PLGA nanoparticle-based docetaxel/LY294002 drug delivery system enhances antitumor activities against gastric cancer
Juan Cai1,2,3 , Keyang Qian1, Xueliang Zuo4, Wuheng Yue5, Yinzhu Bian6, Ju Yang2, Jia Wei2, Wenying Zhao3,
Hanqing Qian1,2 and Baorui Liu1,2
Journal of Biomaterials Applications 0(0) 1–13
! The Author(s) 2019
Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/0885328219837683
journals.sagepub.com/home/jba
Abstract
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Docetaxel (TXT) is acknowledged as one of the most important chemotherapy agents for gastric cancer (GC). PI3K/ AKT signaling is frequently activated in GC, and its inhibitor LY294002 exerts potent antitumor effects. However, the hydrophobicity of TXT and the poor solubility and low bioavailability of LY294002 limit their clinical application. To overcome these shortcomings, we developed poly(lactic acid/glycolic) (PLGA) nanoparticles loaded with TXT and LY294002. PLGA facilitated the accumulation of TXT and LY294002 at the tumor sites. The in vitro functional results showed that PLGA(TXT LY294002) exhibited controlled-release and resulted in a markedly reduced proliferative capacity and an elevated apoptosis rate. An in vivo orthotopic GC mouse model and xenograft mouse model confirmed the anticancer superiority and tumor-targeting feature of PLGA(TXT LY294002). Histological analysis indicated that PLGA(TXT LY294002) was biocompatible and had no toxicity to major organs. Characterized by the combined slow release of TXT and LY294002, this novel PLGA-based TXT/LY294002 drug delivery system provides controlled release and tumor targeting and is safe, shedding light on the future of targeted therapy against GC.
Keywords
Gastric cancer, nanoparticles, docetaxel, LY294002, PLGA
Introduction
Gastric cancer (GC) is a global health burden, ranking as the fifth most common cancer and the third leading cause of cancer-related death worldwide.1 Given that GC is usually asymptomatic until an advanced stage, it is difficult to diagnose this illness in the early stages. Chemotherapy and molecular-targeted therapy have been acknowledged as the fundamental therapeutic modalities for advanced GC.
Being a class of important chemotherapy agents, taxane disrupts microtubule assembly and induces cell apoptosis. Docetaxel (TXT), in the taxane family, is highly efficacious for advanced GC.2 Aberrant PI3K/AKT activation enhances the proliferation and motility and inhibits the apoptosis of GC cells. Pathway inhibitors have exhibited promising clinical values in various cancers.3,4 LY294002, a widely used selective PI3K inhibitor, has been shown to possess antitumor activities towards GC.5 However, the hydro- phobic nature of TXT and the poor solubility and
1The Comprehensive Cancer Centre of Nanjing Drum Tower Hospital, Clinical College of Nanjing Medical University, Nanjing, China
2The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University & Clinical Cancer Institute of Nanjing University, Nanjing, China
3Department of Oncology, The First Affiliated Hospital, Yijishan Hospital of Wannan Medical College, Wuhu, China
4Department of Gastrointestinal Surgery, The First Affiliated Hospital, Yijishan Hospital of Wannan Medical College, Wuhu, China
5The Comprehensive Cancer Centre of Nanjing Drum Tower Hospital, Clinical College of Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, China
6Department of Oncology, The First People’s Hospital of Yancheng, Yancheng, China
Corresponding authors:
Baorui Liu, The Comprehensive Cancer Centre of Nanjing Drum Tower Hospital, Clinical College of Nanjing Medical University, 321 Zhongshan Road, Nanjing 210008, China.
Email: [email protected]
Hanqing Qian, The Comprehensive Cancer Centre of Nanjing Drum Tower Hospital, Clinical College of Nanjing Medical University, 321 Zhongshan Road, Nanjing 210008, China.
Email: [email protected]
low bioavailability of LY294002 overshadow their clin- ical application.6–8 Thus, it might be promising to include TXT and LY294002 in a combined targeted therapy for GC.
Nanoparticles (NPs) provide a practical approach to
delivering drugs to tumor sites via their enhanced per- meability and retention (EPR) effects.9 The poorly developed leaky vasculature and the lack of functional lymphatic drainage of tumors result in an enhanced accumulation of NPs at tumor sites.10 Efficient tumor penetration is a critical prerequisite for targeting tumor cells. Poly(lactic acid/glycolic) (PLGA) is a biocompat- ible and degradable material that can form NPs and has been widely adopted for drug delivery.11 PLGA- based NPs also exhibit high stability that prevents the encapsulated drugs from premature degradation.12 Therefore, we designed PLGA NPs loaded with TXT
and LY294002, named PLGA(TXTþLY294002), to
overcome the abovementioned shortcomings. We,
for the first time, achieved the slow release of these two drugs in combination for GC treatment. We then characterized the properties of this compound and evaluated the uptake profiles and its anticancer effi- cacy. Both in vivo xenograft and orthotopic mouse
models were established to systematically investigate the tumor-suppressive effect of PLGA(TXTþ LY294 002) in GC.
In the present study, we proposed that the combined treatment of TXT and LY294002, delivered by PLGA, could enhance the antitumor activities against GC. This PLGA-based drug delivery system provides con- trolled release and tumor targeting and is safe and bio- compatible in vivo. This novel TXT/LY294002-loaded PLGA system enabled the slow release of these two drugs and might cast light on therapeutic strategies for GC.
Materials and methods
Cell lines and animals
The human GC cell line MKN45 was obtained from the Shanghai Institute of Cell Biology (Shanghai, China) and maintained in RPMI 1640 medium (Wisent, Nanjing, China) containing 10% FBS
(Gibco, Carlsbad, CA, USA) at 37◦C with an atmo-
sphere of 5% CO2. BALB/c nude mice were purchased from the Department of Experimental Animals, Yangzhou University (Yangzhou, China). All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, approved by the Nanjing Drum Tower Hospital Laboratory Animals Welfare & Ethical Committee.
Preparation of PLGA NPs
PLGA (molecular weight: 40 kDa) was purchased from DaiGang Biomaterial Co. Ltd (Jinan, China). TXT/ LY294002-loaded PLGA NPs were formed by the emulsion-solvent evaporation method. PLGA (10 mg), TXT (0.3 mg; Aladdin Industrial Corp., Shanghai, China), and LY294002 (0.3 mg; Selleck Chemicals, Houston, TX, USA) were dissolved in
500 lL of dichloromethane (Aladdin Industrial
Corp.). The mixture was added dropwise into 4 mL of an aqueous phase containing 1% polyvinyl alcohol (Aladdin Industrial Corp.), emulsified using probe son- ication (VCX 130; Sonics & Materials, Inc., Newtown, CT, USA), and stirred in open air to remove the organ- ic solvent. The upper solution was centrifuged, and the NPs were then washed and redissolved. Dynamic light scattering (DLS) with a Nano Particle Analyzer (Zetasizer Nano ZSE, Malvern Instruments Ltd, UK) was used to examine the size, zeta potential, and poly- dispersity index (PDI) of the NPs. Transmission elec- tron micrograph (TEM) of the NPs was acquired by placing a drop of the sample onto a copper mesh and drying in room temperature. To determine the encap- sulation efficiency (EE%), the TXT and LY294002 encapsulated in PLGA were measured using high- performance liquid chromatography (HPLC; Agilent Technologies, Santa Clara, CA, USA). The EE%
and loading efficiency (LE%) were calculated by the following equations: EE% ¼ (weight of drug in the NPs/weight of feeding drug) ×100%, LE% ¼ (weight of drug in the NPs/weight of the NPs) ×100%.
In vitro drug release
The drug release was examined by dialyzing specimens against PBS at pH 7.4 with a dialysis bag at 37◦C for five days, mimicking the in vivo situation. The molec-
ular weight threshold of the dialysis bag was 14 kDa. HPLC was used to detect the TXT and LY294002. The initial concentrations of TXT and LY294002 were 72 lg/mL and 41 lg/mL, respectively.
Cellular uptake of NPs
We labeled PLGA NPs with the fluorescent probe Coumarin-6 (Sigma-Aldrich, St. Louis, MO, USA) to assess the intracellular distribution. MKN45 cells were seeded into 6-well plates and cultured over- night. Free Coumarin-6 or Coumarin-6-NP was added into the culture medium at a concentration
of 2 lg/mL of Coumarin-6. The nuclei were stained
with DAPI. The cellular uptake of Coumarin-6 was observed under a fluorescence microscope (EVOS FL Auto, Tokyo, Japan).
In vitro cytotoxicity assay
For a negative control, MKN45 cells were incubated with different concentrations of bare PLGA to assess the cytotoxicity of bare PLGA. For the experimental
groups, we treated the cells with an equivalent concen- tration of TXT or LY294002. A total of 10 ll of Cell Counting Kit-8 (CCK-8, Beyotime, Nantong, China) was added to the medium. The cells were incubated
for 2 h, and the absorption at 450 nm was recorded. The cell viability (%) and cell death (%) were calculat- ed by the following equations: cell viability (%) ¼ [Abs (sample) – Abs (background)]/[Abs (control) – Abs
(background)] × 100%, cell death (%) ¼ 1 – cell viabil- ity (%). Abs (control) represents the absorbance of the cells without any treatment and was considered as the
blank control.
Cell apoptosis assay
Cell apoptosis was detected using an Annexin V-FITC/ PI apoptosis detection kit (MultiSciences, Hangzhou, China). In the experimental groups, the concentration of TXT was 25 lg/mL, and the concentration of LY294002 was 25 lM. After 24 h or 48 h, the cells were harvested and incubated with 3 lL of Annexin V-FITC and 3 lL of PI. The cell apoptosis rate was quantified with a flow cytometer (Becton Dickinson, San Jose, CA, USA). Cells subjected to no treatment were set as the blank control.
Cell cycle analysis
In all experimental groups, the concentration of TXT was 10 lg/mL, and the concentration of LY294002 was 10 lM. MKN45 cells were subjected to different treat- ments for 24 h and fixed in 75% alcohol. After staining with PI for 15 min in the dark, the cells were collected for a flow cytometry analysis. Cells with no treatment were set as the blank control.
Biodistribution of NPs by systemic injection
The in vivo tumor-targeting capacity of the NPs was examined using a lipophilic carbocyanine dye DiR (MaokangBio, Shanghai, China). Xenograft mouse models were constructed by subcutaneously injecting
100 lL of a cell suspension that contained 2 × 106
MKN45 cells into the groin of the mice. Tail intrave-
nous administration of free DiR or DiR-loaded NPs was performed to evaluate the targeting efficiency. Images were captured after 2, 6, 24, 48, 72, and 120 h (IVIS Lumina XRMS Series III, PerkinElmer, Waltham, MA, USA).
Orthotopic nude mouse model construction
MKN45 cells stably expressing firefly luciferase (MKN45-luc; Genechem, Shanghai, China) were sub- cutaneously implanted into four-week-old BALB/c nude mice. The xenografts were harvested three weeks later, and cut into fragments of 1 mm3 in size. The nude mice were anaesthetized and tumor fragments were then implanted into the serosa of the
stomach. On days 7, 14, and 21 after the procedure, the mice were treated with normal saline, TXT, TXTþ LY294002, or PLGA(TXTþLY294002). The dosages of TXT and LY294002 were 10 mg/kg and 15 mg/kg,
respectively. On days 8, 15, 22, and 29 after the surgery, the mice were subjected to bioluminescent imaging (BLI).
In vivo antitumor efficacy and toxicity evaluation
The xenograft mouse models were constructed as described before. When the tumor volume reached approximately 100 mm3 (10 days after inoculation), the mice were treated with normal saline, TXT,
TXTþLY294002 or PLGA(TXTþLY294002) via
intravenous administration on days 0, 4, and 8. The
concentrations of TXT and LY294002 were 10 mg/kg and 15 mg/kg, respectively. The weights of the mice and tumor volumes were monitored regularly. The tumor volume was recorded using the following equa-
tion: Volume ¼ 1/2 × length × width2. The xenograft
tumors were excised and weighed and fixed with 4%
paraformaldehyde. The major organs, including the heart, liver, spleen, lung, and kidney, were also obtained for further histological examination. The par- affin sections were subjected to hematoxylin-eosin (H&E) staining. TUNEL assays were performed to determine the apoptotic cells using an in situ apoptosis detection kit (KEYGEN, Nanjing, China).
Western blotting and immunohistochemistry
The protein levels of AKT, p-AKT, and GAPDH were detected by Western blotting assays. The Ki-67 levels in xenografts were determined using IHC. The detailed procedures were previously described.13
Statistical analysis
The data are presented as the mean SEM. Student’s t test and one-way ANOVA were employed to examine the differences as appropriate. The data were analyzed
using GraphPad Prism 8.0 (San Diego, CA, USA) and SPSS 22.0 (IBM Corp., Armonk, NY, USA). P < 0.05
is considered statistically significant.
Results
Preparation and characterization of PLGA (TXTþLY294002)
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As an attractive drug delivery material, PLGA could function as the core and entrap the drugs. A W/O emulsion was used to prepare PLGA(TXT LY294002) (Figure 1(a)). The concentrations of TXT and LY294002 were both 0.067 g/L in the PLGA NPs. According to the DLS results, the average diameter of the NPs was 155.3 3.5 nm, with a PDI of 0.146
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—
0.171 (Figure 1(b) and (c)). The PLGA(TXT LY294002) NPs dispersed uniformly with no aggrega- tion according to the TEM image (Figure 1(d)). Due to the shrinking of the PLGA shell during the drying pro- cess, the mean diameter observed from TEM was smaller than the reported DLS values.14 The zeta potential was 16.9 1.6 mV (Figure 1(e)). The EE
% of TXT and LY294002 were 60.2 2.0% and 34.2 1.3%, respectively. The LE% of TXT and LY294002 in the PLGA NPs were 1.70 0.06% and
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1.00 0.04%, respectively. We then examined the in vitro releases of TXT and LY294002 from the PLGA (TXT LY294002) NPs in PBS containing 0.5% Tween
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80 at 37 0.5◦C (Figure 1(f)). The release of TXT from the PLGA(TXT LY294002) NPs was character-
ized by a fast-initial release during the first 48 h (52.33 3.21%). In the subsequent 120 h, a steady and slow release of TXT from the NPs was observed (61.00 2.65%). Similarly, the release rates of LY294002 from the NPs were 54.67 3.06% and
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67.67 3.51% at 48 h and 120 h, respectively. In con- trast, the release rates of free TXT and free LY294002 were 91.33 4.04% and 93.33 2.52% at 24 h, respectively, indicating a relatively rapid release behav- ior. Thus, these results show that the PLGA (TXT LY294002) drug delivery system provides con- trolled release.
Cellular uptake of NPs in vitro
After incubating the tumor cells with Counmarin- 6-NP, the green signal (Counmarin-6) around the nucleus (stained with DAPI) was very strong. In con- trast, the control group presented a weak Counmarin-6 signal in the cells (Figure 1(g)). Collectively, the results reveal that PLGA possesses the capacity of enhancing the cellular drug uptake.
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Cellular cytotoxicity of PLGA(TXT LY294002) in vitro
We performed Western blotting to evaluate the effects of PLGA(TXTþLY294002) on the PI3K/AKT signaling.
The p-AKT level was lower in the TXTþLY294002 and PLGA(TXTþLY294002) groups than in the blank con-
trol and bare PLGA groups, confirming that the addi- tion of LY294002 could inhibit the AKT activation (Figure 2(a)). To evaluate the in vitro antitumor activity, the human GC cell line MKN45 was selected for a CCK-8 analysis. As shown in Figure 2(b), we first evaluated the cytotoxicity of bare PLGA against the MKN45 cells after 48 h incubation. The results indicate that the bare PLGA was nontoxic to cancer cells. We then treated MKN45 cells with different concentrations
of TXT, LY294002, TXTþLY294002, or PLGA
(TXTþLY294002) and incubated the cells for 24 h or 48 h (Figure 2(c) and (d)). When TXT was used in com-
bination with LY294002, the cell death rate was increased in a dose-dependent way. The results show that the TXTþLY294002 group presented a slightly better antiproliferative efficacy than that of the PLGA (TXTþLY294002) group at 24 h. However, the death
rates of these two groups became comparable at 48 h,
further confirming the controlled-release feature of PLGA(TXTþLY294002).
Cellular apoptosis in vitro
To further confirm the antiproliferative effect of PLGA (TXTþLY294002) in GC, we examined the cell apo- ptosis in MKN45 cells subjected to different treat-
ments. Compared with the mono-drug groups, the apoptosis rates in the TXTþLY294002 and PLGA (TXTþLY294002) groups were higher at both 24 h and 48 h (Figure 3(a) and (b)). In the first 24 h, the
TXTþLY294002 group induced more cell apoptosis than the PLGA(TXTþLY294002) group, but the apo- ptosis rate presented no significant difference at 48
h between these two groups.
Cellular cycle in vitro
We then performed a cell cycle analysis using flow cytometry. Compared with the control group, the TXT group presented G2/M cell cycle arrest, while the cells treated with LY294002 exhibited G1/S arrest. According to the results, the cell cycle arrests
in the TXTþLY294002 and PLGA(TXTþLY294002)
groups were both evident (Figure 3(c)).
In vivo distribution of the NPs
After confirming the potent antitumor effect of the NPs in vitro, we further investigated their targeting proper- ties in vivo. Intravenous DiR-NPs led to a higher accu- mulation of the fluorescence signal in the tumor sites compared with that in the free DiR group (Figure 4(a)).
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Figure 1. Preparation, characterization, and cellular uptake of PLGA(TXT LY294002). (a) Representative photograph showing the preparation of PLGA(TXT LY294002). (b) The size distribution profile of the PLGA(TXT LY294002) NPs was measured using DLS.
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(c) DLS measurements of the size and PDI of the PLGA(TXT LY294002) NPs for 7 days. (d) Representative transmission electron micrograph of PLGA(TXT LY294002). (e) The zeta potential of PLGA(TXT LY294002) showing a negative charge. (f) The releases of TXTor LY294002 from the PLGA(TXT LY294002) NPs at 37◦C for five days were determined. The releases of free TXT and free LY294002 were also examined as the controls. The data are shown as the mean SEM. (g) The fluorescent probe Coumarin-6 was
used to show the cellular uptake of PLGA in vitro. MKN45 cells were incubated with Courmarin-6-NP or free Courmarin-6, respectively. PLGA: poly(lactic acid/glycolic); TXT: docetaxel; DLS: dynamic light scattering; PDI: polydispersity index; NP: nanoparticle.
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AKT GAPDH
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Figure 2. Cytotoxicity of the NPs. (a) Western blotting assays were performed to determine the levels of p-AKT and AKT in MKN45 cells treated with blank control, bare PLGA, TXT LY294002 or PLGA(TXT LY294002). (b) Cytotoxicity test of bare PLGA against MKN45 cells after 48 h incubation. (c) The in vitro cytotoxicity of PLGA(TXT LY294002) in comparison with TXT, LY294002, and TXT LY294002 after incubation for 24 h. (d) The in vitro cytotoxicity of PLGA(TXT LY294002) in comparison with TXT,
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LY294002, and TXT LY294002 after incubation for 48 h. The data are shown as the mean SEM. ***P < 0.001 compared to the TXT LY294002 group, ###P < 0.001 compared to the PLGA(TXT LY294002) group. NP: nanoparticle; PLGA: poly(lactic acid/gly- colic); TXT: docetaxel.
The fluorescence climbed to its highest level at 48 h and gradually declined thereafter (Figure 4(b)).
In vivo growth inhibitory effects of NPs using orthotopic mouse model
Orthotopic mouse models were employed to detect the in vivo antitumor activities of PLGA(TXTþLY294002) in GC. The orthotopic mouse models were successfully
constructed, as confirmed by macroscopic observation (Figure 5(a)) and H&E staining (Figure 5(b)). On days 8, 15, 22, and 29, the mice were subjected to BLI
(Figure 5(c)). As shown in Figure 5(d) and (e), the weakest signal intensity was observed in the PLGA (TXTþLY294002) group.
Antitumor efficacy and toxicity evaluation
The antitumor efficacy and systemic toxicity of PLGA (TXTþLY294002) were evaluated by a xenograft nude mouse model. As depicted in Figure 6(a) and (b), the
normal saline group showed a rapid rate of tumor growth, and the tumor volume exceeded 700 mm3 on day 30 after the injection. The body weight of the mice
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Cai et al.
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Figure 3. Effects of PLGA(TXT LY294002) on cell apoptosis and cell cycle distribution. (a) Analysis of cell apoptosis in MKN45 cells treated with blank control, TXT, LY294002, TXT LY294002 or PLGA(TXT LY294002) at an equivalent TXT concentration of 25 lg/mL and LY294002 concentration of 25 lM for 24 h. (b) Analysis of cell apoptosis in MKN45 cells treated with blank control, TXT, LY294002, TXT LY294002 or PLGA(TXT LY294002) at an equivalent TXT concentration of 25 lg/mL and LY294002 concentration of 25 lM for 48 h. (c) Effects of blank control, TXT, LY294002, TXT LY294002 or PLGA(TXT LY294002) on cell cycle of MKN45 cells subjected to different treatments for 24 h. The data are shown as the mean SEM. **P < 0.01, ***P < 0.001 compared to the TXT LY294002 group, #P < 0.05, ##P < 0.01, ###P < 0.001 compared to the PLGA(TXT LY294002) group. PLGA: poly(lactic acid/glycolic); TXT: docetaxel.
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Figure 4. Biodistribution of the NPs in vivo. (a) The in vivo dynamic fluorescence imaging after intravenous injection of free DiR or DiR-NPs. The red arrows indicate the sites of the xenograft tumors. (b) Fluorescence intensities of the tumor sites were quantified by IVIS Living Imaging Software. The data are shown as the mean SEM. ***P < 0.001. NP: nanoparticle.
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in the three experimental groups did not significantly change when compared to that in the normal saline group (Figure 6(c)), suggesting that the PLGA(TXT LY294002) NPs had no obvious toxicity. As shown in Figure 6(d), the tumor weight was 73.82 18.66 mg for the PLGA(TXT LY294002) group, whereas the tumor weights were 124.5 25.68, 195.89 36.8, and
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734.7 170.55 mg for the TXT LY294002, TXT, and normal saline groups, respectively. As expected, the PLGA(TXT LY294002) group had the highest tumor inhibitory efficacy. Ki-67 staining showed that the PLGA(TXT LY294002) group had the lowest proliferative capacity (Figure 6(e)). TUNEL analysis further indicated that the PLGA(TXT LY294002) group exhibited the highest apoptosis rate (Figure 6(e)). Together, our results show that PLGA(TXT LY294002) substantially strengthened the antitumor activities of TXT and LY294002.
Additionally, H&E staining demonstrated that the administration of the drugs resulted in no significant damage to major organs including the heart, liver, spleen, lung, and kidney (Figure 7). These results
indicate that the PLGA(TXTþLY294002) NPs have satisfactory safety in vivo.
Discussion
Herein, we constructed a controlled-release and tumor- targeting drug delivery system using PLGA NPs loaded with TXT and the PI3K/AKT inhibitor LY294002 for the first time. NPs with size < 400 nm could preferen- tially accumulate in tumors and present an optimal
EPR effect, while NPs larger than 100 nm could avoid being engulfed by the mononuclear phagocyte system and excreted with the urine.15 PLGA (TXTþLY294002) have a favorable size (155.3 nm according to the DLS data) for cellular internalization
and tumor penetration. Moreover, the zeta potential of PLGA(TXTþLY294002) indicated a slightly negative surface charge (—16.9 mV), which is desirable for the higher accumulation of PLGA(TXTþLY294002) at the tumor sites.
To date, TXT-based chemotherapy remains to become a crucial strategy for advanced GC treatment.
(a) (b)
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Figure 5. The in vivo growth inhibitory effects of NPs using orthotopic mouse model. (a) Representative photograph of the orthotopic gastric tumor in mouse. The red arrow indicates the site of the orthotopic tumor. (b) H&E staining of the orthotopic gastric tumor tissue. (c) The time points of the drug administration and bioluminescence imaging for gastric cancer orthotopic mouse models. (d) The in vivo bioluminescence images showing the growth of orthotopic gastric tumors. (e) The quantification of biolu- minescence signals in each group. Normal saline was set as the control. NP: nanoparticle; H&E: hematoxylin-eosin.
TXT has been demonstrated to have poor aqueous sol- ubility.16 Currently, high amounts of nonionic surfac- tants (Tween 80 and ethanol) are used for commercial TXT formulation due to its hydrophobicity. However, the addition of Tween 80 and ethanol might result in considerable side effects.17 LY294002, a PI3K/AKT inhibitor, could inhibit GC metastasis by downregulat- ing MMP2, MMP9, and VEGF.18 Nevertheless, LY294002 exhibits poor solubility and low bioavail- ability in vivo. Previous studies have shown that the combination of TXT with a PI3K/AKT inhibitor leads to increased tumor cell apoptosis.19,20 However, the hydrophobicity of TXT and the poor solubility and low bioavailability of LY294002 limit their application in clinical practice. Our novel NP system could overcome these obstacles using PLGA NPs. In our study, a PLGA NP-based drug delivery
system is shown to enhance the cellular uptake of TXT and LY294002.
NP-based drug delivery systems have been devel- oped to increase the therapeutic efficacy of chemother- apy.21,22 Liposomes, viral vectors, and silica-based NPs have been implicated to be potential nanoplatforms for drug delivery.23–25 However, such delivery systems have several unneglectable drawbacks, including high immunogenicity and slow degradation.26 To overcome these flaws, we adopted PLGA in our study to enhance the stability and biocompatibility. The internalization mechanisms of the PLGA-based nanoplatform are pos- sibly through three pathways: endocytosis, direct deliv- ery, and membrane disruption.12 With its advantages of low immunogenicity and low toxicity, PLGA could protect TXT and LY294002 from being degraded too soon. Several other combinations with LY294002 have
(a)
Normal saline TXT
TXT+LY294002 PLGA(TXT+LY294002)
(c) 40
Body weight (g)
30
20
10
0
0 7 10 14 16 18 20 22 24 26 28 30
Time (day)
(b) 1000
Tumor volume (mm3)
800
600
400
200
0
Normal saline TXT
TXT+LY294002 PLGA(TXT+LY294002)
***
***
***
(d)
1000
Tumor weight (mg)
800
600
400
200
0
0 7 10 14 16 18 20 22 24 26 28 30
Day (d)
(e)
Normal saline
TXT TXT+LY294002 PLGA(TXT+LY294002)
Ki-67
TUNEL
þ
Figure 6. The in vivo anticancer effect of PLGA(TXT LY294002) on xenograft mouse model. (a) Photographs of the xenograft tumors. (b) The tumor growth curves. (c) The body weights of the mice. (d) The weights of the xenografts. (e) The Ki-67 and TUNEL staining of the xenografts. Normal saline was set as the control. The data are shown as the mean SEM. **P < 0.01, ***P < 0.001. PLGA: poly(lactic acid/glycolic); TXT: docetaxel.
NOTE: Tumor volume ¼ Tumor volume on the day of measurement – Tumor volume on the day of first drug administration.
also been previously reported. LY294002 and 5-fluoro- uracil-loaded PEGylated NPs have been constructed to target esophageal squamous cell carcinomas.27 LY294002, everolimus, and TXT-loaded PEG-PCL NPs have been developed for the treatment of
metastatic melanomas.28 Our NP system provides con- trolled release and is safe, which is an important and indispensable prerequisite for a drug delivery system. As demonstrated in multiple malignancies, NPs enhance the drug solubility, prolong the drug
TXT
Normal saline
Heart Liver Spleen Lung Kidney
PLGA(TXT+LY294002)
TXT+LY294002
þ þ
Figure 7. Representative H&E staining of the major organs including the heart, liver, spleen, lung, and kidney at the end of the experiment in the normal saline, TXT, TXT LY294002, and PLGA(TXT LY294002) groups. PLGA: poly(lactic acid/glycolic); TXT: docetaxel; H&E: hematoxylin-eosin.
þ
duration in vivo, target the drug delivery, and reduce the toxicity.29,30 Herein, PLGA(TXT LY294002) resulted in a lower cell proliferative capacity and higher apoptosis rate than those using the mono-drug groups and thus could enhance tumor- suppressive activities.
þ
þ
To investigate the antitumor activities of our drug delivery system, we constructed two in vivo mouse models. Xenograft nude mouse models were used, and the results showed that the PLGA (TXT LY294002) group had the lowest proliferative capacity and the highest apoptosis rate. Furthermore, we employed in situ GC mouse models to validate the results derived from the xenograft models. For the assessment of the efficiency of drugs, an in situ mouse model is a preferable in vivo model compared to the xenograft model. In situ GC models resemble the actual tumor microenvironment in human gastric carci- nogenesis, while xenograft mouse models merely assess the subcutaneous tumor formation. According to the results from the in situ GC models, a similar inhibitory effect of PLGA(TXT LY294002) on tumor growth was detected. The tumor-targeting and controlled-release features were also observed, suggesting the promising utility of this drug system in vivo. Despite the promising
antitumor effect of the PLGA(TXTþLY294002) system
and its controlled-release characteristic, the in vivo drug uptake results showed that a proportion of the drug was retained in the liver in addition to the tumor. Many NP- based drug delivery systems exhibit high enrichment in the liver31,32 because the liver is the primary organ for drug biotransformation. Given the high accumulation of NPs in liver, we performed H&E staining and verified the absence of obvious drug-induced hepatotoxicity. In further investigation, GC mouse models induced by car- cinogens should be taken into consideration. Clinical trials on humans are also crucial to evaluate the actual utility of the current drug system.
Conclusions
In this study, we developed a TXT/LY294002-loaded PLGA drug delivery system with controlled-release and tumor-targeting features and anticancer superiority in GC. The in vitro functional experiments showed that the combination of TXT with LY294002 led to higher antitumor activities. The in vivo studies demonstrated that the PLGA-based system enhances the accumula- tion of drugs at the tumor sites, and this system pro- vides controlled release and is safe and biocompatible. We expect this NP system, as a promising new
therapeutic option, could be translated into clinical application of GC treatment eventually.
Authors’ contributions
Juan Cai, Keyang Qian, and Xueliang Zuo contribut- ed equally.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial sup- port for the research, authorship and/or publication of this article: This study was supported by the National Key Research and Development Program of China (No. 2017YFC1308900), the National Natural Science Foundation of China (No. 81602106), and the Natural Science Foundation of Jiangsu Province (BK20150103).
ORCID iD
Juan Cai http://orcid.org/0000-0001-7996-2525
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