Synthesis and Evaluation of a Paclitaxel-Binding Tripeptide Micelle for Lung Cancer Therapy

Abstract

A C₁₀CO-NalLeuVal (C10NLV) tripeptide was synthesized and explored as a carrier for paclitaxel (TAX) delivery. Five types of TAX-loaded micelles were produced by loading TAX with different doses of C10NLV. 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay showed that TAX-loaded micelles dramatically reduced TAX IC₅₀ values of TAX-resistant A549 (A549/TAX) and Lewis lung carcinoma (LLC) cells in a C10NLV-dose-dependent manner, with micelles 4 and 5 exhibited comparable inhibitory effects on A549/TAX proliferation. Flow cytometry analysis showed that TAX-loaded micelles 4 promoted lung cancer cell apoptosis in a TAX-dose-dependent manner. Immunofluorescent staining and Western blotting revealed that TAX-loaded micelles 4 dramatically reduced the protein levels of F-actin, p53, Bcl-2, and LC3A/B in A549/TAX cells. Wound healing, cell adhesion, migration, and invasion assays demonstrated that TAX-loaded micelles 4 suppressed the metastatic abilities of lung cancer cells. Furthermore, compared with the same dose of free TAX, TAX-loaded micelles 4 significantly reduced the volumes and weights of A549/TAX-generated tumors as well as the numbers of LLC-generated pulmonary metastatic foci in mice, without affecting the organ/body weight ratios, body weights, and blood cell counts. Histological analysis demonstrated that TAX-loaded micelles 4 administration resulted in tubulin and CD206 downregulation as well as cytoplasm disappearance and nuclear shrinkage in xenograft tumors. These data suggest that TAX-loaded micelles 4 inhibits the proliferative and metastatic capacity of lung cancer cells, despite TAX resistance. TAX-loaded micelles 4 suppresses lung tumor growth and metastasis in vivo without inducing systemic toxicity. Thus, the C10NLV-based TAX delivery is effective and safe to combat TAX resistance and metastasis in lung cancer.

Introduction

Despite the declining incidence and mortality in recent years, lung cancer, comprising 85% non-small cell lung cancer (NSCLC) and 15% small cell lung cancer (SCLC), remains the second most commonly diagnosed cancer and the leading cause of cancer-related deaths worldwide.¹⁾ The dismal prognosis of lung cancer is primarily because more than 50% of patients present with locally advanced disease or metastasis (stages III and IV) at diagnosis and are ineligible for curative surgery.²⁾ Thus, prevention and inhibition of metastasis may help improve the survival statistic of lung cancer.

Paclitaxel (TAX) is one of the most effective anticancer drugs for the treatment of various solid tumors, including lung cancer.³⁾ By promoting the assembly of tubulin into microtubules and stabilizing microtubules, TAX blocks cell cycle progression, mitosis, and proliferation of cancer cells.⁴⁾ TAX in combination with carboplatin or cisplatin is the first-line palliative treatment of locally advanced or metastatic NSCLC, aiming at improving survival and alleviating symptoms.⁵⁾ However, only 10–30% of patients respond to this treatment, and many patients acquire resistance after the initial response.^6,7) The clinical use of TAX is limited by poor aqueous solubility, low bioavailability, severe systemic toxicity, and drug resistance.⁸⁾ To address these concerns, the encapsulation of anticancer drugs in nanoparticles, such as micelles, liposomes, nanocapsules, and nanospheres, has been developed.⁹⁾ Polymeric micelle is one of the most investigated drug delivery systems for TAX, represented by polyethylene glycol (PEG)–polylactide micelle that has been approved in Korea for the treatment of NSCLC and other cancers.^10–12)

Recently, peptide-based micellar carriers have shown promising results in increasing solubility, accelerating drug release, reducing the side effects, and enhancing the therapeutic efficacy of chemotherapeutic drugs. For example, the stearyl-peptide-based micelle has been developed for the codelivery of hydrophobic doxorubicin and negatively charged microRNA-34a for the treatment of androgen-independent prostate cancer, achieving a fast drug delivery into the nucleus and a synergistic antitumor effect in vivo.¹³⁾ The PEG-b-PLeu-based micelles encapsulate hydrophobic 17-allylaminodemethoxygeldanamycin and TAX, exhibiting a strong synergistic antitumor efficacy in ErbB2⁺ orthotopic breast cancer xenografts without inducing acute toxicity.¹⁴⁾ These findings prompted us to investigate whether TAX encapsulated in a peptide-based micelle carrier could overcome drug resistance and prevent metastasis in lung cancer therapy.

In our previous experimental studies, we identified a series of compounds that reversed resistance, with a 4-fold increase in reversal of resistance for compound 4 and a 6-fold increase in reversal of resistance for compound 6e in MES-SA/Dx5 cells, structures of compounds 4 and 6e as shown in Fig. 1.

Fig. 1. Structures of Compounds 4 and 6e with Drug Resistance Reversal Activity

On this basis, we designed the NalLeuVal tripeptide. In this study, we prepared (S)-2-decanoylamino-3-(1-naphthyl)propionyl-leucyl-valine(C10-CO-NalLeuVal; C10NLV) tripeptide as the micelle carrier for TAX delivery. We evaluated the antitumor effects of TAX-loaded micelles in TAX-resistant A549 cells (A549/TAX) and Lewis lung carcinoma (LLC) cells as well as in xenograft mouse models. Our novel micellar system showed promising therapeutic activity against drug resistance and tumor metastasis in lung cancer.

Experimental

Synthesis of C10NLV and TAX-Loading Micelles

The animal study was approved by the Ethics Committee of Capital Medical University (the Ethics Number AEEI-2018-174). All procedures were conducted following the guidelines of laboratory animal welfare (P. R. China National Standard 1 GB/T 35892-2018).

L-Val-OBzl and tert-butoxycarbonyl (Boc)-Leu underwent a condensation reaction to produce L-Boc-Leu-Val-OBzl, followed by a reaction with 4 N hydrogen chloride–ethyl acetate solution in an ice bath to generate HCl·Leu-Val-OBzl. HCl·Leu-Val-OBzl and Boc-3-(1-naphthyl)-L-alanine underwent a condensation reaction to produce Boc-3-(1-naphthyl)-L-propionyl-leucyl-valine benzyl ester, followed by a reaction with 4 N hydrogen chloride–ethyl acetate solution in an ice bath to generate HCl·NH2-3-(1-naphthyl)-L-propionyl–leucyl–valine benzyl ester. The latter was condensed with capric acid to produce (S)-2-decylamide amino-3-(1-naphthyl)propanediol–leucyl–valine benzyl ester, followed by Pd/C-catalyzed hydrogenation to synthesize C10NLV.

To synthesize C10NLV-TAX micelles (1–5), 0.85 mg paclitaxel was mixed with 0.58, 2.9, 5.8, 11.6, and 17.4 mg C10NLV, respectively, and dissolved in 10 mL dichloromethane. The solution was transformed into a thin film using a rotary evaporator (Heidolph Laborota 4001). After adding 1 mL of 75% ethanol, the film was ultrasonically dispersed, dissolved in 9 mL of distilled water, and then lyophilized to obtain TAX-loaded micelles 1–5 in white powder.

Cell Lines and Cell Culture

Human alveolar basal adenocarcinoma A549/TAX cell line was purchased from keyGEN BioTECH (Nanjing China). LLC cell line were obtained from American Type Culture Collection (ATCC). LLC cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (keyGEN BioTECH) supplemented with 10% fetal bovine serum (FBS; Gibco by Life Technologies, U.S.A.) in a humidified atmosphere of 5% CO₂ at 37 °C. A549/TAX cells were cultured in RPMI 1640 Medium (keyGEN BioTECH) with 10% fetal bovine serum (FBS), containing 200 ng/mL paclitaxel (J&K Scientific Ltd., China). The surviving cells were cultured by drug-free culture in RPMI 1640 Medium with 10% FBS.

Animals and Xenograft Tumor Models

The animal study was approved by the Ethics Committee of Capital Medical University (the Ethics Number AEEI-2018-174). All procedures were conducted following the guidelines of laboratory animal welfare (P. R. China National Standard 1 GB/T 35892-2018). Male C57 mice aged 5-week-old (20 ± 2 g) and male Balb/c nude mice aged 5-week-old (15–17 g) were purchased from Charles River Laboratories (Beijing, China) and maintained under pathogen-free conditions at laboratory animal department of Capital Medical University. To investigate the anti-tumor effect of TAX-loaded micelles in vivo, we established xenograft tumor models using A549/TAX and LLC cells, respectively.

For LLC mouse model, the C57 mice were subcutaneously inoculated with 200 µL phosphate-buffered saline (PBS) containing 1 × 10⁷ LLC cells at the right armpit. The length and width of the tumors were monitored daily. The tumor volume was calculated as (length × width²)/2. Mice were euthanized at 2 weeks after inoculation when the tumor volumes reached 2000 mm³. The tumors were immediately collected and cut into 1 mm³ small pieces. After filtration, tumor cells were resuspended in PBS. Mice were inoculated with 200 µL PBS containing 1 × 10⁷ LLC cells at the right armpit. When the average volume of tumors reached 100 mm³ at 6 d after inoculation, mice were randomly divided into 5 groups (n = 10) and administered 200 µL PBS, micelle 4 (27 mg/kg), TAX (5 mg/kg), 2 mg/kg TAX-loaded micelle 4, or 0.5 mg/kg TAX-loaded micelle 4 via intraperitoneal injection once daily for 9 d. The length and width of the tumors were measured daily using a vernier caliper. Mice were euthanized at 10 d after treatment. The heart, liver, spleen, kidneys, brain, and tumor from each mouse were immediately collected and weighed. The number of metastatic foci on the lung surface was counted.

For A549/TAX cell xenograft tumor model, the Balb/c nude mice were subcutaneously inoculated with 100 µL PBS containing 0.5 × 10⁷ A549/TAX cells at the right armpit. The length and width of the tumors were measured twice a week using a vernier caliper. When the volumes of tumors reached 100–200 mm³ at 12 d after inoculation, mice were randomly divided into 5 groups (n = 10) and administered 200 µL PBS, micelle 4 (55 mg/kg), TAX (4 mg/kg), 4 mg/kg TAX-loaded micelle 4, or 1 mg/kg TAX-loaded micelle 4 via intraperitoneal injection every other day for 18 d. On day 31 after inoculation, mice were anesthetized with ether. The orbital sinus blood sample, the organs (heart, liver, spleen, kidneys, and brain), and the tumor from each mouse were immediately collected. The tumor inhibition rate (%) was calculated as

  

Cell Proliferation Assay

A549, A549/TAX, or LLC cells were seeded in a 96-well plate at a density of 5000 cells/well and incubated at 37 °C for 6 h. Cells were treated with 5‰ dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louise, MO, U.S.A.), TAX, or TAX-loaded micelles for 48 h. Twenty-five µL of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (5 mg/L; Beyotime, Shanghai, China) was added to each well. After 4 h of incubation at 37 °C, the MTT-containing medium was removed, and 150 µL of DMSO was added to each well. The optical density was measured using a Multiskan plate reader (Thermo Fisher Scientific, Waltham, MA, U.S.A.) at a wavelength of 570 nm. The IC₅₀ was calculated using GraphPad Prism5.

Cell Apoptosis Analysis

A549/TAX cells were seeded in a 6-well plate at a density of 1 × 10⁶ cells/well and incubated at 37 °C for 6 h. Cells were treated with 5‰ DMSO or micelle 4 loaded with different concentrations of TAX (2, 5, or 10 µM) for 24 h. Cells were collected and resuspended in 1 mL binding buffer (Beijing Solarbio Science & Technology Co., Ltd., China). Cells were then stained with fluorescein isothiocyanate (FITC)-annexin V antibody and propidium iodide (Beijing Solarbio Science & Technology Co., Ltd.), then the fluorescence intensity was measured on a flow cytometer (Becton Dickinson FACS Calibur, U.S.A.).

Wound Healing Assay

A549/TAX cells were seeded in a 12-well plate at a density of 5 × 10⁵ cells/well and incubated at 37 °C for 6 h. A 200 µL micropipette tip was used to generate a 2 mm-wide scratch line in the cell monolayer. Then, cells were treated with 5‰ DMSO, 20 µM Arg-Gly-Asp-Ser (RGDS), 20 µM micelle 4, 1 µM TAX, or 1 µM TAX-loaded micelle 4. The widths of the scratches were measured at 0, 12, 24, 36, and 48 h after treatment, respectively, using Image J software (NIH, Bethesda, MD, U.S.A.). Images were captured under an inverted light microscope (Leica, Germany). The percentage of wound closure was calculated as

  

Cell Migration and Invasion Assays

For cell migration assay, 5 × 10⁴ A549, A549/TAX, or LLC cells in 100 µL serum-free medium were added in the upper Transwell chamber (8-µm pore size; Corning, Corning, NY, U.S.A.). The upper chamber was coated with Matrigel for the invasion assay. A total of 600 µL medium containing 10% FBS was added to the bottom chambers. A549 cells were treated with PBS, 0.5 µM TAX, 1 µM TAX, 20 µM RGDS, 0.5 µM TAX-loaded micelle 4, or 1 µM TAX-loaded micelle 4. A549/TAX cells were treated with PBS, 20 µM RGDS, 20 µM micelle 4, 1 µM TAX, or 1 µM TAX-loaded micelle 4. LLC cells were treated with PBS, 1 µM TAX, or 1 µM TAX-loaded micelle 4, 20 µM RGDS, or 20 µM micelle 4. Cells were allowed to migrate for 8 h or invade for 12 h. The migrating or invading cells at the lower surface were fixed with 4% formaldehyde for 30 min and stained with 0.1% crystal violet for 15 min. Cells were counted in 9 randomly selected fields, and images were acquired using a Leica microscope at magnification × 10.

Cell Adhesion Assay

Cell adhesion assay was performed using an extracellular matrix (ECM) adhesion array kit (Solarbio) according to the manufacturer’s protocol. Each well of a 96-well plate was pre-coated with Fibronectin. A total of 5 × 10⁴ A549/TAX or LLC cells were seeded each well and treated with PBS, 20 µM RGDS, 1 µM TAX, or 20 µM micelle 4, or 1 µM TAX-loaded micelle 4. Cells were incubated at 37 °C for 2 h. The optical density was measured at 490 nm using a microplate reader. Images were acquired using a Leica microscope at magnification × 10.

Western Blot Analysis

A total of 2 × 10⁶ A549 or A549/TAX cells were seeded in a 10-cm dish and incubated at 37 °C for 6 h. A549/TAX cells were treated with PBS, 100 µM TAX, 10 µM TAX, or micelle 4 loaded with different concentrations of TAX (10, 5, or 2 µM) for 48 h. Cells were collected and lysed using radio immunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology). After centrifugation at 12000 rpm for 15 min at 4 °C, the total proteins were obtained, and the concentration was determined using a bicinchoninic acid (BCA) kit (keyGEN BioTECH). The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, U.S.A.). After blocking with 5% non-fat dry milk, the membrane was incubated overnight at 4 °C with primary antibody against P53 (Beyotime Biotechnology), Bax (Cell Signaling Technology), Bcl-2 (Cell Signaling Technology), LC3A/B (Cell Signaling Technology), or actin (Cell Signaling Technology), followed by incubation with the secondary antibody (Beyotime Biotechnology) for 1 h at room temperature. The blots were developed using ECL reagent (Applygen) and visualize using the Amersham Imager 680 system (GE).

Immunofluorescent Staining

A total of 1 × 10⁶ A549/TAX cells were grown on a sterile coverslip and incubated at 37 °C for 6 h. Cells were treated with 5‰ DMSO, 5 µM TAX, or 5 µM TAX-loaded micelle 4 for 24 h. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature. For filamentous actin (F-actin) detection, the non-specific background was blocked with 1% bovine serum albumin, and cells were incubated with phalloidin-FITC working solution (ab235137, Abcam, U.K.) for 30 min at room temperature. The fluorescence was visualized using a laser scanning confocal microscope (Leica, TCS SP8, Germany).

Histological Examination

The tumor tissue samples were fixed with 4% paraformaldehyde for 48 h. The paraffin-embedded sections (5-µm thick) were prepared and dehydrated in a graded series of alcohol. The sections were stained with hematoxylin-eosin (H&E) and mounted with neutral gum. To detect tubulin and CD206 expression in tumor tissue samples, the paraffin-embedded sections were dewaxed in xylene and dehydrated in ethanol, followed by antigen retrieval with ethylenediaminetetraacetic acid (EDTA). The sections were blocked with 5% bovine serum albumin. The protein expression of tubulin and CD206 were detected using anti-tubulin (Servicebio) and anti-CD206 (Abcam), respectively. The results were examined under Pannoramic scan at a magnification of 20×. Images were acquired using a CaseViewer system.

Statistical Analysis

Data were presented as the means ± standard deviation. Statistical analysis was performed using the Prism 5 software (GraphPad, San Diego, CA, U.S.A.). The two groups were compared using the two-tailed unpaired Student t-test. A p value <0.05 was considered statistically significant.

Results

TAX-Loaded Micelles Inhibit Cell Proliferation While Promoting Cell Apoptosis of Lung Cancer Cells

To develop a novel paclitaxel delivery system, we synthesized a C10NLV tripeptide (Fig. 2A) following the schematic diagram in Fig. 2B. Five types of TAX-loaded micelles (1–5) were generated by loading different doses of C10NLV with TAX. As shown in Figs. 3A–C, micellization with C10NLV dramatically reduced TAX IC₅₀ values of A549/TAX and LLC cells but not A549 cells in a C10NLV-dose-dependent manner compared with free TAX. No significant difference was observed between micelle 4 and micelle 5 in terms of the reduction in IC₅₀ in A549/TAX cells. Thus, we selected micelle 4 for the following investigation. The physicochemical properties of C10NLV and TAX-loaded micelle 4 were shown in Table 1. Flow cytometry analysis showed that compared with vehicle treatment, TAX-loaded micelle 4 remarkably elevated the percentages of apoptotic A549/TAX cells in a TAX-dose-dependent manner (Figs. 3D, E). These results suggest that TAX-loaded micelle is a promising therapeutic agent for the treatment of TAX-resistant lung cancer.

Fig. 2. Synthesis of (S)-2-Decylamide Amino-3-(1-naphthyl)propanediol–Leucyl–Valine (C10-COOHNalLeuVal)

(A) C10-COOHNalLeuVal. (B) Synthesis of C10-COOHNalLeuVal.

Fig. 3. Paclitaxel (TAX)-Loaded Micelles Inhibited Cell Proliferation While Promoting Cell Apoptosis of Lung Cancer Cells

(A–C) A549, A549/TAX, and LLC cells were treated with TAX (100, 50, 25, 12.5, 6.25, 3.125, 1.56 µM) or TAX-loaded micelles 1–5 (40, 20, 10, 5, 2.5, 1.25, 0.625 µM) for 48 h. MTT assay was performed to measure cell viability. The IC₅₀ was calculated. (D, E) A549/TAX cells were treated with 5‰ DMSO(a), micelle 4 (TAX 2 µM)(b), micelle 4 (TAX 5 µM)(c), micelle 4 (TAX 10 µM)(d) for 24 h. Flow cytometry analysis was conducted to examine cell apoptosis. Data are expressed as the mean ± standard deviation (S.D.). * p < 0.05, ** p < 0.01, n = 9.

Table 1. Particle Size and Zeta Values of C10NLV and TAX-Loaded Micelle 4

Group	PDI	Particle size (nm)	Zeta (mV)
C10NLV	0.238	255.0 ± 45.87	−22.0 ± 4.29
TAX-loaded micelle 4	0.096	151.0 ± 46.82	−31.3 ± 6.51

TAX-Loaded Micelle 4 Modulates the Expression of F-actin, P53, and Cell Apoptosis- and Autophagy-Related Proteins

Considering the importance of actin filaments in the cytoskeleton and cell mobility,¹⁵⁾ we assessed the effect of TAX-loaded micelle 4 treatment on the organization and distribution of F-actin in A549/TAX cells. Immunofluorescent staining showed that compared with vehicle and TAX treatment, TAX-loaded micelle 4 treatment resulted in a substantial loss of F-actin (Figs. 4A, B). In addition, Western blot analysis revealed that A549/TAX cells had remarkably elevated protein levels of P53, Bcl-2, and LC3A/B (Autophagy microtubule-associated protein light chain 3A/3B) compared with parental A549 cells. Compared with TAX alone, 10 µM TAX-loaded micelle 4 completely reversed the upregulation of these proteins (Figs. 4C, D). No significant difference was observed in the protein levels of BAX among different groups. Taken together, these results suggest that the antitumor role of TAX-loaded micelle 4 in lung cancer is associated with cytoskeleton disruption, P53 downregulation, cell apoptosis enhancement, and cell autophagy inhibition.

Fig. 4. TAX-Loaded Micelle 4 Modulated the Expression of Filamentous Actin (F-Actin), p53, and Cell Apoptosis- and Autophagy-Related Proteins

(A) A549/TAX cells were treated with 5‰ DMSO (a), 5 µM TAX (b), or 5 µM TAX-loaded micelle 4 (c) containing for 24 h. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Immunofluorescent staining was performed to detect F-action expression. (B) Quantification of (A). (C) A549/TAX cells were treated with PBS, 100 µM TAX, 10 µM TAX, or micelle 4 loaded with different concentrations of TAX (10, 5, or 2 µM) for 48 h. Western blot analysis was performed to determine protein expression of P53, Bax, Bcl-2, and LC3A/B. Actin was used as an internal reference. A549 cells were used as a negative control. (D) Quantification of (C). Data are expressed as the mean ± S.D. * p < 0.05, ** p < 0.01; n = 9.

TAX-Loaded Micelle 4 Inhibits the Metastatic Ability of Lung Cancer Cells

Next, we evaluated the effect of TAX-loaded micelle 4 treatment on the metastatic ability of lung cancer cells. RGDS has been used as a positive control in many studies to assess the ability to inhibit tumor metastasis.¹⁶⁾ Wound healing assay showed that micelle 4 and TAX-loaded micelle 4 treatment significantly reduced the percentages of wound closure in A549/TAX cells, compared with free TAX (Figs. 5A, B). Micelle 4 and TAX-loaded micelle 4 treatment also remarkably inhibited cell adhesion of A549/TAX and LLC cells to ECM (Figs. 6A, B). Furthermore, micelle 4 and TAX-loaded micelle 4 treatment markedly inhibited cell migration and invasion of A549, A549/TAX, and LLC cells, compared with free TAX (Figs. 7, 8 and Supplementary Tables 1–6). Taken together, these data suggest that TAX-loaded micelle 4 suppresses the metastatic ability of lung cancer cells and that this ability was conferred by C10NLV.

Fig. 5. Wound Healing Assay

A549/TAX cells were treated with 5‰ DMSO, 20 µM RGDS, 20 µM micelle 4, 1 µM TAX, or 1 µM TAX-loaded micelle 4. The widths of the scratches were measured at 0, 12, 24, 36, and 48 h after treatment, respectively, using Image J software. (A) Representative images are shown. (B) The percentage of wound closure was calculated as . Data are expressed as the mean ± S.D. * p < 0.05, ** p < 0.01; n = 9.

Fig. 6. Cell Adhesion Assay

A549/TAX (A) or LLC (B) cells were treated with PBS, 20 µM RGDS, 1 µM TAX, or 20 µM micelle 4, or 1 µM TAX-loaded micelle 4 for 4 h. Cell adhesion assay was performed. The optical density was measured at 450 nm using a microplate reader. Representative images are shown. Magnification 10×.

Fig. 7. Cell Migration Assay

A549 (A), A549/TAX (B), or LLC (C) cells were treated as indicated for 8 h. Cell migration assay was performed. Representative images are shown. Magnification 10×.

Fig. 8. Cell Invasion Assay

A549 (A), A549/TAX (B), or LLC (C) cells were treated as indicated for 24 h. Cell invasion assay was performed. Representative images are shown. Magnification 10×.

TAX-Loaded Micelle 4 Suppresses A549/TAX Tumor Growth and LLC Metastasis in Vivo

To evaluate the antitumor effect of TAX-loaded micelle 4 in vivo, we established two xenograft tumor models using LCC and A549/TAX cells, respectively. As shown in Fig. 9B and Table 2, compared with the same dose of TAX (4 mg/kg), TAX-loaded micelle 4 administration resulted in significant reductions in the volumes and weights of A549/TAX cell-generated tumors in mice. Although TAX-loaded micelle 4 failed to suppress the growth of LCC-generated tumors in mice compared with vehicle treatment (Fig. 9E and Table 3), TAX-loaded micelle 4 administration dramatically reduced the numbers of pulmonary metastatic foci in LLC-inoculated mice in a TAX-dose dependent manner (Table 3). Systemic toxicity and bone marrow suppression are the main side effects of TAX.¹⁷⁾ We did not observe a significant difference in the organ/body weight ratios (Figs. 9A, D), body weights (Figs. 9C, F), PLT, WBC, and RBC counts between TAX-loaded micelle 4- and vehicle-treated mice (Supplementary Table 7). Taken together, these data suggest that TAX-loaded micelle 4 suppresses lung tumor growth and metastasis in vivo without inducing systemic toxicity and myelosuppression.

Fig. 9. TAX-Loaded Micelle 4 Suppressed A549/TAX Tumor Growth and LLC Metastasis in Vivo

Mouse lung tumor models were established by subcutaneously inoculating A549/TAX cells or LLC cells. Mice bearing A549/TAX tumors were administered 200 µL NS, micelle 4 (55 mg/kg), TAX (4 mg/kg), 4 mg/kg TAX-loaded micelle 4, or 1 mg/kg TAX-loaded micelle 4 via intraperitoneal injection every other day for 18 d. Mice were sacrificed on day 31 after inoculation. Mice bearing LLC tumors mice were administered 200 µL NS, micelle 4 (27 mg/kg), TAX (5 mg/kg), 2 mg/kg TAX-loaded micelle 4, or 0.4 mg/kg TAX-loaded micelle 4 via ntraperitoneal injection once daily for 9 d. Mice were euthanized at 10 d after treatment. (A and D) Organ/body weight ratios. (B and E) Tumor volumes. (C and F) Body weights.

Table 2. Comparison of the Tumor Weights of A549/TAX Tumor Mouse Model

Group	Tumor weight (X̄ ± S.D., g)	Tumor inhibition rate (%)
Control	1.58 ± 0.27	0
TAX 4 mg/kg	1.44 ± 0.43b)	8.86
C10NLV (55 mg/kg)	1.14 ± 0.52b)	27.85
Micelle 4 (TAX 1 mg/kg)	1.33 ± 0.57b)	15.82
Micelle 4 (TAX 4 mg/kg)	0.73 ± 0.40a), c)	53.8

TAX, paclitaxel; S.D., standard deviation; a) p < 0.01 vs. PBS; b) p > 0.05 vs. PBS; c) p < 0.01 vs. TAX.

Table 3. Comparison of the Tumor Weights and Lung Metastasis of Lewis Lung Carcinoma Mouse Model

Group	Tumor weight (X̄ ± S.D., g)	The number of pulmonary metastatic foci (X̄ ± S.D.)
Control	3.915 ± 0.994	27.363 ± 7.365
C10NLV 27 mg/kg	2.981 ± 1.477	9.333 ± 1.75
TAX 5 mg/kg	2.515 ± 0.607	4.833 ± 3.431
Micelle 4 (TAX 2 mg/kg)	2.227 ± 0.857	4.166 ± 3.061
Micelle 4 (TAX 0.5 mg/kg)	3.533 ± 1.756	6.4 ± 6.228

TAX-Loaded Micelle 4 Inhibits Tubulin and CD206 Expression While Promoting Cell Death in Xenograft Tumors

M2-polarized macrophages promote lung cancer metastasis.^18,19) Immunofluorescent staining showed that treatment with TAX-loaded micelle 4 considerably attenuated CD206 protein expression in tumor tissue samples compared with free TAX (Figs. 10A, B). Similar results were observed in tubulin expression. In addition, HE analysis showed that treatment with TAX-loaded micelle 4 but not TAX led to cytoplasm disappearance and nuclear shrinkage in the xenograft tumors (Fig. 10C), suggesting that TAX-loaded micelle 4 might cause lung cancer cell death. Taken together, these findings suggest that TAX-loaded micelle 4 inhibits M2-like polarization of macrophages and disrupts cell skeleton while promoting tumor cell death in lung cancer, thereby suppressing lung cancer metastasis.

Fig. 10. TAX-Loaded Micelle 4 Inhibited Tubulin and CD206 Expression While Promoting Cell Death in Xenograft Tumors

(A) Immunofluorescent staining was performed to detect tubulin and CD206 expression in A549/TAX tumor tissue samples. (B) Quantification of (A). (C) H&E.

Discussion

In this study, we synthesized a novel NLV tripeptide with a molecular weight of 581.3829 g/mol from L-Val-OBzl and Boc-Leu via 5-step reactions. TAX was loaded with different doses of C10NLV to generated five types of drug-loaded micelles. We found that the TAX-loaded micelles did not significantly alter the TAX IC₅₀ values of A549 cells compared to free TAX. This suggests that the liposomes prepared by combining C10NalLeuVal with TAX did not mask the anti-proliferative activity of TAX. We found that TAX-loaded micelles dramatically reduced TAX IC₅₀ values of A549/TAX and LLC cells in a C10NLV-dose-dependent manner compared with free TAX. Because micelles 4 and 5 exhibited comparable inhibitory effects on A549/TAX cell proliferation, we selected micelle 4 containing 11.6 mg C10NLV in the following investigation. We demonstrated that compared with the same dose of free TAX, TAX-loaded micelle 4 promoted cell apoptosis while inhibiting the metastatic capacity of lung cancer cells, despite TAX resistance. Furthermore, administration of TAX-loaded micelle 4 remarkably suppressed A549/TAX tumor growth and LLC metastasis in mouse models, without inducing significant systemic toxicity. Our results suggest that C10NLV-based TAX delivery is a promising therapeutic strategy against TAX resistance and metastasis in lung cancer treatment.

Mutations and dysregulated expression of cytoskeletal proteins, such as F-actin and tubulin, facilitate cancer cells to resist chemotherapy and metastasize.²⁰⁾ Tubulin inhibitors including TAX and docetaxel are important components of chemotherapy regimens for advanced NSCLC treatment.²¹⁾ F-actin is also a therapeutic target for lung cancer treatment due to its important role in cancer cell migration and invasion.^22,23) Our results showed that compared with the equivalent dose of free TAX, TAX-loaded micelle 4 treatment caused an almost complete loss of F-actin and significant suppression of cell adhesion, migration, and invasion in A549/TAX cells. Furthermore, TAX-loaded micelle 4 treatment dramatically suppressed tubulin expression in A549/TAX-generated tumors and reduced lung metastasis in LLC-implanted mice, whereas free TAX exhibited negligible effects. These data suggest that micelle 4-mediated TAX delivery overcomes drug resistance and prevents metastasis of lung cancer by disrupting the cytoskeleton and suppressing the metastatic capacity of lung cancer cells.

Apoptosis and autophagy that induce cell death or degradation of proteins and organelles play critical roles in the pathophysiology of lung cancer.²⁴⁾ Amplification of the anti-apoptotic Bcl-2 has been observed in NSCLC and is associated with clinical drug resistance.²⁵⁾ Chen and Shi have found that TAX activates Beclin-1 in A549 cells in a dose-dependent manner and that inhibition of autophagy increases the sensitivity of NSCLC cells to TAX treatment.²⁶⁾ Inhibition of autophagy has been shown to prevent the development of TAX resistance in A549 cells and potentiates the effect of TAX by inducing oxidative stress in the cells. Inhibition of autophagy also promotes apoptosis and G0/G1 phase arrest while attenuating the metastatic potential of TAX-treated A549 cells,²⁷⁾ suggesting a complicated interplay among cell proliferation, apoptosis, and autophagy in lung cancer cells. Consistent with these reports, our results showed that compared with the same dose of free TAX, TAX-loaded micelle 4 substantially downregulated protein expression of Bcl-2 and LC3A/B in A549/TAX cells. The protein level of pro-apoptotic BAX remained unchanged. These data suggest that micelle 4-mediated TAX delivery overcomes drug resistance of lung cancer cells by promoting cell apoptosis and inhibiting autophagy. TAX-loaded micelle 4 loaded TAX into A549/TAX cells via C10NalLeuVal, avoiding the efflux caused by TAX alone, which leads to TAX resistance in A549/TAX cells. M2-polarized macrophages promote the metastatic behavior of LLC cells by inducing vascular endothelial growth factor-C expression.¹⁸⁾ High infiltration of M2-polarized macrophages in tumors is associated with tumor progression and poor overall survival in NSCLC.²⁸⁾ Inhibition of M2 polarization of macrophages can suppress the M2-conditioned medium-induced invasion, migration, and angiogenesis of A549 cells and inhibit tumor growth and metastasis in LLC tumor-bearing mice.²⁹⁾ Our results showed that compared with free TAX, micelle 4-mediated TAX delivery may inhibit M2 polarization of macrophages in lung cancer, as illustrated by reduced expression of CD206 in A549/TAX-generated tumors.

The in vitro release results for paclitaxel-binding tripeptide micelles in the blood stream were profiled in Supplementary Fig. S1. The release was shown to be sustained and followed first-order kinetics could be primarily attributed to hydrophobic interactions between the extremely hydrophobic paclitaxel molecule and tripeptide derivative. The inference above is further supported by characterization data (such as differential scanning calorimetry (DSC) studies) that confirms the amorphous nature of entrapped paclitaxel at the core of the micelles. This observation and conclusion is further supported by and aligns with similar studies reported in the literature.³⁰⁾ The results of the in vivo pharmacokinetic studies in mice were shown in Supplementary Fig. S2. According to the references and the in vivo pharmacokinetic studies, the paclitaxel-binding tripeptide micelles did release some of paclitaxel in the blood stream, and the reason of the initial burst release of the drug (first 1–2 h) can be attributed to “the immediate dissolution of free paclitaxel in the formulation (outside the polymeric micelles) as well as the drug.”³¹⁾

Changes in body weights and blood cell counts reflect systemic and hematological toxicity. To evaluate the safety and tolerance of TAX-loaded micelle 4, we measured the organ/body weight ratios, body weights, and blood cell counts in the mice. Our results showed that compared with other groups, TAX-loaded micelle 4 treat-mice did not show significant changes in the above indicators, suggesting that TAX-loaded micelle 4 treatment is a well-tolerated and safe therapeutic approach in lung cancer treatment.

In this study, we present a tripeptide-based TAX delivery system for enhanced chemotherapy efficacy and minimal side effects in lung cancer treatment. Compared with the equivalent dose of TAX, the TAX-loaded micelles significantly inhibited the proliferative and metastatic capacity while promoting cell apoptosis in A549/TAX and LLC cells. In vivo study showed that the micelle-mediated TAX delivery was well-tolerated and safe to suppress A549/TAX tumor growth and LLC metastasis in mice. Mechanistically, the antitumor effect of TAX-loaded micelles was associated with disruption of the cytoskeleton, enhancement of cell apoptosis, and inhibition of autophagy of cancer cells as well as suppression of M2 polarization of macrophages in tumors. Therefore, the C10NLV-based TAX delivery system is highly beneficial to combat chemoresistance and metastasis in lung cancer.

Acknowledgments

We are grateful to Yaonan Wang of the Capital Medical University for helping us with this project. This work was supported by the Beijing Excellent Talent Training Fund, the Great Wall Scholar Program of Beijing Municipal Education Commission (CIT & TCD20180332), the 863 Program (2015AA020902), and the Beijing Highly Qualified Talent Introduction and Development Program.

Author Contributions

Yuji Wang conceived and designed the study. Jie Gao, Yijiang Jia, Taledaohan Ayijiang, Tuohan Marmar, Xi Hu, Li Li, Yuanming Li, performed and analyzed the experiments, wrote the paper. All authors reviewed the results and approved the final version of the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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