A novel long-sustaining system of apatinib for long-term inhibition of the proliferation of hepatocellular carcinoma cells

Figures & data

Table 1 The primers used in this work

Targets	Primer sequence (5′–3′)
E-cadherin	Forward: 5′-AAGGCACGCCTGTCGAAGCA-3′ Reverse: 5′-ACGTTGTCCCGGGTGTCATCCT-3′
N-cadherin	Forward: 5′-TGCGCGTGAAGGTTTGCCAGT-3′ Reverse: 5′-TGGCGTTCTTTATCCCGGCGT-3′
Vimentin	Forward: 5′-ACCGCACACAGCAAGGCGAT-3′ Reverse: 5′-CGATTGAGGGCTCCTAGCGGTT-3′
β-actin	Forward: 5′-ACCGCACACAGCAAGGCGAT-3′ Reverse: 5′-CGATTGAGGGCTCCTAGCGGTT-3′

Table 2 The loading efficiency of apatinib formulations

Groups	Loading efficiency (mg/mL)
Solvent control	–
Control	0.03±0.02
Apa-Sol	1.93±0.16
Apa-Cyc	1.99±0.08

Notes: Data are presented as mean±SD.

Abbreviations: Apa-Cyc, apatinib–cyclodextrin inclusion complex; Apa-Sol, apatinib solution; Control, apatinib suspension.

Figure 1 Preparation of apatinib formulations.

Notes: Apatinib was 1) simply mixed with ddH₂O to form an apatinib suspension (named Control); 2) dissolved in organic solvents and diluted in ddH₂O to form an apatinib solution (named Apa-Sol); and 3) mixed with cyclodextrin to form an apatinib–cyclodextrin inclusion complex (named Apa-Cyc). (A) Apatinib formulations were scanned with a microplate reader to obtain the OD values of apatinib formulations (Control, Apa-Sol, and Apa-Cyc) measured under a series of wavelengths. Cyclodextrin solution in ddH₂O was used as a solvent control. (B) Apatinib suspension was filtered with a 0.22 µm pore diameter filter membrane. Apatinib formulations (Control, Apa-Sol, and Apa-Cyc) were extracted by ACN for LC-MS/MS examination. (C–F) The typical photographs of Solvent Control (C), Control (apatinib suspension) (D), Apa-Sol (E), and Apa-Cyc (F) by LC-MS/MS. *P<0.05.
Abbreviations: ACN, acetonitrile; Apa-Cyc, apatinib–cyclodextrin inclusion complex; Apa-Sol, apatinib solution; ddH₂O, double-distilled H₂O; LC-MS/MS, liquid chromatography mass spectrometry/mass spectrometry; MRM, multi reaction monitoring.

Figure 2 Antitumor effect of apatinib formulations on the subcutaneous growth of MHCC97-H cells.

Notes: MHCC97-H cells were injected into nude mice to form subcutaneous tumors. The mice that received solvent control PBS was the control group. The indicated doses of apatinib were orally administered or injected via the tail vein with an indicated dose of Apa-Cyc to the mice. The results are shown as (A) photographs of tumors, (B) tumor volumes of nude mice receiving orally administered apatinib, (C) tumor volumes of nude mice receiving tail vein-injected Apa-Cyc, (D) tumor weights of nude mice receiving orally administered apatinib, and (E) tumor weights of nude mice receiving tail vein-injected Apa-Cyc. *P<0.05 vs apatinib oral administration group with control group (untreated group); *P<0.05 vs apatinib injection group with control group (untreated group).
Abbreviation: Apa-Cyc, apatinib–cyclodextrin inclusion complex.

Table 3 Phase-transition temperature of Apa-Gel containing different poloxamer 407 concentrations

Poloxamer 407 (%)	Phase-transition temperature (t1/2; °C)
5.0	–
7.5	41.38±0.18
10.0	39.21±0.44
12.5	37.05±0.22
15.0	32.11±0.27
17.5	25.50±0.33
20.0	19.83±0.54

Note: Data are presented as % or mean±SD.

Figure 3 In vitro release of Apa-Gel.

Notes: Apa-Cyc was prepared to form a temperature-sensitive phase-change hydrogel (named Apa-Gel). Apa-Gel was incubated in ddH₂O at 37°C. The ddH₂O was collected at the indicated time points for LC-MS/MS experiments. (A) Results are shown as the drug-sustaining curve of Apa-Gel. (B–E) The typical photographs of Apa-Gel at (B) 0-hour time point, (C) 24-hour time point, (D) 96-hour time point, and (E) 408-hour time point.
Abbreviations: ACN, acetonitrile; Apa-Gel, a temperature-sensitive phase-change hydrogel of apatinib; ddH₂O, double-distilled H₂O; LC-MS/MS, liquid chromatograph mass spectrometry/mass spectrometry; MRM, multi reaction monitoring.

Table 4 Half-life of apatinib in HCC tissues injected with apatinib formulations

Apatinib formulations	Half-life of apatinib in HCC tissues (t1/2 value; hours)
Apa-Sol	16.55±1.66
Apa-Cyc	18.65±2.07
Apa-Gel	99.16±7.67

Notes: Data are presented as mean±SD.

Abbreviations: Apa-Cyc, apatinib–cyclodextrin inclusion complex; Apa-Sol, apatinib solution; HCC, hepatocellular carcinoma.

Figure 4 In vivo release of apatinib formulations in HCC tumor tissues.

Notes: Apa-Gel, Apa-Sol, and Apa-Cyc were injected into subcutaneous tumor tissues formed by MHCC97-H cells. At the indicated time points, tumor tissues were harvested and apatinib sustaining in tumor tissues was extracted by ACN for LC-MS/MS examination. (A–D) Apatinib sustaining in HCC subcutaneous tumor tissues injected with Apa-Gel formed by MHCC97-H cells was examined at (A) 0-hour time point, (B) 24-hour time point, (C) 96-hour time point, and (D) 408-hour time point with LC-MS/MS. (E–G) Apatinib sustaining in HCC subcutaneous tumor tissues injected with Apa-Sol formed by MHCC97-H cells was examined at (E) 0-hour time point, (F) 24-hour time point, and (G) 96-hour time point with LC-MS/MS. (H–J) Apatinib sustaining in HCC subcutaneous tumor tissues injected with Apa-Cyc formed by MHCC97-H cells was examined at (H) 0-hour time point, (J) 24-hour time point, and (I) 96-hour time point with LC-MS/MS. (K) The sustaining curve of apatinib in subcutaneous tumor tissues formed by MHCC97-H cells is shown. *P<0.05 vs Apa-Gel group with Apa-Sol group; *P<0.05 vs Apa-Gel group with Apa-Cyc group.
Abbreviations: ACN, acetonitrile; Apa-Gel, a temperature-sensitive phase-change hydrogel of apatinib; Apa-Sol, apatinib solution; Apa-Cyc, apatinib–cyclodextrin inclusion complex; LC-MS/MS, liquid chromatography mass spectrometry/mass spectometry.

Figure 5 Long-acting antitumor effect of Apa-Gel formulations on the subcutaneous growth of MHCC97-H cells.

Notes: MHCC97-H cells were injected into nude mice to form subcutaneous tumors. The control group was the mice that received intratumor injection of solvent control. Mice were administered with one intratumor injection of Apa-Sol, Apa-Cyc, or Apa-Gel. The results are shown as (A) photographs of tumors, (B) tumor volumes, and (C) tumor weights. *P<0.05 vs Apa-Gel group with control group; *P<0.05 vs Apa-Gel group with Apa-Sol group; *P<0.05 vs Apa-Gel group with Apa-Cyc group.
Abbreviations: Apa-Gel, a temperature-sensitive phase-change hydrogel of apatinib; Apa-Sol, apatinib solution; Apa-Cyc, apatinib–cyclodextrin inclusion complex.

Figure 6 Long-acting antitumor effect of Apa-Gel formulations on the EMT process of MHCC97-H cells.

Notes: MHCC97-H cells were injected into nude mice to form subcutaneous tumors. The control group was the untreated group. Mice were administered with one intratumor injection of Apa-Sol, Apa-Cyc, or Apa-Gel. Tumors were harvested for qPCR experiments. The expression of (A) E-cadherin (an epithelial indicator), (B) N-cadherin (a mesenchymal indicator), and (C) vimentin (a mesenchymal indicator) are shown. *P<0.05 vs Apa-Gel group with control group; *P<0.05 vs Apa-Gel group with Apa-Sol group; *P<0.05 vs Apa-Gel group with Apa-Cyc group.
Abbreviations: Apa-Cyc, apatinib–cyclodextrin inclusion complex; Apa-Gel, a temperature-sensitive phase-change hydrogel of apatinib; Apa-Sol, apatinib solution; EMT, epithelial–mesenchymal transition; qPCR, quantitative polymerase chain reaction.

Figure S1 Poloxamer 407 hydrogel did not inhibit subcutaneous growth of MHCC97-H in nude mice.

Notes: MHCC97-H cells were injected into nude mice to form subcutaneous tumors. The control group mice received intratumor injection of solvent control. Mice were once administered with 12.5% concentration of poloxamer 407 hydrogel. The results are shown as (A) photographs of tumors, (B) tumor volumes, and (C) tumor weights.