Study On The Interaction Mechanism Of Pegylated Salvianolic Acid B Liposomes And Pegylated Docetaxel Liposomes Based On Tumor Microenvironment

The clinical treatment failure of nanoparticles and the formation of immunosuppressive TME are closely related to the high degree of fibrosis and abundant collagen in desmoplastic breast cancer.TAFs are abundant stromal cells in TME.TAFs not only increase IFP by secreting ECM to reduce the penetration of nanoparticles in tumors,but also contribute to the immunosuppressive microenvironment.Therefore,remodeling TME by inhibiting the activation of TAFs is an effective strategy to improve the anti-tumor efficiency of nanoparticles.Objective:The project was to research whether PEG-SAB-Lip can inhibit TAFs activation by interfering with TGF-?1/Smad signaling.PEG-SAB-Lip promote the penetration of nanoparticles in tumors and enhance tumor immune response by remodeling TME.PEG-SAB-Lip improve the chemotherapeutic effect of PEG-DTX-Lip.Methods:Firstly,PEG-SAB-Lip were prepared by ethanol injection,and the optimal prescription was screened through single factor investigation and orthogonal experiment.Particle size,zeta potential,EE%,drug loading,morphology,storage stability,and in vitro release were used to characterize PEG-SAB-Lip.Secondly,the effects of SAB and PEG-SAB-Lip on the TGF-?1/Smad signaling in NIH3T3 cells activated by TGF-?1were studied in vitro.The mixed 3D spherical model was used to study whether SAB and PEG-SAB-Lip can enhance the penetration and anti-tumor effects of nanoparticles in the 3D spherical model.Then,100?L of PBS containing 4T1 cells(5×10⁵)and NIH3T3 cells(2.5×10⁵)and matrigel was subcutaneously injected into the right side of the mouse at a ratio of 1:1(v/v)to establish breast cancer model.Immunofluorescence and masson's trichrome staining were used to verify the desmoplastic tumor model.To study the regulation of tumor fibrosis microenvironment and immune microenvironment,as well as the influence of nanoparticles penetration in breast cancer after repeated three times tail vein injection of SAB and PEG-SAB-Lip.Western blot experiments were used to research the changes in the expression of TGF-?1/Smad signal-related proteins(TGF-?1,Smad2,Smad3,pSmad2,pSmad3,Smad4,and?-SMA)in tumor tissues after the action of SAB and PEG-SAB-Lip.Immunofluorescence experiment and masson's trichrome staining were used to study the effects of SAB and PEG-SAB-Lip on?-SMA and collagen deposition in tumor areas.To study the impact of SAB and PEG-SAB-Lip on the tumor immune microenvironment,immunofluorescence and RT-qPCR experiments were used to investigate the changes in the expression of immune cell populations,cytokines,and chemokines in the tumor area.PEGylated liposomes loaded with the fluorescent dye DiD(PEG-DiD-Lip)were used as model nanoparticles to study whether SAB and PEG-SAB-Lip pretreatment can increase the penetration and distribution of nanoparticles in tumors.Finally,we investigated whether the combined use of PEG-SAB-Lip and PEG-DTX-Lip can increase the anti-tumor efficiency of PEG-DTX-Lip in tumor models.Among them,the trend of tumor volume change,tumor weight,and tumor tissue cell apoptosis were used to evaluate the anti-tumor efficiency of PEG-SAB-Lip and PEG-DTX-Lip in combination.H&E staining,mouse body weight changes,and liver and kidney function indexes were used for safety assessment.Results:1 Characterization and quality evaluation of PEG-SAB-LipThe particle size,zeta potential,EE%and drug loading of PEG-SAB-Lip prepared by the best prescription and best process were 166.2±13.35 nm,-37.8±2.51 m V,82.04±2.91%,and 17.21±0.61%,respectively.Transmission electron microscopy results showed that PEG-SAB-Lip were spherical structure with smooth surface.In addition,PEG-SAB-Lip were relatively stable after 15 days of storage at 4°C,and there was no obvious drug leakage.In vitro release studies have shown that PEG-SAB-Lip can prolong the release time of SAB.2 SAB and PEG-SAB-Lip enhance the penetration and anti-tumor effect of nanoparticles in three-dimensional multicellular tumor spheres2.1 SAB and PEG-SAB-Lip inhibit TAFs activationThe expression of?-SMA in NIH3T3 cells was increased after activation of TGF-?1.SAB and PEG-SAB-Lip reversed this increase in a concentration-dependent manner.After confirming that SAB and PEG-SAB-Lip have the function of inactivating TAFs,the potential mechanism was further explored.The results of western blot showed that after low,medium,and high concentrations of SAB and PEG-SAB-Lip acted on activated NIH3T3 cells,the expression of TGF-?1/Smad signal-related proteins was significantly reduced.2.2 SAB and PEG-SAB-Lip enhance the penetration and cytotoxicity of nanoparticles in three-dimensional multicellular tumor spheresFirstly,the culture plan of three-dimensional multicellular tumor spheroids was screened.When the total number of NIH3T3 cells and 4T1 cells was 15000 and the culture time was 3 days,the globularity was better.After different concentrations of SAB and PEG-SAB-Lip act on the three-dimensional multicellular tumor spheroid,PEG-DiD-Lip can penetrate the sphere.Also,as the concentration of SAB and PEG-SAB-Lip increased,the penetration of PEG-DiD-Lip into the sphere increased.Then,we further researched whether SAB or PEG-SAB-Lip pretreatment can increase the cytotoxicity of PEG-DTX-Lip to multicellular tumor spheroid.The results showed that the IC₅₀value of PEG-DTX-Lip alone was 111.97?M,while the IC₅₀value of SAB and PEG-DTX-Lip was significantly reduced to 16.54?M after combined use.The IC₅₀value of PEG-SAB-Lip and PEG-DTX-Lip in combination was 11.95?M.This result indicated that SAB and PEG-SAB-Lip can significantly increase the cytotoxicity of PEG-DTX-Lip in multicellular tumor spheroid.3 PEG-SAB-Lip remodel the TME3.1 PEG-SAB-Lip improved the tumor fibrosis microenvironmentTo study whether PEG-SAB-Lip can inhibit TAFs activation,immunofluorescence staining was used to observe the expression of TAFs activation marker?-SMA in tumor tissues.The results showed that the expression of?-SMA in the tumor area was significantly down-regulated after SAB and PEG-SAB-Lip pretreatment,and the effect of PEG-SAB-Lip was stronger than SAB.The results of masson's tricolor staining showed that the collagen deposition in the normal saline(NS)and PEGylated blank liposomes(PEG-B-Lip)groups was severe,while the collagen in the SAB and PEG-SAB-Lip groups significantly reduced(P<0.05,P<0.01).Besides,PEG-SAB-Lip has a stronger regulatory effect on collagen deposition than SAB(P<0.05).Since most TAFs differentiated from resident fibroblasts in response to TGF-?1,the expression of TGF-?1 and downstream signaling proteins was investigated.PEG-SAB-Lip can significantly reduce the expression of?-SMA,TGF-?1,Smad2,Smad3,p Smad2,p Smad3,and Smad4(compared with the NS group,P<0.05 or P<0.01).SAB can significantly down-regulate the protein levels of TGF-?1,Smad3,p Smad2 and p Smad3(compared with the NS group,P<0.05 or P<0.01).Notably,PEG-SAB-Lip has a stronger regulatory effect on TGF-?1 signaling than SAB.3.2 PEG-SAB-Lip improved tumor immune microenvironmentRT-qPCR was used to study the effects of PEG-SAB-Lip treatment on Th1 cytokines(including IFN-?,TNF-?,IL-12?,and IL-2),Th2 cytokines(including IL-4,IL-6,IL-10,and IL-13),inflammatory factors(IL-1?),and chemokines(CCL2,CCL5,CXCL9,CXCL10,CXCL12,and CXCL13)in the tumor area.The results showed that the expression of Th1 cytokine IL-2 increased after SAB pretreatment(P<0.05),while the expression of Th2 cytokine IL-4 decreased(P<0.001).The expression of other cytokines IFN-?,TNF-?,IL-12?,IL-6,IL-10,and IL-13 has no effect.After the action of PEG-SAB-Lip,the expression of Th1 cytokines IFN-?,TNF-?,IL-12?,and IL-2 in the tumor area increased,while the expression of Th2 cytokines IL-4,IL-6,and IL-10decreased compared to the NS group.In addition,the expression of inflammatory factors IL-1?,chemokines CCL5 and CXCL13 in the PEG-SAB-Lip treatment group decreased,and the expression of CXCL9 and CXCL10 increased.Compared with the NS group,PEG-SAB-Lip had no obvious effect on the expression of CCL2 and CXCL12.Immunofluorescence staining analyzed the changes of the immune cell population in the tumor area after each treatment.The results showed that after SAB treatment,CD4⁺T cells were recruited in the tumor area but had no effect on CD8⁺T cells.The infiltration of CD8⁺and CD4⁺T cells into tumor tissues increased after PEG-SAB-Lip treatment.Compared with the NS group,MDSCs and Tregs in the free SAB or PEG-SAB-Lip treatment group were significantly reduced(P<0.01 or P<0.001).SAB or PEG-SAB-Lip treatment can increase M1 macrophages and decrease M2macrophages in tumors.3.3 PEG-SAB-Lip enhanced the distribution and penetration of PEG-DiD-Lip in tumorsPEG-DiD-Lip were used as model nanoparticles to research whether PEG-SAB-Lip can enhance the distribution and penetration of nanoparticles in tumors.The results showed that after SAB and PEG-SAB-Lip pretreatment,the distribution of PEG-DiD-Lip increased significantly in the tumor area,indicating that SAB and PEG-SAB-Lip pretreatment enhanced the distribution of subsequently injected nanoparticles in the tumor.In addition,fluorescence microscopy observed the penetration of PEG-DiD-Lip in the tumor.The results showed that the PEG-DiD-Lip in the tumor pretreated with SAB or PEG-SAB-Lip penetrated into the area away from the blood vessels,indicating that the penetration of PEG-DiD-Lip in the tumor was enhanced.More importantly,compared with the SAB group,the penetration distance of PEG-DiD-Lip in the PEG-SAB-Lip group was longer.Therefore,PEG-SAB-Lip were chosen for the following research.4 PEG-SAB-Lip enhanced the anti-tumor efficiency of PEG-DTX-Lip in desmoplastic breast tumor models4.1 The effect of PEG-SAB-Lip on the anti-tumor efficiency of PEG-DTX-LipA sequential treatment plan was used to study the inhibitory effect of PEG-SAB-Lip and PEG-DTX-Lip on tumor growth.The results showed that compared with NS treatment alone,PEG-DTX-Lip treatment alone produced weaker tumor growth inhibitory effects,while PEG-SAB-Lip treatment alone did not find significant tumor growth inhibitory effects.As expected,the combination therapy of PEG-SAB-Lip and PEG-DTX-Lip can significantly inhibit tumor growth,which indicated that PEG-SAB-Lip enhanced the anti-tumor effect of PEG-DTX-Lip.The results of TUNEL staining showed that when PEG-DTX-Lip was used alone,tumor cells showed obvious apoptosis(P<0.05).However,when PEG-SAB-Lip and PEG-DTX-Lip were used in combination,obvious cell apoptosis appeared in the tumor area(P<0.001).Furthermore,compared with the single use of PEG-SAB-Lip or PEG-DTX-Lip,the combined use of PEG-SAB-Lip and PEG-DTX-Lip caused more significant tumor cell apoptosis(P<0.05,P<0.001).This result indicated that the combined use of PEG-SAB-Lip and PEG-DTX-Lip can enhance the anti-tumor efficiency of PEG-DTX-Lip.4.2 The effect of PEG-SAB-Lip and PEG-DTX-Lip combination therapy on TME The results of immunofluorescence showed that the expression of?-SMA decreased in tumors treated with single PEG-SAB-Lip or PEG-SAB-Lip and PEG-DTX-Lip combined,indicating that the TAFs in the tumor area were reduced and the fibrotic microenvironment was improved.The results of masson's tricolor staining showed that the collagen in the tumor tissue was reduced after PEG-SAB-Lip treatment alone or the combined use of PEG-SAB-Lip and PEG-DTX-Lip.However,there was no significant change in collagen in tumor tissues after PEG-DTX-Lip treatment alone.These suggested that the combined use of PEG-SAB-Lip and PEG-DTX-Lip can reduce tumor tissue collagen deposition,and this effect was produced by PEG-SAB-Lip.Both PEG-SAB-Lip monotherapy or PEG-SAB-Lip and PEG-DTX-Lip combination therapy resulted in a significant decrease in the expression of TGF-?1 and its downstream related proteins(P<0.05 or P<0.01).After PEG-SAB-Lip and PEG-DTX-Lip combined treatment,the level of Th1 cytokines increased,and the expression of CCL5,CXCL12and CXCL13 decreased.Moreover,CD4⁺,CD8⁺T cells,and M1 macrophages increased,while M2 macrophages,MDSCs,and Tregs decreased in tumors treated with PEG-SAB-Lip and PEG-DTX-Lip.4.3 Safety assessment of PEG-SAB-Lip and PEG-DTX-Lip combination therapyDuring the treatment,the weight of the mice did not decrease significantly.The levels of AST,ALT,BUN,and Cre in plasma were within normal values,indicating that each treatment group had no damage to liver and kidney function.H&E staining results showed that no obvious damage was observed in the heart,liver,spleen,lung and kidney tissues after treatment,indicating that each treatment group had no obvious toxicity to other tissues.Conclusions1.The results showed that PEG-SAB-Lip can inhibit TAFs activation by interfering with TGF-?1/Smad signaling in vitro.This result caused the increased penetration of PEG-DiD-Lip in three-dimensional multicellular tumor spherical,and increased the cytotoxicity of PEG-DTX-Lip to three-dimensional multicellular tumor spherical.2.Three repeated injections of PEG-SAB-Lip in vivo can inhibit TAFs activation,thereby remodeling TME and increasing the distribution and penetration of nanoparticles in tumors.Besides,PEG-SAB-L can improve the immunosuppressive microenvironment and enhance the anti-tumor efficiency of PEG-DTX-Lip.