Phenethyl Isothiocyanate and Cisplatin Co-Encapsulated in a Liposomal Nanoparticle for Treatment of Non-Small Cell Lung Cancer

Lung cancer is the leading cause of cancer-related death in the Unites States, and approximately 85% of all lung cancers are classified as non-small cell lung cancer (NSCLC), which is extremely difficult to treat and its survival rate is low. After decades of clinical trials, the most effective treatments are still those that implement the first-generation platinum anticancer agent cisplatin (CDDP) in combination with other drugs. We previously demonstrated that the naturally-occurring compound phenethyl isothiocyanate (PEITC) can be used to sensitize NSCLC cells to CDDP. Furthermore, co-encapsulation of PEITC and CDDP in liposomes enhances their toxicity toward NSCLC cells. We here optimize liposomal-PEITC-CDDP, demonstrate the release of PEITC and CDDP from the nanoparticle, and show that liposomal-PEITC-CDDP is much more toxic toward both A549 and H596 human NSCLC cell lines than toward WI-38 and BEAS-2B human normal lung cell lines. Thus, we have prepared an efficacious therapy that has significantly higher toxicity toward cancer cell lines than normal cell lines.


Introduction
Lung cancer is the leading cause of cancer-related death in the United States [1]. Approximately 85% of all lung cancers are classified as non-small cell lung cancer (NSCLC), which is extremely difficult to treat and its survival rate is low [1]. After decades of clinical trials, the most effective treatments are still those that implement the first-generation platinum anticancer agent cisplatin (CDDP) in combination with other drugs [2]. Tubulin-binding agents, such as paclitaxel, are often used in combination with CDDP for treatment [3]. Microtubules, which are assembled from dimers of α-and β-tubulin, are critical for cell division, making tubulin proteins an important target in rapidly dividing NSCLC cells. Unfortunately, both CDDP and the agents used in combination with CDDP are toxic to the patient. Furthermore, tumors often gain resistance to CDDP. One of the major issues associated with CDDP is its nephrotoxicity, and when this is coupled with the neurotoxicity associated with the typically used tubulin binding agents, taxanes and vincas [3,4], the patient experiences significant side-effects. Thus, a treatment modality that aggressively decreases tumor volume, but has less toxicity to the patient has been sought. It has been demonstrated that the liposomal CDDP formulation LipoPlatin TM is less toxic to patients than free CDDP is, but has a similar efficacy against NSCLC [5]. In a randomized clinical trial, the effectiveness of LipoPlatin TM with the βx-tubulin binding agent paclitaxel was similar to that Particle size distributions and zeta potentials of blank and PEITC and/or CDDP loaded liposomes were characterized using a Zetasizer. As shown in Table 2, the average diameter of these liposomes ranged from 116.3 (blank liposomes) to 173.4 nm (Lipo-PEITC-CDDP). The average polydispersity indexes (PDI) of these nanoparticles indicate uniform particle size and good dispersion. The stability of the blank liposomes and drug-encapsulated liposomes was verified by measuring their zeta potentials, which ranged from −40 to −60 mV. It is shown in Figure 1A that Lipo-PEITC-CDDP has a narrow size distribution, which indicates uniformity. Field Emission Scanning Electron Microscope (FESEM) was used to characterize the morphology of Lipo-PEITC-CDDP. SEM observation confirmed that the Lipo-PEITC-CDDP exhibits spherical morphology, with an average particle size of approximately 150 nm.  Particle size distributions and zeta potentials of blank and PEITC and/or CDDP loaded liposomes were characterized using a Zetasizer. As shown in Table 2, the average diameter of these liposomes ranged from 116.3 (blank liposomes) to 173.4 nm (Lipo-PEITC-CDDP). The average polydispersity indexes (PDI) of these nanoparticles indicate uniform particle size and good dispersion. The stability of the blank liposomes and drug-encapsulated liposomes was verified by measuring their zeta potentials, which ranged from −40 to −60 mV. It is shown in Figure 1(A) that Lipo-PEITC-CDDP has a narrow size distribution, which indicates uniformity. Field Emission Scanning Electron Microscope (FESEM) was used to characterize the morphology of Lipo-PEITC-CDDP. SEM observation confirmed that the Lipo-PEITC-CDDP exhibits spherical morphology, with an average particle size of approximately 150 nm.    Figure 2. The amount of CDDP released from Lipo-CDDP and Lipo-PEITC-CDDP over 24 h was measured using inductively-coupled plasma-mass spectrometry (ICP-MS). As is shown in Figure 2A and 2C, Lipo-CDDP and Lipo-PEITC-CDDP release 76.5 ± 3.9% and 75.2 ± 3.3% of their CDDP, respectively, in 2 h. Over 24 h, the amounts of CDDP released reached 86.1 ± 4.3% and 91.1 ± 2.9% for Lipo-CDDP and Lipo-PEITC-CDDP, respectively. A 1,2-benzenedithiol (BDT) assay was used to obtain the PEITC release profile of Lipo-PEITC and Lipo-PEITC-CDDP [19]. As is shown in Figure 2B, Lipo-PEITC showed a relatively sustained PEITC release, with 40.2 ± 2.5% PEITC released gradually from Lipo-PEITC in the first 8 h. The percent release remained almost the same up to 24 h. A similar PEITC release profile was observed in Lipo-PEITC-CDDP ( Figure 2C) with 44.8 ± 2.6% of the PEITC released gradually within 8 h. The release rate of CDDP in Lipo-PEITC-CDDP was higher than that of PEITC.
The benzenedithiol (BDT) assay was used to obtain the PEITC release profile of Lipo-PEITC and Lipo-PEITC-CDDP [19]. As is shown in Figure 2B, Lipo-PEITC showed a relatively sustained PEITC release, with 40.2 ± 2.5% PEITC released gradually from Lipo-PEITC in the first 8 h. The percent release remained almost the same up to 24 h. A similar PEITC release profile was observed in Lipo-PEITC-CDDP ( Figure 2C) with 44.8 ± 2.6% of the PEITC released gradually within 8 h. The release rate of CDDP in Lipo-PEITC-CDDP was higher than that of PEITC.

Effect of CDDP and PEITC Combination Therapy
In the cytotoxicity study using A549 cells ( Figure 3A), the percent cell survival when treated with CDDP (5 µM) alone was 55.9 ± 3.4%, while treatment with PEITC (15 µM) alone resulted in a percent cell survival of 79.2 ± 3.8%. When treated with a combination of 5 µM CDDP and 15 µM PEITC (CDDP + PEITC), A549 cells had a percent survival of 46.2 ± 2.7%. The cytotoxicity associated with the combination of CDDP and PEITC was greater than those associated with CDDP or PEITC alone. As is shown in Figure 3B, the viability of H596 cells after treatment was similar to that of A549 cells. The percent survival after treatment with CDDP (5 µM) alone was 74.6 ± 9.2%, and the percent survival after treatment with PEITC (15 µM) alone was 84.9 ± 8.9%. Cells treated with a combination of 5 µM CDDP and 15 µM PEITC had a percent survival of 55.0 ± 9.5%. There are significant differences among groups CDDP + PEITC, CDDP and PEITC, as determined using a one-way ANOVA in R, for both A549 (p = 1.1 × 10 −14 ) and H596 cell lines (p = 1.55 × 10 −7 ) [20]. A Dunnett's post hoc test accounting for multiple comparisons shows that for both cancer cell lines, the percent survival for the cells treated with a combination of CDDP + PEITC is significantly lower than free drug CDDP and PEITC, respectively ( Table 3). The significant difference between the percent survival of CDDP + PEITC treatment groups and free CDDP treatment groups in both A549 and H596 cells confirmed the reported sensitization role of PEITC [8].

Effect of Lipo-PEITC-CDDP on Cancer Cell Lines
When treated with liposomes containing 5 µM of CDDP and 15 µM of PEITC (Lipo-PEITC-CDDP), the A549 cells had a percent survival of 33.3 ± 2.9%, which was greater than that of CDDP + PEITC, Lipo-CDDP and Lipo-PEITC. Like A549 cells, the lowest percent survival of H596 cells (28.6 ± 6.3%) Molecules 2019, 24, 801 7 of 13 occurred when treated by 5 µM of CDDP and 15 µM of PEITC loaded in liposomes (Lipo-PEITC-CDDP), which was lower than that of CDDP + PEITC, Lipo-CDDP and Lipo-PEITC. There is a significant difference among groups Lipo-PEITC-CDDP, CDDP + PEITC, Lipo-CDDP, and Lipo-PEITC as determined using a one-way ANOVA for both A549 (p = 1.26 × 10 −14 ) and H596 cell lines (p = 5.58 × 10 −10 ). A Dunnett's post hoc test accounting for multiple comparisons shows that for both cancer cell lines, the percent survival of the cells treated with Lipo-PEITC-CDDP is significantly lower than the free drug combination (CDDP + PEITC), as well as liposomal formulations of either single drug (Lipo-CDDP and Lipo-PEITC) ( Table 5). Loaded with CDDP and PEITC, Lipo-PEITC-CDDP has the advantages of enhanced CDDP efficacy and effective liposomal drug delivery and showed the highest cytotoxicity against the two NSCLC cell lines. A Welch two-sample t-test found that there is a significant difference (p = 1.16 × 10 −14 ) between no treatment group and Lipo-PEITC-CDDP group for the A549 cell line. The t-value capturing the effect size of the difference is 68.0. Similarly, for H596, a difference between the two groups was found with a t-value of 23.9 and a p-value of 4.22 × 10 −8 . On the other hand, the effect sizes of the difference on normal cell lines were much lower (WI-38: t = 4.23, p = 5.10 × 10 −3 ; BEAS-2B: t = 4.72, p = 4.07 × 10 −3 ) ( Table 6). This result shows that the Lipo-PEITC-CDDP drug therapy leads to a more significant cytotoxicity toward cancer cell lines as compared with normal cell lines.   Because of our 3:1 PEITC:CDDP ratio, we were able to use less cisplatin in the Lipo-PEITC-CDDP against NSCLC cells than in our last formulation, which was 2:1 PEITC: CDDP [16]. We were able to achieve similar toxicity profiles using less CDDP and a higher concentration of PEITC. Cisplatin is a  Because of our 3:1 PEITC:CDDP ratio, we were able to use less cisplatin in the Lipo-PEITC-CDDP against NSCLC cells than in our last formulation, which was 2:1 PEITC: CDDP [16]. We were able to achieve similar toxicity profiles using less CDDP and a higher concentration of PEITC. Cisplatin is a known nephrotoxic agent, so reducing its concentration to get a similar toxicity profile in NSCLC cells is advantageous. For the two normal lung cell lines ( Figure 4A,B), while the Lipo-PEITC-CDDP caused the lowest survival percentages, 59.0 ± 15.7% and 71.5 ± 11.0% for WI-38 and BEAS-2B, respectively, the cytotoxic effect of Lipo-PEITC-CDDP was much greater in NSCLC cells. Therefore, the PEITC and CDDP combined liposomal therapy has a high therapeutic index.

Determination of PEITC Loading in Liposomal-PEITC and Liposomal-PEITC-CDDP
The amount of PEITC loaded in the liposomes was determined using a 1,2-benzenedithiol (BDT) assay reported by Zhang with minor modifications [19]. To 1 mL of liposomal-PEITC suspended in potassium phosphate solution (100 mM, pH 8.5) or 1 mL of liposomal-PEITC-CDDP suspended in the same, was added 1 mL of 4 mM BDT solution in methanol. Then the 2 mL reaction solution was heated in a closed glass vial at 65 • C for 2 h, and its absorbance at 365 nm was measured using a UV-Vis spectrophotometer SpectraMax (Molecular Devices, Sunnyvale, CA, USA). A standard curve was established using PEITC (R 2 = 0.98) in the same manner, and concentrations of PEITC in samples were determined based on the standard curve.
The PEITC drug loading was calculated using the equation: PEITC loading(%) = Weight of PEITC in liposomes Total weight of lipids and PEITC × 100% (1)

Determination of CDDP Loading in Liposomal-CDDP and Liposomal-PEITC-CDDP
Cisplatin loading was determined using inductively-coupled plasma-mass spectrometry (ICP-MS, PerkinElmer 350D, Waltham, MA, USA). Liposomal-CDDP and liposomal-PEITC-CDDP were digested with 70% nitric acid and heated at 70 • C overnight to evaporate. Then, the sample was re-dissolved in 70% nitric acid and diluted using deionized water, and the final samples for the ICP-MS test contained 2% nitric acid. Lutetium (20 ppb) was used as an internal standard.
The CDDP drug loading was calculated using the equation: CDDP loading(%) = Weight of CDDP in liposomes Total weight of lipids and CDDP × 100%. (2)

In Vitro Drug Release Studies
Different amounts of PEITC or CDDP were used to prepare liposomal-PEITC or liposomal-CDDP. The formulation that achieved the highest loading (%) of PEITC is referred to as "Lipo-PEITC", the highest loading (%) of liposomal-CDDP is referred to as "Lipo-CDDP" and the liposomal formulation which combined the highest loading (%) of both PEITC and CDDP is referred to as "Lipo-PEITC-CDDP". These three liposomal formulations were tested for their in vitro drug release and cytotoxicity. Drug release was studied using a dialysis method. Dialysis sacks were rinsed thoroughly using deionized water for 30 min, then 1 mL of Lipo-PEITC, Lipo-CDDP or Lipo-PEITC-CDDP was placed in the dialysis sack. The sack was fully immersed in a capped glass vial containing 20 mL of 1× PBS with 25% v/v methanol. A spin bar was added in the vial to achieve 100 rpm rotation speed. The temperature was set at 37 • C, and aliquots (100 µL) of the release medium were withdrawn for analysis at different time points (up to 24 h) and replaced with fresh medium. The absorbance of PEITC released from Lipo-PEITC and Lipo-PEITC-CDDP was measured using the same BDT assay as described above. The amount of CDDP released from Lipo-CDDP and Lipo-PEITC-CDDP was determined using ICP-MS.
The percentage of release of CDDP or PEITC at different time points was calculated using the equation: Cumulative amount released Total amount in liposomes × 100% (3)

Cytotoxicity Studies
All cell studies were carried out in a humidified 37 • C, 5% CO 2 (standard conditions) atmosphere incubator. For the two normal cell lines, the culture medium used for the WI-38 cells was the minimum essential medium (MEM) containing 100 µg/mL streptomycin, 10% fetal bovine serum (FBS), 2.0 mM L-glutamine and 100 IU/mL penicillin; the culture medium used for the BEAS-2B cells was bronchial epithelial basal medium (BEBM) with 10% FBS and supplements. The culture medium used for the two cancer cell lines, H596 and A549 cells, was Roswell Park Memorial Institute (RPMI) medium containing 100 µg/mL streptomycin, 100 IU/mL penicillin, 10% FBS, and 2.0 mM L-glutamine.
Nine groups (n = 6) were included to test the cytotoxicity toward each cell line. Two control groups were included: group 1, control group with only medium; group 2, control group with non-treated cells. Group 3-9 were cells with treatment: group 3, free CDDP; group 4, free PEITC; group 5, CDDP and PEITC (CDDP + PEITC); group 6, blank liposomes; group 7, Lipo-CDDP; group 8, Lipo-PEITC; group 9, Lipo-PEITC-CDDP. The cells were seeded at 5 × 10 3 cells/mL (100 µL/well) in 96-well plates and allowed to grow for 24 h; after that, the medium was removed and replaced with 100 µL of medium containing the treatment suspensions, which was removed after an exposure time of 24 h and replaced with 100 µL of fresh medium. To each well, 20 µL of MTS solution was added and incubated for 2 h, and the UV-Vis absorbance was read at 490 nm. The percent survival of cells treated was calculated using the following equation: where A t is the absorbance of cells in treatment groups, A m is the absorbance of medium alone and A c is the absorbance of cells without treatment. All p values were calculated using Microsoft Excel t-test function (Redmond, WA, USA).

Statistical Analyses
All statistical analyses were conducted using R [20]. For the comparison of two group means, two-sample Welch's t-tests were conducted using the t.test() function. The Levene test was carried out to test for homogeneity of variances using leveneTest() from the car package [21]. For comparison of more than two group means, one-way ANOVAs were conducted using the aov() function with a post-hoc Dunnett test carried out using the glht() function from the multcomp package [22].

Conclusions
Cisplatin is commonly used against NSCLC in the clinic; however, the negative side-effects associated with this metal-based drug urgently need to be addressed. We encapsulated CDDP and PEITC in liposomes, as this strategy has been shown to increase the circulation time of encapsulated CDDP and reduce its side-effects [17]. Furthermore, co-encapsulation of PEITC and CDDP in liposomes enhances their cytotoxicity toward NSCLC cells [16]. Release of PEITC and CDDP was studied and the cytotoxicity of this formulation against human NSCLC and normal lung cells determined. Lipo-PEITC-CDDP was significantly more toxic toward both NSCLC cell lines than toward the normal lung cell lines tested. Thus, we have prepared an efficacious therapy that is more toxic toward cancer cells than toward normal cells.