Encapsulation of chloroquine and doxorubicin by MPEG-PLA to enhance anticancer effects by lysosomes inhibition in ovarian cancer

Abstract

Purpose: As the deadliest gynecological malignancy, ovarian cancer ranks as a major cause of disease-related deaths to women worldwide and is treated with transurethral resection or systemic chemotherapy. However, traditional chemotherapeutic drug in antitumor therapy has shown unavoidable limitations, such as poor curative effects, systemic toxicity and development of drug resistance, leading to failure of tumor inhibition and recurrence. This study aims to explore an innovative method to enhance the clinical efficiency of ovarian cancer.

Materials and methods: Using MTT assay, the cell viability was detected under different culture systems. Western blot was used to examine the expression of P-gp in doxorubicin-resistant and wild-type A2780/SKOV3 cells. We used confocal to examine the drug concentration under different culture conditions. Also, flow cytometry was used to detect the drug absorption at the determined time points under different culture systems. Using nude mice model, we evaluated the killing efficacy of chemotherapeutic drugs with or without nanoparticle encapsulation. ELISA was used to examine the levels of creatinine, alanine aminotransferase and aspartate aminotransferase in plasma.

Results: We found that pretreatment of chloroquine (CQ) as chemosensitizer markedly enhanced the anticancer effects in ovarian cancer. We also provided evidence that CQ efficiently increase the pH value of lysosomes in tumor cells, leading to the reverse of drug sequestration induced by lysosomes. To further improve the pharmacokinetics profiles and avoid the systemic toxicity caused by chemotherapeutic agents, we encapsulated CQ and chemotherapeutic drugs by polymeric nanoparticles methoxy poly(ethylene glycol)-poly(l-lactic acid). Codelivery of CQ and chemotherapeutic agents by nanocarrier revealed enhanced anticancer effects compared with the free drug delivery by tail vein injection. More importantly, accumulated drugs, prolonged drug circulation and reduced organic damages were observed in nanoparticles delivery.

Conclusion: Codelivery of CQ and chemotherapeutic drugs by methoxy poly(ethylene glycol)-poly(l-lactic acid) could significantly improve the anticancer effects and might have important potency in clinical applications for ovarian cancer therapy.

Keywords: MPEG-PLA; chloroquine; cisplatin; nanoparticles; ovarian cancer.

Figure 1

CQ sensitized the ovarian cancer cells to chemotherapeutic drugs in vitro. Notes: (A) Cell viability of A2780 cells was analyzed by MTT methods after treatment with gradient doses of CQ (5/10/20/50/100/200 µM) for 48 hours. (B) Cell viability of SKOV3 cells was analyzed by MTT methods after treatment with gradient doses of CQ (5/10/20/50/100/200 µM) for 48 hours. (C) Cell viability of A2780 cells was analyzed by MTT methods after treatment with gradient doses of DOX (0.0001/0.001/0.01/0.1/1 µM) pretreated with or without CQ (10 µM, 2 hours) for 48 hours. (D) Cell viability of SKOV3 cells was analyzed by MTT methods after treatment with gradient doses of DOX (0.0001/0.001/0.01/0.1/1 µM) pretreated with or without CQ (10 µM, 2 hours) for 48 hours. (E) Cell viability of A2780 and SKOV3 cells was analyzed by MTT methods after treatment with PTX (4 µM for A2780 and 40 nM for SKOV3 cells) pretreated with or without CQ (10 µM, 2 hours) for 48 hours. (F) Cell viability of A2780 and SKOV3 cells was analyzed by MTT methods after treatment with DDP (20 µM for both A2780 and SKOV3 cells) pretreated with or without CQ (10 µM, 2 hours) for 48 hours. (G) Cell viability of A2780/DOX^R cells was analyzed by MTT methods after treatment with gradient doses of DOX (0.0001/0.001/0.01/0.1/1 µM) for 48 hours. (H) Using Western blot, we examined the expression of P-gp in wild-type and DOX-resistant A2780 cells. (I) Cell viability of A2780/DOX^R cells was analyzed by MTT methods after treatment with gradient doses of DOX (0.0001/0.001/0.01/0.1/1 µM) pretreated with or without CQ (10 µM) for 48 hours. ^*P<0.05; ^**P<0.01; ^***P<0.001. Abbreviations: CQ, chloroquine; DOX, doxorubicin; ns, no significant difference; PTX, paclitaxel; ET, wild type.

Figure 2

CQ sensitized the ovarian cancer cells to chemotherapy through reversing the drug sequestration of lysosomes. Notes: (A) A2780 cells were treated with DOX (4 µM, 4 hours) pretreated with or without CQ (10 µM, 2 hours), followed by LAMP2 staining. The result was analyzed under two-photon confocal microscope. Scale bar, 10 µm. (B) The cell viability of A2780 cells knocking down TFEB after treated with 0.1 µM DOX for 48 hours. (C) The cell viability of SKOV3 cells knocking down TFEB after treated with 0.1 µM DOX for 48 hours pretreated with or without CQ (10 µM, 2 hours). (D) Silencing TFEB or mock A2780 cells were treated with DOX (4 µM) for 4 hours, followed by LAMP2 staining. The result was analyzed under two-photon confocal microscope. Scale bar, 10 µm. (E) A2780 cells were treated with or without CQ (10 µM, 2 hours), followed by stained with lysosome sensor (1 µM) for 30 minutes. The result was analyzed under two-photon confocal microscope. Scale bar, 10 µm. (F) The lysosomal pH values of A2780 cells treated with or without CQ (10 µM) for 2 hours. (G) The metabolisms of DOX under different pH conditions were examined by HPLC. (H) The co-locations of LAMP2 and P-gp in A2780/DOX^R cells were analyzed by using two-photon confocal microscope. Scale bar, 10 µm. (I) A2780/DOX^R cells were pretreated with or without verapamil (5 µM, 2 hours), followed by DOX treatment (4 µM, 4 hours). The cells were stained with LAMP2 and analyzed under two-photon confocal microscope. Scale bar, 10 µm. (J) The cell viability of A2780/DOX^R cells was detected after DOX (0.1 µM) treatment with or without verapamil (5 µM, 2 hours). (K) The cell viability of A2780/DOX^R cells was measured after DOX treatment with or without CQ (10 µM, 2 hours). ^*P<0.05; ^**P<0.01; ^***P<0.001. Abbreviations: CQ, chloroquine; DOX, doxorubicin; ns, no significant difference; TFEB, transcription factor EB.

Figure 3

CQ combined with chemotherapeutic drugs inhibited the tumor growth in ovarian cancer mice model along with organ damage. Notes: (A) Tumor volume of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), DDP (6 mg/kg) or DDP (6 mg/kg) combining with CQ (5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), DDP (6 mg/kg) or DDP (6 mg/kg) combining with CQ (5 mg/kg) in each group (n=6) (right). (B) Tumor volume of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), PTX (15 mg/kg) or PTX (15 mg/kg) combining with CQ (5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), PTX (15 mg/kg) or PTX/PP (15 mg/kg) combining with CQ (5 mg/kg) in each group (n=6) (right). (C) Tumor volume of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), DOX (5 mg/kg) or DOX (DOX 5 mg/kg) combining with CQ (5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), DOX (5 mg/kg) or DOX (5 mg/kg) combining with CQ (5 mg/kg) in each group (n=6) (right). (D) Tumor volume of mice model bearing SKOV3 cells treated with PBS, CQ (5 mg/kg), DDP (6 mg/kg) or DDP (6 mg/kg) combining with CQ (5 mg/kg) were measured (n=6) (left). Survival time of mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), DDP (6 mg/kg) or DDP (5 mg/kg) combining with CQ (5 mg/kg) in each group (n=6) (right). (E) Tumor volume of nude mice model bearing SKOV3 cells treated with PBS, CQ (5 mg/kg), PTX (15 mg/kg) or PTX (15 mg/kg) combining with CQ (5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), PTX (15 mg/kg) or PTX (15 mg/kg) combining with CQ (5 mg/kg) in each group (n=6) (right). (F) Tumor volume of nude mice model bearing SKOV3 cells treated with PBS, CQ (5 mg/kg), DOX (5 mg/kg) or DOX (5 mg/kg) combining with CQ (5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), DOX (5 mg/kg) or DOX (5 mg/kg) combining with CQ (5 mg/kg) in each group (n=6) (right). (G) Tumor volume of nude mice model bearing A2780/DOX^R cells treated with PBS, CQ (5 mg/kg), DOX (5 mg/kg) or DOX (5 mg/kg) combining with CQ (5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), DOX (5 mg/kg) or DOX (5 mg/kg) combining with CQ (5 mg/kg) in each group (n=6) (right). (H) The body weight of C57 mice was measured in control, DOX and DOX-CQ groups (n=6). (I) The levels of alanine aminotransferase/glutamic-pyruvic transaminase (ALT/GPT) were detected in C57 mice received DOX (5 mg/kg) with or without CQ (5 mg/kg) (n=6). (J) The levels of aspartate aminotransferase/glutamic oxalacetic transaminase (AST/GOT) were detected in C57 mice received DOX (5 mg/kg) with or without CQ (5 mg/kg). (K) The concentrations of CRE were detected in C57 mice received DOX (5 mg/kg) with or without CQ (5 mg/kg). PP was short for MPEG-PLA. ^*P<0.05; ^**P<0.01; ^***P<0.001. Abbreviations: CRE, creatinine; CQ, chloroquine; DOX, doxorubicin; ns, no significant difference; PTX, paclitaxel.

Figure 3

CQ combined with chemotherapeutic drugs inhibited the tumor growth in ovarian cancer mice model along with organ damage. Notes: (A) Tumor volume of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), DDP (6 mg/kg) or DDP (6 mg/kg) combining with CQ (5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), DDP (6 mg/kg) or DDP (6 mg/kg) combining with CQ (5 mg/kg) in each group (n=6) (right). (B) Tumor volume of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), PTX (15 mg/kg) or PTX (15 mg/kg) combining with CQ (5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), PTX (15 mg/kg) or PTX/PP (15 mg/kg) combining with CQ (5 mg/kg) in each group (n=6) (right). (C) Tumor volume of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), DOX (5 mg/kg) or DOX (DOX 5 mg/kg) combining with CQ (5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), DOX (5 mg/kg) or DOX (5 mg/kg) combining with CQ (5 mg/kg) in each group (n=6) (right). (D) Tumor volume of mice model bearing SKOV3 cells treated with PBS, CQ (5 mg/kg), DDP (6 mg/kg) or DDP (6 mg/kg) combining with CQ (5 mg/kg) were measured (n=6) (left). Survival time of mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), DDP (6 mg/kg) or DDP (5 mg/kg) combining with CQ (5 mg/kg) in each group (n=6) (right). (E) Tumor volume of nude mice model bearing SKOV3 cells treated with PBS, CQ (5 mg/kg), PTX (15 mg/kg) or PTX (15 mg/kg) combining with CQ (5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), PTX (15 mg/kg) or PTX (15 mg/kg) combining with CQ (5 mg/kg) in each group (n=6) (right). (F) Tumor volume of nude mice model bearing SKOV3 cells treated with PBS, CQ (5 mg/kg), DOX (5 mg/kg) or DOX (5 mg/kg) combining with CQ (5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), DOX (5 mg/kg) or DOX (5 mg/kg) combining with CQ (5 mg/kg) in each group (n=6) (right). (G) Tumor volume of nude mice model bearing A2780/DOX^R cells treated with PBS, CQ (5 mg/kg), DOX (5 mg/kg) or DOX (5 mg/kg) combining with CQ (5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, CQ (5 mg/kg), DOX (5 mg/kg) or DOX (5 mg/kg) combining with CQ (5 mg/kg) in each group (n=6) (right). (H) The body weight of C57 mice was measured in control, DOX and DOX-CQ groups (n=6). (I) The levels of alanine aminotransferase/glutamic-pyruvic transaminase (ALT/GPT) were detected in C57 mice received DOX (5 mg/kg) with or without CQ (5 mg/kg) (n=6). (J) The levels of aspartate aminotransferase/glutamic oxalacetic transaminase (AST/GOT) were detected in C57 mice received DOX (5 mg/kg) with or without CQ (5 mg/kg). (K) The concentrations of CRE were detected in C57 mice received DOX (5 mg/kg) with or without CQ (5 mg/kg). PP was short for MPEG-PLA. ^*P<0.05; ^**P<0.01; ^***P<0.001. Abbreviations: CRE, creatinine; CQ, chloroquine; DOX, doxorubicin; ns, no significant difference; PTX, paclitaxel.

Figure 4

MPEG-PLA nanoparticles encapsulated CQ and DOX enhanced cell apoptosis in vitro and drug accumulation in tumor sites in vivo. Notes: (A) Size distribution spectrum of DOX- and CQ-loaded MPEG-PLA nanoparticles was examined. (B) TEM image of DOX- and CQ-loaded MPEG-PLA nanoparticles was shown (Scale bar, 40 nm). (C) Zeta potential spectrum of DOX- and CQ-loaded MPEG-PLA micelles. (D) Drug release of free DOX and DOX-CQ/PP under different pH conditions (pH 5.5 and pH 7.4) (n=3). (E) Drug release of free CQ and DOX-CQ/PP under different pH conditions (pH 5.5 and pH 7.4) (n=3). (F) The EE and DL of CQ/DOX/DDP/PTX encapsulated by MPEG-PLA were examined. (G) A2780 cells were treated with DOX (1 µM), DOX/PP (DOX concentration 1 µM) and DOX-CQ/PP for 4 hours. The drug uptake was detected by concofol. (H) A2780 cells were treated with DOX (1 µM), MPEG-PLA encapsulated DOX (DOX concentration 1 µM) with or without CQ (10 µM). The MFI of the A2780 treated with DOX (1 µM), MPEG-PLA encapsulated DOX (DOX concentration 1 µM) with or without CQ (10 µM) was detected by flow cytometry at different time points. (I) The cell viability of A2780 cells treated with PBS, DOX (0.1 µM), DOX/PP (DOX concentration 0.1 µM), and DOX-CQ/PP (DOX concentration 0.1 µM and CQ concentration 10 µM) for 48 hours. (J) The cell viability of A2780 cells treated with PBS, DDP (20 µM), DDP/PP (DDP concentration 20 µM) and DDP-CQ/PP (DDP concentration 20 µM and CQ concentration 10 µM). (K) The cell viability of A2780 cells treated with PBS, PTX (4 µM), PTX/PP (PTX concentration 4 µM) and PTX-CQ/PP (PTX concentration 4 µM and CQ concentration 10 µM) for 48 hours. (L) The cell viability of A2780/DOX^R cells treated with PBS, DOX (0.1 µM), DOX/PP (DOX concentration 0.1 µM) and DOX-CQ/PP (DOX concentration 0.1 µM and CQ concentration 10 µM) for 48 hours. (M) Nude mice bearing A2780 cells treated with DOX (5 mg/kg), DOX/PP (DOX concentration 5 mg/kg) and DOX-CQ/PP (DOX concentration 5 mg/kg, CQ concentration 5 mg/kg) every 2 days for 10 days, then sacrificed the mice and obtained blood, tumor tissue and organs. HPLC was used to detect the concentration of DOX (n=6). PP was short for MPEG-PLA. ^*P<0.05; ^**P<0.01; ^***P<0.001. Abbreviations: CQ, chloroquine; DL, drug loading; DOX, doxorubicin; EE, encapsulation efficiency; MFI, mean fluorescence intensity; MPEG-PLA, methoxy poly(ethylene glycol)-poly(l-lactic acid); ns, not statistically significant; TEM, transmission electron microscope.

Figure 5

Encapsulated CQ and DOX by MPEG-PLA nanoparticles enhanced killing efficiency and reduced systemic toxicity in vivo. Notes: (A) Tumor volume of nude mice model bearing A2780 cells treated with PBS, MPEG-PLA, DDP (6 mg/kg) or DDP/PP (DDP concentration, 6 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, MPEG-PLA, DDP (6 mg/kg) or DDP/PP (DDP concentration, 5 mg/kg) in each group (n=6) (right). (B) Tumor volume of nude mice model bearing A2780 cells treated with PBS, MPEG-PLA, DOX (5 mg/kg) or DOX/PP (DOX concentration, 5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, MPEG-PLA, DOX (5 mg/kg) or DOX/PP (DOX concentration, 5 mg/kg) in each group (n=6) (right). (C) Tumor volume of nude mice model bearing A2780 cells treated with PBS, MPEG-PLA, PTX (15 mg/kg) or PTX/PP (PTX concentration, 15 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, MPEG-PLA, PTX (15 mg/kg) or PTX/PP (PTX concentration, 15 mg/kg) in each group (n=6) (right). (D) Tumor volume of nude mice model bearing A2780 cells treated with PBS, DDP (6 mg/kg) combining with CQ (5 mg/kg) or DDP-CQ/PP (DDP concentration, 6 mg/kg and CQ, 5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, DDP (6 mg/kg) combining with CQ (5 mg/kg) or DDP-CQ/PP (DDP concentration, 6 mg/kg and CQ, 5 mg/kg) in each group (n=6) (right). (E) Tumor volume of nude mice model bearing A2780 cells treated with PBS, DOX (5 mg/kg) combining with CQ (5 mg/kg) or DOX-CQ/PP (DOX concentration, 5 mg/kg and CQ, 5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, DOX (5 mg/kg) combining with CQ (5 mg/kg) or DOX-CQ/PP (DOX concentration, 5 mg/kg and CQ, 5 mg/kg) in each group (n=6) (right). (F) Tumor volume of nude mice model bearing A2780 cells treated with PBS, PTX (15 mg/kg) combining with CQ (5 mg/kg) or PTX-CQ/PP (PTX concentration, 15 mg/kg and CQ, 5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, PTX (15 mg/kg) combining with CQ (5 mg/kg) or PTX-CQ/PP (PTX concentration, 15 mg/kg and CQ, 5 mg/kg) in each group (n=6) (right). (G) Tumor volume of nude mice model bearing A2780/DOX^R cells treated with PBS, MPEG-PLA, DOX (5 mg/kg) or DOX/PP (DOX concentration, 5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, MPEG-PLA, DOX (5 mg/kg) or DOX/PP (DOX concentration, 5 mg/kg) in each group (n=6) (right). (H) The body weight of C57 mice was measured in control, DOX-CQ and DOX-CQ/PP groups (n=6). (I) The levels of alanine aminotransferase/glutamic-pyruvic transaminase (ALT/GPT) were detected in C57 mice received DOX (5 mg/kg)-CQ (5 mg/kg) or DOX-CQ/PP (n=6). (J) The levels of aspartate aminotransferase/glutamic oxalacetic transaminase (AST/GOT) were detected in in C57 mice received DOX (5 mg/kg)–(5 mg/kg) or DOX-CQ/PP. (K) The concentrations of CRE were detected in C57 mice received DOX (5 mg/kg) with or without CQ (5 mg/kg). PP was short for MPEG-PLA. ^*P<0.05; ^**P<0.01; ^***P<0.001. Abbreviations: CQ, chloroquine; DOX, doxorubicin; MPEG-PLA, methoxy poly(ethylene glycol)-poly(l-lactic acid); CRE, creatinine; ns, not statistically significant.

Figure 5

Encapsulated CQ and DOX by MPEG-PLA nanoparticles enhanced killing efficiency and reduced systemic toxicity in vivo. Notes: (A) Tumor volume of nude mice model bearing A2780 cells treated with PBS, MPEG-PLA, DDP (6 mg/kg) or DDP/PP (DDP concentration, 6 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, MPEG-PLA, DDP (6 mg/kg) or DDP/PP (DDP concentration, 5 mg/kg) in each group (n=6) (right). (B) Tumor volume of nude mice model bearing A2780 cells treated with PBS, MPEG-PLA, DOX (5 mg/kg) or DOX/PP (DOX concentration, 5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, MPEG-PLA, DOX (5 mg/kg) or DOX/PP (DOX concentration, 5 mg/kg) in each group (n=6) (right). (C) Tumor volume of nude mice model bearing A2780 cells treated with PBS, MPEG-PLA, PTX (15 mg/kg) or PTX/PP (PTX concentration, 15 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, MPEG-PLA, PTX (15 mg/kg) or PTX/PP (PTX concentration, 15 mg/kg) in each group (n=6) (right). (D) Tumor volume of nude mice model bearing A2780 cells treated with PBS, DDP (6 mg/kg) combining with CQ (5 mg/kg) or DDP-CQ/PP (DDP concentration, 6 mg/kg and CQ, 5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, DDP (6 mg/kg) combining with CQ (5 mg/kg) or DDP-CQ/PP (DDP concentration, 6 mg/kg and CQ, 5 mg/kg) in each group (n=6) (right). (E) Tumor volume of nude mice model bearing A2780 cells treated with PBS, DOX (5 mg/kg) combining with CQ (5 mg/kg) or DOX-CQ/PP (DOX concentration, 5 mg/kg and CQ, 5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, DOX (5 mg/kg) combining with CQ (5 mg/kg) or DOX-CQ/PP (DOX concentration, 5 mg/kg and CQ, 5 mg/kg) in each group (n=6) (right). (F) Tumor volume of nude mice model bearing A2780 cells treated with PBS, PTX (15 mg/kg) combining with CQ (5 mg/kg) or PTX-CQ/PP (PTX concentration, 15 mg/kg and CQ, 5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, PTX (15 mg/kg) combining with CQ (5 mg/kg) or PTX-CQ/PP (PTX concentration, 15 mg/kg and CQ, 5 mg/kg) in each group (n=6) (right). (G) Tumor volume of nude mice model bearing A2780/DOX^R cells treated with PBS, MPEG-PLA, DOX (5 mg/kg) or DOX/PP (DOX concentration, 5 mg/kg) were measured (n=6) (left). Survival time of nude mice model bearing A2780 cells treated with PBS, MPEG-PLA, DOX (5 mg/kg) or DOX/PP (DOX concentration, 5 mg/kg) in each group (n=6) (right). (H) The body weight of C57 mice was measured in control, DOX-CQ and DOX-CQ/PP groups (n=6). (I) The levels of alanine aminotransferase/glutamic-pyruvic transaminase (ALT/GPT) were detected in C57 mice received DOX (5 mg/kg)-CQ (5 mg/kg) or DOX-CQ/PP (n=6). (J) The levels of aspartate aminotransferase/glutamic oxalacetic transaminase (AST/GOT) were detected in in C57 mice received DOX (5 mg/kg)–(5 mg/kg) or DOX-CQ/PP. (K) The concentrations of CRE were detected in C57 mice received DOX (5 mg/kg) with or without CQ (5 mg/kg). PP was short for MPEG-PLA. ^*P<0.05; ^**P<0.01; ^***P<0.001. Abbreviations: CQ, chloroquine; DOX, doxorubicin; MPEG-PLA, methoxy poly(ethylene glycol)-poly(l-lactic acid); CRE, creatinine; ns, not statistically significant.