*Article* **Adenosine Conjugated Docetaxel Nanoparticles—Proof of Concept Studies for Non-Small Cell Lung Cancer**

**Hibah M. Aldawsari 1,2,\* , Sima Singh <sup>3</sup> , Nabil A. Alhakamy 1,2 , Rana B. Bakhaidar <sup>1</sup> , Abdulrahman A. Halwani <sup>1</sup> , Nagaraja Sreeharsha 4,5,\* and Shaimaa M. Badr-Eldin 1,2**


**Abstract:** Non-small cell lung cancer, a molecularly diverse disease, is the most prevalent cause of cancer mortality globally. Increasing understanding of the clinicopathology of the disease and mechanisms of tumor progression has facilitated early detection and multimodal care. Despite the advancements, survival rates are extremely low due to non-targeted therapeutics and correspondingly increased risk of metastasis. At some phases of cancer, patients need to face the ghost of chemotherapy. It is a difficult decision near the end of life. Such treatments have the capability to prolong survival or reduce symptoms, but can cause serious adverse effects, affecting quality of life of the patient. It is evident that many patients do not die from burden of the disease alone, but they die due to the toxic effect of treatment. Thus, increasing the efficacy is one aspect and decreasing the toxicity is another critical aspect of cancer formulation design. Through our current research, we tried to uncover both mentioned potentials of the formulation. Therefore, we designed actively targeted nanoparticles for improved therapeutics considering the overexpression of adenosine (ADN) receptors on non-small cell lung cancer (NSCLC) cells. Docetaxel (DTX), an essential therapeutic as part of combination therapy or as monotherapy for the treatment of NSCLC, was encapsulated in biodegradable poly(lactic-co-glycolic acid) nanoparticles. ADN was conjugated on the surface of nanoparticles using EDC-NHS chemistry. The particles were characterized in vitro for physicochemical properties, cellular uptake, and biocompatibility. The size and zeta potential of DTX nanoparticles (DPLGA) were found to be 138.4 ± 5.45 nm and −16.7 ± 2.3 mV which were found to change after ADN conjugation. The size was increased to 158.2 ± 6.3 nm, whereas zeta potential was decreased to −11.7 ± 1.4 mV for ADN-conjugated DTX nanoparticles (ADN-DPLGA) indicative of surface conjugation. As observed from transmission electron microscopy (TEM), the nanoparticles were spherical and showed no significant change in encapsulation efficiency even after surface conjugation. Careful and systematic optimization leads to ADN-conjugated PLGA nanoparticles having distinctive characteristic features such as particle size, surface potential, encapsulation efficacy, etc., that may play crucial roles in the fate of nanoparticles (NPs). Consequently, higher cellular uptake in the A549 lung cancer cell line was exhibited by ADN-DPLGA compared to DPLGA, illustrating the role of ADN receptors (ARs) in facilitating the uptake of NPs. Further in vivo pharmacokinetics and tissue distribution experiments revealed prolonged circulation in plasma and significantly higher lung tissue distribution than in other organs, dictating the targeting potential of the developed formulation over naïve drug and unconjugated formulations. Further, in vivo acute toxicity was examined using multiple parameters for non-toxic attributes of the developed formulation compared to other nontargeted organs. Further, it also supports the selection of biocompatible polymers in the formulation. The current study presents a proof-of-concept for a multipronged formulation technology strategy

**Citation:** Aldawsari, H.M.; Singh, S.; Alhakamy, N.A.; Bakhaidar, R.B.; Halwani, A.A.; Sreeharsha, N.; Badr-Eldin, S.M. Adenosine Conjugated Docetaxel Nanoparticles—Proof of Concept Studies for Non-Small Cell Lung Cancer. *Pharmaceuticals* **2022**, *15*, 544. https://doi.org/10.3390/ph15050544

Academic Editor: Huijie Zhang

Received: 28 March 2022 Accepted: 24 April 2022 Published: 28 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

that might be used to maximize anticancer therapeutic responses in the lungs in the treatment of NSCLC. An improved therapeutic and safety profile would help achieve maximum efficacy at a reduced dose that would eventually help reduce the toxicity.

**Keywords:** docetaxel; adenosine receptors; PLGA; nanoparticles; lung cancer

#### **1. Introduction**

Lung cancer is the greatest cause of death and illness in the world (1.59 million deaths per year), followed by colon and liver cancer. NSCLC (non-small cell lung cancer) constitutes roughly 85% of all bronchogenic carcinomas [1] that pose a relentless threat to human health [2]. It is marked by a high proliferative rate, a strong predilection for metastasis, and a poor prognosis. More than 70% of NSCLC patients are elderly, current, or past heavy smokers, and the risk rises with increasing duration and intensity of smoking [3]. Although the disease is highly sensitive to chemotherapy and radiation, a higher dose of radiation causes severe damage to normal tissues around the tumor, causing poor patient compliance and therapeutic outcome [4]. On the other side, conventional chemotherapy has its shortcomings, such as nonspecific biodistribution, toxicity, etc. [5,6].

Cancer nanotherapeutics are rapidly evolving to solve several limitations of conventional drug delivery systems [7]. The ideal physicochemical characteristics that jointly confer molecular targeting, immune evasion, and controlled drug release have been a fundamental barrier to effective clinical translation of anticancer nanomedicines. Increasing understanding of the clinicopathology of the disease and mechanisms of tumor progression has proved that adenosine (ADN) receptors (ARs) are over-expressed on tumor cells of NSCLC [8]. There are multiple subtypes of ARs that are being explored, i.e., A1, A2A, A2B, and A3, for cancer research [9], though primarily A3ARs were found to be upregulated in multiple cancers including NSCLS [10]. This is the reason why the A3AR was considered to be the tumor marker. Extracellular ADN induces apoptosis in cancer cells via diverse signaling pathways linked to ARs. ADN and other AR agonists might be effective in preventing or slowing the progression of NSCLC and other cancers [11]. Although there are many studies on the role of ARs in cancer, the ligand potential of ADN in NSCLC is still superficially studied. Chung et al. illustrated the role of ADN, as a component of a polymer chain, in increasing the cellular uptake of the polymeric carrier in cancer cells and elucidated the reduction in cellular uptake of nucleic cargo when cells were pre-treated with free ADN [12]. However, they did not gain a deep understanding of the role of ARs in drug delivery. Later, Swami et al. profoundly reported improved efficacy of and-conjugated solid lipid nanoparticles in prostate and breast cancers vis-à-vis their native counterparts [7]. The results appear consistent with prior research but they need to be validated with NSCLC. Hence, it is mandatory to prove whether ADN ligand conjugated nanoparticles can assist in targeting NSCLC or not.

Poor prognosis in the early stages of cancer makes treatment of NSCLC difficult in later stages with monotherapy with platinum-based drugs. Therefore, Docetaxel (DTX) monotherapy is generally considered a standard line treatment when patients show progression after being treated with platinum-based chemotherapy [13]. DTX is a semisynthetic BCS Class IV, highly potent, water-insoluble taxol-derived broad-spectrum antineoplastic agent, with enhanced activity in malignant and cisplatin-resistant NSCLC. Clinically used DTX contains very high amounts of surfactants and alcohol that may preclude or limit their potential clinical application due to associated toxicities.

To elucidate the ligand potential of ADN in NSCLC, we designed ADN surface decorated PLGA nanoparticles encapsulating DTX. The Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have authorized poly (lactic-co-glycolic acid) (PLGA) as a biodegradable and biocompatible polymer, raising the possibility of PLGA for sustained delivery systems. The polymer provides better control over release and

degradation of the drug delivery system. There are many commercially available products based on PLGA such as Lupron Depot, Zoladex, etc. Their success rate proved the potential of using PLGA for the present research. Moreover, free carboxylic groups at flanking ends serve another advantage for conjugation of ADN without the need for any other excipient or linker. The literature supports many instances in which DTX was used in conjunction with PLGA nanoparticles, which dictate higher efficacy and less toxicity [14]. However, this study is the first-ever report on exploring the ADN-conjugated nanoparticles for effective management of NSCLC. We provided a proof-of-concept for systematically exploring DTXloaded PLGA nanoparticles as a safe, improved, actively targeted therapeutic intervention for NSCLC using in vitro characterizations and in vivo evaluations.

#### **2. Results and Discussion**

#### *2.1. Formulation and Characterization of DPLGA and ADN-DPLGA Nanoparticles*

DTX encapsulation is responsible for increasing the mean particle size of the PLGA nanoparticles from 102.2 ± 3.23 nm to 138.43 ± 5.45 nm (Table 1).


**Table 1.** Mean particle size, surface potential, and entrapment efficiency among different formulations \*.

\* Data represent the mean of six determinations ± SD.

Upon conjugation, an additional increase in the mean particle size was evident owing to the attachment of multiple ADN molecules over the DPLGA nanoparticles surface (158.2 ± 6.3 nm). This is evident by the significant difference in the mean particle size of the two nanoparticles (Table 1). However, practiced peptide chemistry for the conjugation of free carboxylic groups on the surface of the DPLGA nanoparticles with the amine group of the ADN molecules resulted in decreased overall negative charge of ADN-DPLGA nanoparticles. Thus, the zeta potential of conjugated nanoparticles (ADN-DPLGA) was significantly lower (−11.7 ± 1.4) than that of non-conjugated particles (DPLGA, −16.7 ± 2.3 mV). Encapsulation efficiency expressed as % was observed as 80.12 ± 1.98 and 84.4 ± 2.61%, respectively, for DPLGA and ADN-DPLGA nanoparticles. An earlier report on multicomponent PLGA nanoparticles of docetaxel has shown entrapment efficiency of 69–75% [15]. There was no significant change in the DTX encapsulation after ADN decoration over the DPLGA nanoparticles, as illustrated in Table 1. TEM images of DPLGA Nanoparticles and ADN-DPLGA nanoparticles (Figure 1A,B) indicate that nanoparticles are spherical and have uniform size distribution. The conjugation did not affect the size and morphology of the particles. *Pharmaceuticals* **2022**, *15*, x FOR PEER REVIEW 4 of 17

> **Figure 1.** (**A**,**B**) The transmission electron microscopy (TEM) images of DPLGA and ADN-DPLGA nanoparticles, respectively. (**C**,**D**) The in vitro release profiles of DTX from pristine DTX, DPLGA, and ADN-DPLGA nanoparticles in phosphate buffer saline pH 7.4, and sodium acetate buffer (pH

> The extent of conjugation of amine groups of ADN with free carboxylic groups of PLGA was assessed using the colorimetry method [7]. The results were very encouraging, presenting around 75% conjugation efficiency. Higher conjugation efficiency is also the cause of the larger size of ADN-DPLGA nanoparticles observed in size measurements and

TEM images shown in earlier sections. **Figure 1.** *Cont*.

5.0), respectively.

*2.2. Conjugation Efficiency* 

**Figure 1.** (**A**,**B**) The transmission electron microscopy (TEM) images of DPLGA and ADN-DPLGA nanoparticles, respectively. (**C**,**D**) The in vitro release profiles of DTX from pristine DTX, DPLGA, and ADN-DPLGA nanoparticles in phosphate buffer saline pH 7.4, and sodium acetate buffer (pH 5.0), respectively. **Figure 1.** (**A**,**B**) The transmission electron microscopy (TEM) images of DPLGA and ADN-DPLGA nanoparticles, respectively. (**C**,**D**) The in vitro release profiles of DTX from pristine DTX, DPLGA, and ADN-DPLGA nanoparticles in phosphate buffer saline pH 7.4, and sodium acetate buffer (pH 5.0), respectively.

#### *2.2. Conjugation Efficiency 2.2. Conjugation Efficiency*

The extent of conjugation of amine groups of ADN with free carboxylic groups of PLGA was assessed using the colorimetry method [7]. The results were very encouraging, presenting around 75% conjugation efficiency. Higher conjugation efficiency is also the cause of the larger size of ADN-DPLGA nanoparticles observed in size measurements and TEM images shown in earlier sections. The extent of conjugation of amine groups of ADN with free carboxylic groups of PLGA was assessed using the colorimetry method [7]. The results were very encouraging, presenting around 75% conjugation efficiency. Higher conjugation efficiency is also the cause of the larger size of ADN-DPLGA nanoparticles observed in size measurements and TEM images shown in earlier sections.

## *2.3. In Vitro Release Studies*

In vitro release of DTX from pristine DTX suspension in two different pH media, namely pH 7.4 phosphate buffer saline and pH 5.0 sodium acetate buffer, was almost complete within 12 h (>95%) (Figure 1C,D). The developed PLGA nanoparticles showed a biphasic release pattern, indicated by initial burst release followed by sustained and slow release over a prolonged period. Approximately 20 ± 2% and 21.2 ± 1.8% of DTX were released from DPLGA and ADN-DPLGA nanoparticles, respectively. At the end of day 1, only 22.5 ± 1.3 and 23.5 ± 1.2% DTX was released in phosphate buffer saline. However, in the next 4 days, only an additional 13–16% of DTX was released. The initial rapid release of DTX can be attributed to the dissolution of DTX present on the surface

of the nanoparticles. Similar phenomena were seen in sodium acetate buffer as well, for both nanoparticle formulations. However, the release in sodium acetate buffer was slightly faster than the release in phosphate buffer. This may facilitate the faster release of DTX from the nanoparticles once they are taken up by the cancer cells. However, the singlepoint measurement on 8th day denotes around 70% release, illustrating the degradation mechanism becoming the prominent mechanism of drug release. The findings were in accordance with the previously published reports [16,17]. An earlier report on lipid-based DTX particles showed around 80% release in 10 days [18]. When the release profile of DTX from pristine DTX suspension was compared with that of nanoparticles using the *f* <sup>2</sup> similarity factor, the release patterns were dissimilar as the value of *f* <sup>2</sup> was less than 50 (*f* <sup>2</sup> = 15 for DTX vs. DPLGA and *f* <sup>2</sup> = 13.9 for DTX vs. ADN-DPLGA in phosphate buffer saline pH 7.4 and *f* <sup>2</sup> = 13.6 for DTX vs. DPLGA and *f* <sup>2</sup> = 14.2 for DTX vs. ADN-DPLGA). The release profiles of DTX from DPLGA and ADN-DPLGA were similar as the *f* <sup>2</sup> similarity value was 87.8 and 82.9 for phosphate buffer saline pH 7.4 and sodium acetate buffer pH 5.0, respectively. nanoparticle formulations. However, the release in sodium acetate buffer was slightly faster than the release in phosphate buffer. This may facilitate the faster release of DTX from the nanoparticles once they are taken up by the cancer cells. However, the singlepoint measurement on 8th day denotes around 70% release, illustrating the degradation mechanism becoming the prominent mechanism of drug release. The findings were in accordance with the previously published reports [16,17]. An earlier report on lipid-based DTX particles showed around 80% release in 10 days [18]. When the release profile of DTX from pristine DTX suspension was compared with that of nanoparticles using the *f2* similarity factor, the release patterns were dissimilar as the value of *f2* was less than 50 (*f2* = 15 for DTX vs. DPLGA and *f2* = 13.9 for DTX vs. ADN-DPLGA in phosphate buffer saline pH 7.4 and *f2* = 13.6 for DTX vs. DPLGA and *f2* = 14.2 for DTX vs. ADN-DPLGA). The release profiles of DTX from DPLGA and ADN-DPLGA were similar as the *f2* similarity value was 87.8 and 82.9 for phosphate buffer saline pH 7.4 and sodium acetate buffer pH 5.0, respectively.

In vitro release of DTX from pristine DTX suspension in two different pH media, namely pH 7.4 phosphate buffer saline and pH 5.0 sodium acetate buffer, was almost complete within 12 h (>95%) (Figure 1 C,D). The developed PLGA nanoparticles showed a biphasic release pattern, indicated by initial burst release followed by sustained and slow release over a prolonged period. Approximately 20 ± 2% and 21.2 ± 1.8% of DTX were released from DPLGA and ADN-DPLGA nanoparticles, respectively. At the end of day 1, only 22.5 ± 1.3 and 23.5 ± 1.2% DTX was released in phosphate buffer saline. However, in the next 4 days, only an additional 13–16% of DTX was released. The initial rapid release of DTX can be attributed to the dissolution of DTX present on the surface of the nanoparticles. Similar phenomena were seen in sodium acetate buffer as well, for both

#### *2.4. In Vitro Cell-Based Assays 2.4. In Vitro Cell-Based Assays*

*2.3. In Vitro Release Studies* 

#### 2.4.1. In Vitro MTT Assay for Calculation of IC<sup>50</sup> (Half Maximal Inhibitory Concentration) 2.4.1. In Vitro MTT Assay for Calculation of IC50 (Half Maximal Inhibitory Concentration)

A concentration-dependent toxicity profile of the formulation was evident in the MTT assay on the A549 cell line. However, intraformational differences revealed higher activity, in terms of lower IC<sup>50</sup> values, in the case of PLGA nanoparticles compared to pristine DTX treatment (Figure 2). A concentration-dependent toxicity profile of the formulation was evident in the MTT assay on the A549 cell line. However, intraformational differences revealed higher activity, in terms of lower IC50 values, in the case of PLGA nanoparticles compared to pristine DTX treatment (Figure 2).

*Pharmaceuticals* **2022**, *15*, x FOR PEER REVIEW 5 of 17

**Figure 2.** In vitro cell line studies. (**A**) Cell viability (%) of A549 cell lines treated with pristine DTX, DPLGA, and ADN-DPLGA nanoparticles. (**B**) Receptor competition assay outcomes showed an increase in the IC50 of ADN-DPLGA nanoparticles after A549 cells were treated with free ADN (presaturation), causing blockage of ADN receptors on the cells. Thus, causing a reduction in the uptake of nanoparticles by the cells results in decreased efficacy. Values represent the mean of six determinations, and error bars indicate standard deviation. **Figure 2.** In vitro cell line studies. (**A**) Cell viability (%) of A549 cell lines treated with pristine DTX, DPLGA, and ADN-DPLGA nanoparticles. (**B**) Receptor competition assay outcomes showed an increase in the IC<sup>50</sup> of ADN-DPLGA nanoparticles after A549 cells were treated with free ADN (pre-saturation), causing blockage of ADN receptors on the cells. Thus, causing a reduction in the uptake of nanoparticles by the cells results in decreased efficacy. Values represent the mean of six determinations, and error bars indicate standard deviation.

IC50 values were found to be 130.83, 80.72, and 49.50 ng/mL for pristine DTX, DPLGA, and ADN-DPLGA nanoparticles, respectively, after 48 h. The efficacy of DTX was IC<sup>50</sup> values were found to be 130.83, 80.72, and 49.50 ng/mL for pristine DTX, DPLGA, and ADN-DPLGA nanoparticles, respectively, after 48 h. The efficacy of DTX was significantly increased after being encapsulated in nanoparticles. We speculate that this might be due to the small particle size of nanoparticles resulting in higher internalization. Previous literature reported having 16-fold overexpression of ADN receptors in A549 [19]. This overexpression of the ADN receptor might be the reason for the higher retention of ADN-DPLGA nanoparticles in A549 cells due to ligand-mediated internalization. The higher efficacy in ADN-DPLGA was substantiated by the higher/rapid release of DTX in acidic pH, i.e., cancer cells (as presented in the release profile investigation in previous sections).

#### 2.4.2. Receptor Competition Assay

The role of ADN receptors in the uptake of ADN-DPLGA nanoparticles was assessed using a receptor competition assay. Results were found to be in favor of the proposed hypothesis. It is observed that IC<sup>50</sup> values were notably amplified (*p* < 0.001) after saturation of ADN receptors with free ADN, as shown in Figure 2B. Free ADN exposure to A549 cells caused blockage of ARs, causing a reduction in the receptor-mediated endocytosis of the ADN-DPLGA nanoparticles. These findings support the notion that uptake of the ADN-DPLGA nanoparticles is influenced by the overexpression of ADN receptors over A549 cells that represent an example of non-small cells causing lung cancer. Previously chen et al. also documented the effect of folic acid–folic acid receptor interaction

#### 2.4.3. Cellular Uptake of Nanoparticles

A549, an epithelial carcinoma cell line, is commonly used as a model to study nonsmall-cell lung cancer [20]. Moreover, as already discussed in the previous results there is around 16-fold overexpression of ARs over A549 cell lines [19]. Following the literature evidence, we too observed higher uptake in the case of ADN-RhoPLGA nanoparticles due to selective uptake of ADN conjugated nanoparticles through highly specific and effective receptor-mediated endocytosis (Figure 3). These findings also corroborated our previous results obtained in the MTT assay. Similar inferences were drawn in several previous publications, where the authors illustrated the receptor–ligand interaction as the crucial factor for internalization of the nanoparticles to cancer cells [21,22]. Other than ARs, there are many receptors that were highlighted in previous research, assisting in cellular uptake of nanoparticles, i.e., transferrin, lactoferrin, sigma receptors, folic acid receptors, etc. [23–25]. *Pharmaceuticals* **2022**, *15*, x FOR PEER REVIEW 7 of 17

**Figure 3.** Cellular uptake and distribution of rhodamine 6G labeled DPLGA and ADN-DPLGA nanoparticles in A59 cells. (**A**,**B**) Representative CLSM images of A549 cells treated with rhodamine 6G labeled PLGA (Rho-PLGA) and ADN-RhoPLGA nanoparticles for 2 h. The cell nucleus was stained with Hoechst 33342. (**C**) Comparison of average fluorescence intensities for quantitative evaluation. The average fluorescence intensity of rhodamine was calculated using Image J software showing significantly higher uptake of ADN-PLGA nanoparticles by A549 cells. (**D**) represents the hemocompatibility analysis of DPLGA and ADN-PLGA nanoparticles when treated with red blood cells of rats. Positive control represents the treatment with Triton resulting in complete rupture of red blood cells causing maximum hemolysis. Negative control cells were treated with DMSO in phosphate buffer saline. The data represents the mean of 6 determinations, and error bars represent the standard deviation. 2.4.4. Hemocompatibility Analysis to Estimate Biocompatibility of Nanoparticles **Figure 3.** Cellular uptake and distribution of rhodamine 6G labeled DPLGA and ADN-DPLGA nanoparticles in A59 cells. (**A**,**B**) Representative CLSM images of A549 cells treated with rhodamine 6G labeled PLGA (Rho-PLGA) and ADN-RhoPLGA nanoparticles for 2 h. The cell nucleus was stained with Hoechst 33342. (**C**) Comparison of average fluorescence intensities for quantitative evaluation. The average fluorescence intensity of rhodamine was calculated using Image J software showing significantly higher uptake of ADN-PLGA nanoparticles by A549 cells. (**D**) represents the hemocompatibility analysis of DPLGA and ADN-PLGA nanoparticles when treated with red blood cells of rats. Positive control represents the treatment with Triton resulting in complete rupture of red blood cells causing maximum hemolysis. Negative control cells were treated with DMSO in phosphate buffer saline. The data represents the mean of 6 determinations, and error bars represent the standard deviation.

Outlining the interaction of developed nanoparticles with red blood cells is an essential step toward establishing the safety of the product and the plausibility of utilizing the

we expect them to be safe for the blood cells. In the present investigation, the hemolytic effects of DPLGA nanoparticles and ADN-DPLGA nanoparticles were compared with Triton in PBS (1% *w*/*v*, positive control) and DMSO in PBS (0.1% *v*/*v*, negative control) (Figure 3D). In general, hemolysis less than 10% is considered non-hemolytic and, therefore, safe and biocompatible. In the present investigation, the DPLGA nanoparticle formulation and ADN-DPLGA nanoparticles showed 3.1 and 3.2% hemolysis. Therefore, the developed polymeric nanoparticles are considered safe for systemic administration for the treatment

The mean plasma concentration vs. time profiles after single intravenous injection of DPLGA nanoparticles, ADN-DPLGA nanoparticles, and Docepar® (Parenteral Drugs In-

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

dia Ltd., Mumbai, India) in rats are shown in Figure 4.

of NSCLC [26,27].

2.5.1. Pharmacokinetic Studies

#### 2.4.4. Hemocompatibility Analysis to Estimate Biocompatibility of Nanoparticles

Outlining the interaction of developed nanoparticles with red blood cells is an essential step toward establishing the safety of the product and the plausibility of utilizing the polymeric nanoparticles as delivery tools for several other therapeutic and biomedical applications. Formulations are composed of biocompatible and biodegradable. Therefore, we expect them to be safe for the blood cells. In the present investigation, the hemolytic effects of DPLGA nanoparticles and ADN-DPLGA nanoparticles were compared with Triton in PBS (1% *w*/*v*, positive control) and DMSO in PBS (0.1% *v*/*v*, negative control) (Figure 3D). In general, hemolysis less than 10% is considered non-hemolytic and, therefore, safe and biocompatible. In the present investigation, the DPLGA nanoparticle formulation and ADN-DPLGA nanoparticles showed 3.1 and 3.2% hemolysis. Therefore, the developed polymeric nanoparticles are considered safe for systemic administration for the treatment of NSCLC [26,27].
