**1. Introduction**

**\***

> The two major problems in cancer treatment are drug resistance and the spreading of cancer cells in the body or metastasis. Metastatic tumors are difficult to treat because they frequently develop drug resistance, which remains a major cause of cancer-related death. During metastasis, cancer cells float through the blood and lymphatic circulatory system until they reach targeted locations in distant organs [1]. Normal epithelial and endothelial cells require adhesion to their appropriate extracellular matrix (ECM) and neighboring cells for maintaining their survival. Detachment from ECM or adhesion to inappropriate ECM leads to activation of a programmed cell death termed anoikis in the normal epithelial and endothelial cells [2,3]. This phenomenon avoids the misplaced growth of normal cells in other sites. However, some populations of cancer cells are able to develop anoikis resistance to support their survival when they become floating cells during the journey through the circulatory systems [4]. There is considerable interest in developing approaches for managing anoikis-resistant cancer cells.

> Drug resistance of cancer cells in patients can occur before receiving chemotherapy (inherent or intrinsic resistance) or emerge after treatment (acquired resistance), this classi-

**Citation:** Pramual, S.;

Lirdprapamongkol, K.; Atjanasuppat, K.; Chaisuriya, P.; Niamsiri, N.; Svasti, J. PLGA-Lipid Hybrid Nanoparticles for Overcoming Paclitaxel Tolerance in Anoikis-Resistant Lung Cancer Cells. *Molecules* **2022**, *27*, 8295. https:// doi.org/10.3390/molecules27238295

Academic Editor: Rita Cortesi

Received: 2 November 2022 Accepted: 24 November 2022 Published: 28 November 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/).

fication is based on the time when resistance is developed [5]. The effectiveness of current chemotherapeutic drugs is gradually decreased by the acquired resistance after repeated treatments, while the intrinsic resistance of cancer cells causes poor drug response from the first treatment so that patients do not receive the benefit of chemotherapy [6].

New deaths worldwide from lung cancer in 2020 have been estimated to be 1.8 million or 18% of all cancer deaths, making lung cancer the most deadly cancer [7]. A major type of lung cancer is non-small cell lung cancer (NSCLC) which accounts for about 85% of all lung cancer cases [8]. More than 75% of new lung cancer cases were advanced cases at metastatic stage III or IV at the time of diagnosis [9]. Over the past decade, several new treatments for advanced NSCLC have been developed, including targeted therapy and immunotherapy [8]. However, cytotoxic chemotherapy has been the first-line standard regimen for metastatic NSCLC patients who do not have a targetable mutation [10]. Therefore, the improving efficacy of cytotoxic anticancer drugs is still essential for cancer chemotherapy.

Paclitaxel or TaxolTM is a widely used anticancer agent, either as monotherapy or in combination with other drugs, for the treatment of several cancers such as lung, breast, ovarian, prostate, liver, gastric, and bladder cancer [11,12]. Several lines of evidence show that the occurrence of paclitaxel resistance is associated with metastasis in NSCLC patients who have never received paclitaxel treatment. Intrinsic resistance to paclitaxel was observed in 76–79% of the NSCLC patients with metastatic stage III/IV cancer [13,14]. So, we previously used an in vitro non-adherent culture model to mimic floating cancer cells during metastasis and showed that floating H460 lung cancer cells exhibited paclitaxel tolerance that resulted from increased expression of the βIVa-tubulin isotype [15]. Therefore, this model may be used for studying metastasis-associated paclitaxel tolerance in anoikisresistant lung cancer cells.

Since paclitaxel-resistant cancer cells require a higher dose of the drug, it is important to explore approaches that enable the increase in intracellular drug concentrations. Paclitaxel is a drug with low solubility in water which requires assistance to allow desired therapeutic concentrations to be reached in tumors: this is a major problem in enabling paclitaxel to achieve satisfactory results [16]. With regards to this issue, a variety of nanoformulation platforms for paclitaxel and other poorly soluble bioactive compounds have been developed to improve solubility and bioavailability [17–19]. In addition, nano-sized formulations utilize the concept of enhanced permeability and retention (EPR) effect and passively extravasate through the leaky vasculature of tumor tissues [20].

Different platforms of paclitaxel nanoformulations have been investigated for NSCLC treatment such as albumin-bound nanoparticles [21], solid lipid nanoparticles [22], lipidbased nanoparticles [23], and polymeric micelle nanoparticles [24]. Poly(D,L-lactide-coglycolide) or PLGA is an FDA-approved copolymer widely used for producing polymeric nanoparticles with biodegradability and biocompatibility. Over the past two decades, PLGA nanoparticles have been used as a carrier of paclitaxel, either as a single drug or in combination with other agents, for lung cancer treatment. Fonseca et al. prepared paclitaxel-loaded PLGA nanoparticles by interfacial deposition method (presently known as a nanoprecipitation method) and showed the enhancement of paclitaxel cytotoxicity in NCI-H69 lung cancer cells, by the nanoparticles compared to free paclitaxel [25]. Recently, Jiménez-López et al. used a modified nanoprecipitation method to prepare paclitaxelloaded PLGA nanoparticles and demonstrated promising results of the nanoparticles by inhibiting the proliferation of several lung cancer cell lines with an average threefold reduction in paclitaxel IC50 values compared to free drug. Moreover, the paclitaxel nanoparticles also decreased in vitro growth of cancer stem cells and tumor spheroids, as well as resulting in the rapid accumulation of paclitaxel in various tissues including lungs of mice after intravenous administration of paclitaxel nanoparticles, compared with free paclitaxel [26].

Until now, there have been few studies evaluating paclitaxel-loaded PLGA nanoparticles in drug-resistant lung cancer cells. Yuan et al. used PLGA-Tween80 co-polymer to fabricate paclitaxel-loaded PLGA-Tween80 nanoparticles which exhibited a greater effect

than paclitaxel-loaded PLGA nanoparticles for facilitating cellular up-take in paclitaxelresistant A549 lung cancer cells. Furthermore, the IC50 value of paclitaxel-PLGA-Tween80 nanoparticles in the resistant cell line was three-fold and eight-fold lower than IC50 values of paclitaxel-PLGA nanoparticles and free drug, respectively, indicating that the presence of hydrophilic part of Tween80 on the surface of PLGA-Tween80 nanoparticles effectively improved paclitaxel delivery into the drug-resistant cells [27].

Recently, research trends sugges<sup>t</sup> that PLGA-lipid hybrid nanoparticles (PLHNPs) have a wide range of therapeutic applications. The PLHNPs consist of three parts, (i) an inner PLGA core encapsulating the hydrophobic drug, (ii) a lipid monolayer shell of lecithin coating the PLGA core, and (iii) an outer PEGylated lipid hydrophilic stealth layer of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG-COOH) interspersed throughout the lecithin monolayer, which prolongs systemic circulation of the nanoparticles by avoiding clearance by the immune system [28]. Previous studies have shown the effectiveness of PLHNP formulations for the treatment of multiple cancers [29].

In this work, we aimed to apply PLHNPs to improve the effectiveness of the therapeutic effect of paclitaxel towards anoikis-resistant lung cancer cells during metastasis. Thus, we prepared paclitaxel-PLHNPs and demonstrated their potential for overcoming metastasis-associated paclitaxel tolerance in A549 floating lung cancer cells.

## **2. Results and Discussion**

## *2.1. Preparation and Characterization of Paclitaxel-PLHNPs*

First, we investigated the effect of solvent used to dissolve the PLGA polymer for the preparation of PLHNPs. Acetonitrile and tetrahydrofuran were chosen as organic solvents in this study. Empty PLHNPs were prepared using a combination of PLGA and mixed lipid through the nanoprecipitation/self-assembly method to form nanoparticles in the nanometer-sized range with a narrow polydispersity index (PDI). As shown in Figure 1a, the particle size of nanoparticles made with PLGA/acetonitrile was smaller than those made with PLGA/tetrahydrofuran. The more water-miscible solvents tended to have greater polarity which resulted in decreasing the size of nanoparticles [30]. Use of acetonitrile as organic solvent compared with tetrahydrofuran resulted in a more highly negative charged surface (Figure 1b). Therefore, acetonitrile has been chosen as an organic solvent for the formulation of paclitaxel-PLHNPs since smaller particle sizes and higher zeta potential were obtained.

The size of formulated paclitaxel-PLHNPs was 103.0 nm with uniform particle distribution (PDI of 0.11). The paclitaxel-PLHNPs possessed a strong negatively charged surface with carboxylic acid end groups of DSPE-PEG-COOH with zeta potential −52.9 mV, indicating a stable nanoparticle dispersion (Table 1).

To determine the storage stability of nanoparticles, the prepared paclitaxel-PLHNPs were kept at 4 ◦C and room temperature. As shown in Figure 2, the higher storage temperature induced aggregation of paclitaxel-PLHNPs, causing an increase in particle size. Paclitaxel-PLHNPs were stable in suspension when stored at 4 ◦C up to 28 days, compared to storage under room temperature. This was consistent with a previous study reporting that PLGA nanospheres should be stored at 4 ◦C in order to avoid the aggregation [31].

## *2.2. Evaluation of Anoikis Resistance*

In vitro non-adherent culture using polyHEMA-coated plates has been used to obtain floating cancer cells with an anoikis-resistant property that mimics metastasizing cells in the blood and lymphatic circulatory system [15]. Morphological differences were found between A549 attached cells grown as monolayers (Figure 3a) and A549 floating cells cultivated in polyHEMA-coated plates (Figure 3b). The floating cells exhibited a round shape and formed aggregates similar to the aggregated floating lung cancer cells found in lymphatic vessels of lung cancer patients [32].

**Figure 1.** The effect of organic solvents on (**a**) size, PDI, and (**b**) zeta potential of PLGA–lipid hybrid nanoparticles (PLHNPs). Data are reported as mean ± SD from three independent experiments, \* *p* < 0.05 compared between the two organic solvents.

**Table 1.** Characteristics of paclitaxel-PLHNPs. Data are reported as mean ± SD from three independent experiments. \* *p* < 0.05 when comparing empty PLHNPs to paclitaxel-PLHNPs.


**Figure 2.** Storage stability of paclitaxel-PLHNPs. Data are reported as mean ± SD from three independent experiments, \* and \*\* *p* < 0.05 compared to Day 0.

Next, we studied the activation of caspase-3 which is a hallmark of apoptotic cell death [33]. Anoikis is defined as a type of apoptosis specifically induced by loss of cell attachment, and caspase-3 activation has been used to detect anoikis in cancer cells [32]. We, therefore, used an assay of caspase-3 to evaluate anoikis resistance in our floating cancer cells. Normal epithelial cells undergo anoikis with loss of attachment. Caspase-3 activity of HMECs cultured under non-adherent conditions was significantly increased, compared with the attached cells (Figure 4a), indicating anoikis induction in these cells. In contrast, there was no significant difference between the caspase-3 activities of A549 floating cells and A549 attached cells (Figure 4b). These results demonstrated that anoikis resistance emerged in A549 floating cells when cultured under non-adherent conditions. Taken together, the results confirmed that the A549 floating cells in our in vitro model displayed morphology and anoikis resistance property similar to that found in metastasizing lung cancer cells. Therefore, this model was further used for investigating the effect of paclitaxel-PLHNPs.

**Figure 3.** Morphology of (**a**) A549 attached cells cultured under adherent condition and (**b**) A549 floating cells cultured under non-adherent condition. Original magnification of ×200. Scale bar is 100 μm.

**Figure 4.** Caspase-3 activation in (**a**) human mammary epithelial cells/HMECs and (**b**) A549 cells after culturing under adherent condition (attached cells) and non-adherent condition (floating cells) for 24 h. Data are reported as mean ± SD from three independent experiments, \* *p* < 0.05 compared to the attached cells.

### *2.3. Comparison of the Cytotoxic Effect of Paclitaxel-PLHNPs and Free Drug in Anoikis-Resistant A549 Cells*

We applied the model of A549 floating cells to evaluate the potential of our formulated paclitaxel-PLHNPs against anoikis-resistant cancer. The A549 attached and floating cells were treated with free paclitaxel and paclitaxel-PLHNPs at concentrations equivalent to 1–1000 nM of paclitaxel for 72 h. As shown in Figure 5, both free paclitaxel and paclitaxel-PLHNPs displayed a similar pattern of dose-dependent cytotoxicity in attached and floating cells. The anoikis-resistant floating cells showed tolerance to free paclitaxel with 53.1-fold resistance, compared with the attached cells (Table 2). Treatment with paclitaxel-PLHNPs could reduce the paclitaxel tolerance of floating cells to a 10.3-fold resistance (Table 2). Our previous studies demonstrated that the acquisition of paclitaxel tolerance in floating lung cancer cells was not due to overexpression of drug transporters such as MDR1/P-gp, but was associated with upregulation of βIVa-tubulin gene, a paclitaxel-resistant β-tubulin isotype [15,34].

**Figure 5.** Relative survival rate (%) of (**a**) A549 attached cells and (**b**) A549 floating cells treated with paclitaxel and paclitaxel-PLHNPs. \* *p* < 0.05 compared to free paclitaxel.


**Table 2.** IC50 values of paclitaxel in A549 attached and A549 floating cells. \* *p* < 0.05 compared to free paclitaxel.

The remaining 10.3-fold resistance in A549 floating cells after paclitaxel-PLHNPs treatment indicated the contribution of other mechanisms to the paclitaxel tolerance of the floating cells. Several mechanisms can confer paclitaxel resistance in cancer, such as MDR1/P-gp overexpression, altered expression or mutation of β-tubulin isotypes, changes in expression of anti- or pro-apoptotic proteins, and equally important is that each of these mechanisms could separately contribute to the resistance [35]. Since paclitaxel acts by interfering with microtubule dynamics during mitosis in dividing cells, a reduction in cell growth capability may decrease the sensitivity of cells to paclitaxel cytotoxicity. Supporting evidence was reported from gene expression microarray analysis in paclitaxel-resistant MCF-7 breast cancer cells exhibiting slow growth rate, where the cell doubling time and expression of a cell cycle inhibitor gene CDKNA2/p16 were increased in the paclitaxelresistant cells [36]. Similarly, we observed that our A549 floating cells exhibited a slower growth compared to the attached cells (unpublished data). Prolonging cell division reduces the chance of paclitaxel interfering with microtubule dynamics, making the A549 floating cells have less response to paclitaxel. This resistant mechanism could not be overcome by increasing intracellular drug concentration, and the mechanism might contribute to the remaining 10.3-fold resistance in A549 floating cells after paclitaxel-PLHNPs treatment.

In A549 attached cells, paclitaxel-PLHNPs showed significantly enhanced cytotoxicity of the drug with a 71.6-fold decrease in IC50 value compared to free paclitaxel (Table 2). Interestingly, treatment with paclitaxel-PLHNPs showed a greater cytotoxic effect in A549 floating cells compared with attached cells, as demonstrated by a 370.4-fold decrease in IC50 value. These results indicated that the PLHNP platform markedly improved the effectiveness of paclitaxel in anoikis-resistant lung cancer cells. This is in line with a report by Lee et al. [37] demonstrating five-fold higher cytotoxicity of doxorubicin encapsulated in human serum albumin nanoparticles (HSA + DOX NPs), compared with free doxorubicin, in anoikis-resistant MDA-MB-231 breast cancer cells.

The effectiveness of paclitaxel-PLHNPs to overcome paclitaxel tolerance in A549 floating cells might be due to the ability of PLHNPs to deliver the hydrophobic drug into the cells, leading to increased intracellular drug concentration, as proven in our previous study for the use of PLHNPs to deliver a hydrophobic photosensitizer into multidrugresistant lung cancer cells [34].
