Celastrol-polyethylene Glycol-ginsenoside Rh2 Polymeric Micelles For Synergistic Treatment Of Non-small Cell Lung Cancer

Objective Our aim is 1)to optimize the synthesis of celastrol-polyethylene glycolginsenoside Rh2 through screening the key factors,such as the starting material,reaction temperature,reaction time,and purification method;2)To optimize the preparation of tripterine-polyethylene glycol-ginsenoside Rh2 polymer micelles(drug-loaded CG-M)via screening the key factors,such as drug-loading ratio,carrier concentration,water volume and dialysis time,and investigate the main pharmaceutical parameters,such as stability,drug release and entrapment efficiency/drug loading;3)To verify the optimal mass ratio of celastrol and ginsenoside Rh2 and to explore the anti-tumor advantage of CG-M for in vitro synergistic anti-tumor efficacy;4)To investigate the pharmacokinetics,biodistribution and anti-tumor effect of CG-M,and to evaluate the advantage and potential mechanism of CGM in vivo;5)To evaluate the feasibility and effectiveness of CG-M in reversing multidrug resistance NSCLC in vivo and in vitro,and to investigate the potential mechanism of reversing tumor drug resistance.Methods Celastrol and ginsenoside Rh2 were conjugated with different PEG segments via amide and ester bonds,respectively.The effects of reaction temperature,reaction time and purification method on the reaction yield were studied by single factor investigation.The chemical structure of polymeric materials were confirmed by 1H NMR and FT-IR.The critical micelle concentration(CMC)of celastrol-polyethylene glycolginsenoside Rh2 was determined by pyrene entrapment method.Celastrol-loaded celastrolpolyethylene glycol-ginsenoside Rh2(CG-M)was prepared and optimized through the single factor method.The stability of CG-M was investigated by different pH,dilution times and serum conditions.The disassembly and drug release of blank micelles(CG-Mblank)under serum environment were investigated by water-adding method in vitro,respectively.Celastrol and ginsenoside Rh2 with different mass ratios and the two types of micelle were used to evaluate the antiproliferation against A549 non-small cell lung cancer cells.The intracellular drug was quantified by HPLC to investigate the A549 cellular uptake.The A549 cells apoptosis induction of each group was investigated by AnnexinV/7-AAD apoptosis kit.C6-labeled CG-M micelles(C6/CG-M)were prepared using C6 as a fluorescent probe.Lysosomes were stained with Lyso-TrackerRed.The intracellular distribution of C6/CG-M was observed by laser confocal microscope.The time-concentration curve and pharmacokinetic parameters of SD rats treated with CG-M,CG-Mblank and celastrol+ginsenoside Rh2 were investigated by HPLC.The accumulation of tumor and main normal tissues of SD rat treated with CG-M,CG-Mblank and celastrol+ginsenoside Rh2 groups were quantified at 12 hours post tail vein injection towards A549 tumor xenograft-bearing nude mice.Besides,A549 tumor xenograft-bearing nude mice treated with same groups were used to record the tumor volume,survival period,and body weight every day.The antiproliferation of each formulation,such as celastrol,celastrol+ginsenoside Rh2(different mass ratio),CG-M and CG-Mblank,against A549/MDR cells was detected by MTT method.The A549/MDR cellular uptake was quantified by HPLC and qualified through fluorescence labeling method.AnnexinV/7-AAD apoptosis kit was used to investigate the apoptosis induction of each formulation on A549/MDR cells.A549/MDR tumor xenograft-bearing nude mice were treated with celastrol,celastrol+ginsenoside Rh2,CG-M and CG-Mblank once every two days.During the treatments,the tumor volume,survival curve,body weight and biochemical indexes of mice were recorded every day.Results Optimal synthesis condition of celastrol-polyethylene glycol-ginsenoside Rh2 was as follows,anhydrous pNP-PEG5000-pNP was refluxed in chloroform with celastrol and ginsenoside Rh2 for 24 hours,followed by purifying with methanol under pressure after column chromatography and drying under vacuum dryer.1HNMR and FT-IR characterization showed that the characteristic peaks of celastrol-polyethylene glycolginsenoside Rh2 were clearly visible,and the signal of the chemical reaction sites of pNPPEG5000-pNP have changed accordingly,which confirm that the product is successfully synthesized.After coupling the equal molar of celastrol and ginsenoside Rh2 at both ends of PEG,the CMC of celastrol-polyethylene glycol-ginsenoside Rh2 is about 64.6 μg/mL(1×10-5M).The different proportion of the two hydrophobic segments significantly influenced the CMC value.Optimal preparation technology of CG-Mblank was as follows:carrier concentration 50 mg/mL,water volume 5 mL,and dialysis time 24 hours.The in vitro release of CG-Mblank was not affected by pH environment.～50%of celastrol and～40%of ginsenoside Rh2 were released within 24 hours under weak acid+serum condition.The particle size of CG-Mblank was～100 nm,and the zeta potential was about-23mV.CG-Mblank is resistant to pH and liquid dilution,and can be sensitively disassembled in serum.The optimal preparation parameters of CG-Mblank were as follows:drug/carrier weight ratio,2/10;carrier concentration,50 mg/mL;water volume,5 mL and dialysis time,12 hours.The particle size of CG-M was～120 nm and the zeta potential was～-22mV,which was resistant to weak acid environment and liquid dilution,but sensitive disassembly to serum.Celastrol and ginsenoside Rh2 had obvious synergistic anti-tumor effect when the mass ratio was set as 2/5.CG-M with such a mass ratio could significantly inhibit the proliferation against A549 cells.The cellular uptake of CG-M was significantly higher than that of CG-Mblank and the control groups.After entering A549 cells,CG-M mainly distributed in lysosomes,which is helpful to disassemble CG-M and release active components.Compared with CG-Mblank,CG-M can significantly improve the cell apoptosis rate.Compared with the controls,CG-M showed significant advantages in in vivo circulation time,area under the curve and plasma peak concentration.The tumoral distribution of CG-M group was significantly higher than that of CG-Mblank group and other controls.CG-M could significantly inhibited the tumor growth,prolonged the survival time,meanwhile,such treatment presented the better safety.CG-M and CG-Mblank could both effectively inhibit the growth of A549/MDR cells,increase the cellular uptake and promote the apoptosis induction.These mechanisms might be associated with ginsenoside Rh2-based interference with P-gp ATP enzyme activity and micelle-based improvement of internalization.Both CG-M and CG-Mblank can significantly retard the growth of A549/MDR xenograft tumors and prolong the survival period of A549/MDR tumor xenograft-bearing nude mice.The mechanisms might be related to the antiproliferation of intra tumoral cells and the promotion of cell apoptosis.CG-M and CGMblank treatments did not lead to significant weight loss or obvious abnormality in biochemical indexes.Conclusion Celastrol-polyethylene glycol-ginsenoside Rh2 is successfully preparation and characterized using different measurements.CG-M and CG-Mblank are successfully prepared and their pharmaceutical properties as well as drug release meet the requirements of the follow-up work.CG-M has significant advantages in synergistic antilung cancer cell proliferation,cellular uptake and apoptosis induction.CG-M has significant advantages in anti-tumor efficacy in vivo,and the mechanisms might be related to the improved pharmacokinetics and tumor distribution.CG-M holds the promising potential to reverse multidrug resistance of NSCLC in vivo and in vitro,and the mechanisms may be related to the interference with the function of P-gp and increase of tumor distribution.