Preparation Of Gambogic Acid Nanoformulations And Their Effect And Mechanism In Tumor Treatment

Research backgroundCancer is a serious threat to human life and health due to its high morbidity and mortality.The conventional cancer treatments include surgery,radiotherapy and chemotherapy.Among them,chemotherapy,as one of the most effective treatments for cancer,has been widely used in the treatment of cancer at various stages.However,chemotherapy also has its drawbacks which cannot be ignored.In recent years,emerging combination therapy strategies have attracted people’s attention,which jointly treat cancer by using drugs or therapies with different mechanisms,in order to obtain better therapeutic effects and reduce toxic and side effects.Among many anticancer drugs under development,the natural product gambogic acid（GA）has attracted much attention due to its good anticancer activity against a variety of tumor cells.However,in the course of treatment in vivo,the defects of GA result in poor therapeutic effects and adverse reactions.For example,GA has strong hydrophobicity,it is often necessary to add excipients such as L-arginine for dissolution during intravenous injection,and the addition of excipients may generate irritation to organisms;rapid elimination of GA in vivo results in low bioavailability;non-specific distribution of GA in vivo leads to inevitable systemic side effects;in addition,there is a narrow safe treatment window during the treatment of GA,the therapeutic effect is difficult to achieve when the dose of GA is too small,and the normal tissue is relatively damaged with too large dose of GA.Therefore,how to solve the problems of GA in cancer therapy for better anti-tumor activity and less toxic and side effects has become a new research focus.In this thesis,we designed and constructed two nanoformulations of GA for solving the above problems and improving the tumor therapeutic effect of GA.First,the GA-loaded polymer micelles（GA Micelles）were designed and constructed using polymer micelles as drug delivery systems（DDS）,and the therapeutic activities in vitro and in vivo for pancreatic cancer（PC）were studied.The experimental results show that GA Micelles have achieved good therapeutic effects in cancer therapy,however,the occurrence and development of tumor are complex,heterogeneous and diverse,and the therapeutic effect of single chemotherapy needs to be further improved.In addition,there will be drug accumulation toxicity in the course of multiple chemotherapy with large dose,resulting in toxic side effects.The problems caused by chemotherapy can be solved or reduced by emerging combination therapy strategies,also,combination therapy strategies could improve the therapeutic effect of cancer,reduce the dosage of chemotherapy drugs and toxic side effects.Therefore,in subsequent experiments,we envisaged optimizing the chemotherapy based on GA and designing a combination treatment strategy with better therapeutic effect.In the experiment of anti-tumor therapy with GA Micelles,it was found that due to the efflux of tumor cells,the content of drugs ingested by tumor cells to play the role of chemotherapy was obviously reduced,and the therapeutic effect of single chemotherapy was not ideal at last.Studies have shown that P-glycoprotein（P-gp）in tumor cell membrane is a transporter for tumor cells to expel drugs from cells,which can reduce the concentration of drugs in cells and produce drug resistance.Nitric oxide（NO）,as a gas therapeutic molecule,can not only directly kill tumor cells,but also effectively inhibit the activity of P-gp,inhibit the efflux of tumor cells and reverse the occurrence of drug resistance.The combined application of NO and GA will likely enhance the chemotherapeutic effect of GA.However,NO,as a gas molecule with poor chemical stability and short half-life,it is difficult to be delivered to the tumor site.Therefore,diethylenetriamine nitric oxide nucleophilic complex（Diethylenetriamine Diazeniumdiolate,DETA NONOate）,an NO donor with special chemical properties,has become a good choice,which remains relatively stable under physiological conditions.Also,it can release NO rapidly under acidic conditions,which also accords with the characteristics of tumor microenvironment.In addition,studies have shown that GA can not only induce apoptosis and inhibit cell proliferation,but also inhibit the activity of heat shock protein90（HSP90）and reduce the heat resistance of tumor cells,thus having the potential to improve the effect of photothermal therapy（PTT）.At the same time,DETA NONOate also has thermally responsive release behavior,which can be controlled by photothermal action.IR780 iodide（IR780）was used as the photothermal agent,and it is envisaged that the introduction of PTT in combination with the above chemotherapy and gas therapy to form a synergistic enhanced combined therapy system with dual stimulation response.Therefore,it is necessary to deliver the hydrophobic natural product GA,the hydrophobic photothermal agent IR780,and the hydrophilic NO donor DETA NONOate to the tumor site together.According to the characteristics of liposome which can encapsulate hydrophobic drugs and hydrophilic drugs,a dual-stimulation-responsive multifunctional liposome was designed and constructed.It could co-load IR780,GA and DETA NONOate,endow them with stronger tumor targeting ability,and synergistic enhance chemotherapy,PTT and gas therapy for cancer treatment by inhibiting the expression level of tumor resistance proteins（P-gp,HSP90）.Research methodsIn Chapter 2,the amphiphilic polymer MPEG-P（CL-ran-TMC）was synthesized by ring-opening polymerization,and its synthesis was verified by the analysis of proton nuclear magnetic resonance（¹H-NMR）spectrum.Next,the amphiphilic polymers were used to encapsulate GA by a thin-film hydration method to obtain GA Micelles.The characterization experiments of the prepared GA Micelles were conducted,including morphology,particle size distribution andζ-potential,as well as transmission electron microscopy（TEM）.The stability investigations and the drug release behavior of GA Micelles were also investigated.Next,the anti-tumor activity and mechanism of GA Micelles were further studied at the cellular level,including cell uptake experiment,cytotoxicity experiment and apoptosis experiment.In vivo antitumor research,a subcutaneous xenograft model of human pancreatic cancer As PC-1 was established in non-obese diabetic/severe combined immunodeficiency（NOD-SCID）mice,and the tumors were treated with formulations by tail vein injection.The antitumor activity in vivo of GA Micelles was evaluated by analyzing the tumor growth curve of the mice during the treatment.In addition,the hematoxylin-eosin staining（H&E）of tumor tissue sections,the Td T-mediated d UTP nick-end labeling（TUNEL）immunofluorescence experiment and Ki-67 immunohistochemistry experiment were adopted for further verification of the anti-tumor effect of GA Micelles.At the same time,the change of mice body weight during the treatment and the complete blood count test and serum chemistry profile analysis were used to prove the potential toxicity of the application of GA Micelles in vivo.In Chapter 3,1,2-distearoyl-sn-glycero-3-phosphooethanolamine-n-[method（polyethylene glycol）]（u PA-PEG-DSPE）polymer materials were obtained by modifying urokinase plasminogen activator（u PA）polypeptide on the polyethylene glycol(Poly（ethylene glycol）（PEG）block of PEG-DSPE,and the result was verified by ¹H-NMR spectrum.Next,prescription screening was performed on a variety of phospholipid materials such as dioleoyl phosphatidylethanolamine（DOPE）,dipalmitoyl phosphatidylcholine（DPPC）and soybean lecithin(Soy lecithin,SPC₁₀₀),and multifunctional responsive liposomes（IGN Lipo）co-loaded with IR780,GA and NO donor DETA NONOate were prepared by the film hydration method and liposome extrusion method,the optimal proportion was screened out.The particle size distribution,ζ-potential,TEM and UV/Vis absorption spectrum were used to characterize IGN Lipo,and the encapsulation efficiency（EE）and drug loading（DL）of components in IGN Lipo were detected by high-performance liquid chromatography（HPLC）.Next,the double-responsive function of IGN Lipo was verified by the release behaviors of NO and GA and the morphological changes of liposomes under the double stimulation of acid（p H 5.0）and photothermal.In vitro treatment experiment of triple-negative breast cancer（TNBC）,the cellular uptake effect,the cytotoxic effect and apoptosis induction of IGN Lipo on MDA-MB-231 cells were tested.At the same time,the synergistic enhancement effect between the three treatment methods was verified,including MTT assay,rhodamine 123 efflux assay and Western Blot（WB）assay.In vivo anti-tumor treatment experiment,the TNBC subcutaneous xenografts model was established in severe combined immune（SCID）mice to verify the in vivo targeting and anti-tumor activity of IGN Lipo,and the mechanism of synergistic enhanced treatment has been further verified,including the mice imaging experiments in vivo,the mice photothermal imaging experiment,and mice tumor treatment experiment.Also,the mice body weight curve,complete blood count test and serum chemistry profile analysis,and organ H&E staining were used to reflect the potential toxicity of IGN Lipo.The H&E staining,TUNEL immunofluorescence staining,Ki-67,HSP90,and P-gp immunohistochemical staining of mice tumor tissue sections were adopted to further verify the anti-tumor effect and internal mechanism of IGN Lipo.ResultsIn Chapter 2,the synthesis of the amphiphilic polymer MPEG-P（CL-ran-TMC）was verified by the analysis of ¹H-NMR spectrum.GA Micelles with the optimal ratio（mass ratio of 30:70）were finally obtained by prescription screening.From the appearance,the results showed that GA Micelles solution presented as a yellow,clear and transparent solution with opalescence.The results of particle size distribution andζ-potential verified that GA Micelles were nanoparticles with a particle size of about44 nm and electronegativity,transmission electron microscopy（TEM）images further verified that GA Micelles appeared as a uniform spherical,and stability investigations proved that GA Micelles had remained stable for 72 h.It was found that compared with free GA,GA Micelles showed excellent sustained release effect in vitro simulated drug release experiment.In the uptake experiment of human pancreatic cancer As PC-1 cells,the cell uptake of GA Micelles was higher than that of free GA at both 2 h and 4 h,and showed significant difference at 4 h（**,P<0.01）.In the MTT experiment,it was found that GA Micelles group had similar cytotoxic effect with free GA group.At the same time,in the apoptosis induction experiment,it was also proved that the two groups had similar apoptosis induction ratios.At the same time,the cytotoxicity of MPEG-P（CL-ran-TMC）blank micelles was explored,and it was found that blank micelles had low toxicity.In vivo antitumor research,the experimental results of anti-tumor treatment in vivo showed that compared with free GA,GA Micelles could significantly inhibit tumor growth（*,P<0.05）,showing a good anti-tumor effect.Also,the results of H&E staining,TUNEL immunofluorescence experiment and Ki-67immunohistochemistry experiment of tumor tissue sections further confirmed that GA Micelles showed stronger tumor growth inhibition and apoptosis promotion than that of free GA.At last,the change of mice body weight during the treatment and complete blood count test and serum chemistry profile analysis proved that there was no significant toxic effect of GA Micelles in the in vivo anti-tumor treatment.In Chapter 3,the synthesis of uPA-PEG-DSPE was verified by the results of ¹H-NMR spectrum.Next,the liposomes mainly composed of SPC₁₀₀,cholesterol（Chol）,PEG-DSPE and u PA-PEG-DSPE,and the optimal therapeutic ratio of IGN Lipo（IR780:GA:DETA NONOate 8:3:0.3 w/w）were finally selected by prescription screening.The results of particle size distribution,ζ-potential verified that IGN Lipo were stable and uniform nanoparticles with a particle size of about 130 nm andζ-potential of about-10 m V,and the TEM images further proved that IGN Lipo were spherical liposome structure with uniform size.The results of the UV/Vis absorption spectrum demonstrated the successful encapsulation of IR780,GA and DETA NONOate by IGN Lipo.The encapsulation efficiency（EE）and Drug loading（DL）of IR780 in IGN Lipo were 98.8%and 18.2%,respectively,the EE and DL of GA were 94.8%and 6.6%,respectively,and those of DETA NONOate were 35.6%and 0.3%,respectively,as determined by HPLC.The particle size and TEM results of IGN Lipo revealed that IGN Lipo were affected by acid and photothermal effects,and the structure was damaged;the release behavior of NO and GA in IGN Lipo indicate that the release rates of NO and GA in IGN Lipo are significantly increased under dual stimulation.These results indicated that the combined action of the acidic environment in which IGN Lipo entered and contacted with the lysosomes of tumor cells and the thermal energy generated by IR780 stimulated by near-infrared light（NIR）may accelerate the release of the other two therapeutic agents.The results of cell uptake experiment showed that u PA-modified liposomes significantly enhanced the drug uptake of human TNBC MDA-MB-231 cells（***,P<0.001）.The results of MTT experiment and apoptosis experiment proved that compared with other treatment groups,IGN Lipo group had stronger cytotoxicity（***,P<0.001）and apoptosis induction（*,P<0.05）.Furthermore,the synergistic enhancement of three treatments was verified.The results of WB assay and rhodamine 123 efflux assay verified that NO could effectively inhibit P-gp activity,and the results of MTT assay showed that NO could enhance the therapeutic effect of low-dose chemotherapy with GA.Meanwhile,the results of WB assay verified that GA could down-regulate the expression level of HSP90,and the results of MTT assay showed that GA could enhance the therapeutic effect of IR780low temperature photothermal.It can be verified from the imaging experiments in vivo in mice that IGN Lipo was more focused on the tumor site,and has good tumor targeting.The experimental results of anti-tumor treatment in vivo showed that IGN Lipo had a stronger tumor therapeutic effect compared with other treatment groups（*,P<0.05）,which effectively inhibited the growth of mice tumors and exerted the anti-tumor effect of low-dose chemotherapy and low-temperature photothermal and gas therapy well.At the same time,the mice body weight curve,complete blood count test and serum chemistry profile analysis,and organ H&E staining reflected that IGN Lipo had no obvious toxic and side effects during in vivo application.The results of H&E staining,TUNEL immunofluorescence staining,Ki-67,HSP90,and P-gp immunohistochemical staining of mice tumor tissue sections further verified that IGN Lipo can amplify the therapeutic effect by inhibiting tumor resistance proteins,inhibit the growth of tumor cells better and enhance the apoptosis of tumor cells,and achieve better antitumor effect.ConclusionsIn order to solve the defects of GA in the tumor treatment process in vivo,such as strong hydrophobicity,low bioavailability,and lack of tumor targeting,we have designed and constructed two nanoformulations of GA for solving the problems,and optimized the application of GA in cancer therapy for better anti-tumor activity and less toxic and side effects.First,GA Micelles for the treatment of PC were designed and constructed,and achieved good therapeutic effects without obvious toxic and side effects to the body.In order to further improve the therapeutic effect of tumors and solve the problems such as body accumulation toxicity easily occurred during long-term chemotherapy,a synergistic enhanced treatment combining chemotherapy,PTT and gas therapy was adopted.The multifunctional liposomes（IGN Lipo）capable of co-loading IR780,GA and DETA NONOate were designed and constructed.IGN Lipo can responsively release drugs under that co-stimulation of acidic environment and photothermal in tumor cell;at the same time,IGN Lipo effectively inhibited the activity of P-gp and HSP90 tumor resistance proteins during the treatment process,amplifying low-temperature photothermal therapy and low-dose chemotherapy.In a word,IGN Lipo played the role of synergistic enhancement in the treatment of TNBC,achieved excellent therapeutic benefits,and did not cause obvious toxic side effects to the body.This design provided a novel idea and strategy for tumor treatment of GA.