPLGA nanoparticles, and ADN-DPLGA nanoparticles. However, prominent characteristic features were perceived in the liver (hepatocytes degeneration and infiltrations), spleen (splenocytes damage), and kidney (necrotic tubules and debris). alcohol to solubilize the hydrophobic DTX to avoid drug precipitation in vitro and the systemic circulation after intravenous administration. However, the formulation is known for its toxic effects, such as hypersensitivity reactions, tissue toxicity, etc., which coincide with the native side effects of the DTX. Hence, assessing our developed formulations' toxicities and comparing them with the clinically used formulation is of utmost necessity. Variations in serum toxicity markers to analyze the abnormality in the blood hepatobiliary system (ALT, AST) and kidney (BUN, creatinine) exemplified significant improvement (*p* < 0.05) when compared with the developed nanoparticles, as presented in Figure 6.

mulations due to already stated reasons. Though insignificant, (*p* <sup>&</sup>gt; 0.05) a difference **Figure 6.** *Cont*.

**Figure 6.** In vivo toxicity studies. (**A**–**D**) The serum biochemical markers levels representing ALT, AST, BUN, and creatinine, respectively, for four different groups of animals administered with untreated normal control (phosphate buffer saline, PBS), Docepar®, DPLGA, and ADN-DPLGA nanoparticles. Asterisk in biochemical studies signifies statistical limits in biochemical marker graphical representations: \*\*\* represents significant difference at *p* < 0.001, respectively, by Newman–Keuls analysis following ANOVA at 95% confidence limit. (**E**) Histological evaluations of different collected organs from the animals for toxicity investigations. Arrows indicate histological changes.

Docepar® showed significantly higher toxicity (*p* < 0.001) as compared to PLGA for-

Docepar®, a clinically used formulation of DTX, utilizes a cocktail of surfactant and alcohol to solubilize the hydrophobic DTX to avoid drug precipitation in vitro and the systemic circulation after intravenous administration. However, the formulation is known for its toxic effects, such as hypersensitivity reactions, tissue toxicity, etc., which coincide with the native side effects of the DTX. Hence, assessing our developed formulations' toxicities and comparing them with the clinically used formulation is of utmost necessity. Variations in serum toxicity markers to analyze the abnormality in the blood hepatobiliary system (ALT, AST) and kidney (BUN, creatinine) exemplified significant improvement (*p* < 0.05) when compared with the developed nanoparticles, as presented in Figure 6.

**Figure 6.** In vivo toxicity studies. (**A**–**D**) The serum biochemical markers levels representing ALT, AST, BUN, and creatinine, respectively, for four different groups of animals administered with untreated normal control (phosphate buffer saline, PBS), Docepar®, DPLGA, and ADN-DPLGA nanoparticles. Asterisk in biochemical studies signifies statistical limits in biochemical marker graphical representations: \*\*\* represents significant difference at *p* < 0.001, respectively, by Newman–Keuls analysis following ANOVA at 95% confidence limit. (**E**) Histological evaluations of different collected organs from the animals for toxicity investigations. Arrows indicate histological changes. **Figure 6.** In vivo toxicity studies. (**A**–**D**) The serum biochemical markers levels representing ALT, AST, BUN, and creatinine, respectively, for four different groups of animals administered with untreated normal control (phosphate buffer saline, PBS), Docepar®, DPLGA, and ADN-DPLGA nanoparticles. Asterisk in biochemical studies signifies statistical limits in biochemical marker graphical representations: \*\*\* represents significant difference at *p* < 0.001, respectively, by Newman– Keuls analysis following ANOVA at 95% confidence limit. (**E**) Histological evaluations of different collected organs from the animals for toxicity investigations. Arrows indicate histological changes.

#### Docepar® showed significantly higher toxicity (*p* < 0.001) as compared to PLGA formulations due to already stated reasons. Though insignificant, (*p* > 0.05) a difference **3. Materials and Methods**

2.5.3. In Vivo Toxicity Evaluations

#### *3.1. Materials*

TherDose Pharma Pvt Ltd. generously provided docetaxel (DTX) and poly(d,l-lacticco-glycolic acid) (PLGA) with a free carboxyl end group (uncapped) and an L/G molar ratio of 50:50, (Hyderabad, Andhra Pradesh, India) and Evonik (Mumbai, Maharashtra, India), respectively. ADN, Tween 80, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), Formaldehyde, Hoechst blue 33342 Rhodamine 6G, chloroform, methanol, acetone, dichloromethane, phosphotungstic acid, mannitol, dimethyl sulfoxide (DMSO), and acetonitrile were of HPLC grade (Merck, Mumbai, Maharashtra, India). The A549 cell line was obtained from the National Centre for Cell Science (NCCS, Pune, Maharashtra, India). Dulbecco's modified Eagle's Medium (DMEM), fetal bovine serum MTT (3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide), trypsin, EDTA, 2-(N-morpholino) ethanesulfonic acid (MES), Triton, and 96-well flat bottom tissue culture plates were purchased from Himedia (Mumbai, Maharashtra, India). Dialysis tubes were purchased from Spectrum (Float-A-Lyzer (G2, Spectrum, Repligen, MA, USA).

#### *3.2. Preparation of DTX-Loaded PLGA Nanoparticles (DPLGA)*

DTX (10 mg) was dissolved in 2 mL of acetone and dichloromethane mixture (1:1). PLGA (100 mg) was added to the DTX solution. This oil phase was emulsified for two minutes in an ice bath with an aqueous solution containing 0.25% Tween® 20 using a probe sonicator (VCX 130, Sonic and Materials, Newtown, CT, USA). After emulsification, the oil-in-water emulsion was magnetically stirred for eight hours to evaporate the organic solvent [32]. The dispersion of nanoparticles was centrifuged at 15,000 rpm for 20 min at 4 ◦C, then washed three times with deionized water, lyophilized (5% mannitol as a freeze-drying agent), and kept at 2–8 ◦C. The freeze-dried nanoparticles were characterized. Similarly, blank PLGA nanoparticles were also prepared.

#### *3.3. Conjugation of ADN on the Surface of DPLGA Nanoparticles*

Ten milligrams of DPLGA nanoparticles were distributed in five milliliters of 0.1 M MES buffer and incubated with NHS and EDC (1:5 *w*/*w*). The dispersion was kept under gentle stirring for 2 h at room temperature, protected from light to activate free carboxylic acid groups on the PLGA nanoparticles' surfaces. To this, 1 mg ADN was added, mixed well, and kept for further stirring for 4 h. ADN-conjugated DTX-loaded PLGA (ADN-DPLGA) nanoparticles were collected after centrifugation (Sigma Laborentrifugen GMBH, Osterode am Harz, Germany) at 15,000 rpm for 20 min and washed thrice with distilled water to remove unconjugated ADN in the supernatant. Prepared ADN–DPLGA pellets were recollected and freeze-dried (Lab Conco, Mumbai, Maharashtra, India). Freeze-dried nanoparticles were characterized further.

The conjugation efficiency of ADN to PLGA nanoparticles was quantified using phenol-sulphuric acid calorimetry assay as reported by Swami et al. [7] and expressed as a percentage of ADN bound to DPLGA nanoparticles. For cellular uptake, the nanoparticles were prepared using the same method along with rhodamine 6G as the fluorescent marker.

#### *3.4. In Vitro Characterization of DPLGA and ADN-DPLGA Nanoparticles*

The prepared nanoparticles, namely DPLGA and DTX-DPLGA, were characterized for several physicochemical parameters as stated below.
