The Accurate Diagnosis And Treatment Of Rat Glioma By Ultrasound-targeted Microbubble Destruction Technique Combined With L19-(Q-D-lip)

Research background:Glioblastoma(GBM)is the most common primary malignant tumor in the central nervous system,accounting for 40%-50%of brain tumors.At present,the main problems of GBM treatment are as follows:①the precise location in the surgery;②targeted therapy of drug;③accurate evaluation of therapeutic effect and prognosis.Quantum dots(QDs),as a new kind of inorganic nanometer fluorescent labeling probe,has the advantages of strong fluorescence signal and good stability,and can realize biological polychromatic imaging.Lipo-soluble QDs has poor bio-compatibility and it is difficult to be excreted and easily be accumulated in the body.Water-soluble QDs has high bio-compatibility,but their core and shell structures are easily affected by the environment,easy to be oxidized,and generate cytotoxicity,which limits their application in organisms.In our research group,water-soluble QDs contained in liposomes with independent intellectual property rights were applied to improve physical stability and to retain the fluorescence characteristics of QDs,thus reducing biological toxicity.Recent studies have found that L19 and its derivatives can specifically bind to extracellular fibronectin(FN)in extra-cellular matrix components to achieve GBM targeted tracer and drug delivery.Based on the existing literatures,CA(short peptide of amino acid,key fragment of L19 antibody binding EDB-FN)was selected as the targeted ligand,which was modified on drug excipients PEG-DSPE to prepare a new liposome containing water-soluble QDs,and the anti-tumor drug of docetaxel(DTX)was loaded[abbr.CA-(Q-D-lip)].After administration of the tail vein,the rat brain was positioned by ultrasound to open BBB reversibly using the ultrasound-targeted microbubbles destruction technology(UTMD),delivering QDs nanoparticles through BBB into the brain,and CA-(Q-D-lip)will effectively completed the combination of"QDs efficient tracer technology","tumor-targeted nanoparticles preparation technology" and" BBB opened by UTMD mediated technology",give full play to the new material,new preparation and ultrasound-mediated means such as technical superiority,realize the effective,targeted therapy and specific tracer of GBM.MRI routine and enhanced MRI scan can show the characteristics of tumor enhancement,distinguish the features of tumor tissue,surrounding edema area and normal tissue,as well as the characteristics of tumor blood supply,liquefaction,necrosis,bleeding and calcification.MRI perfusion imaging(PWI)is commonly used to evaluate the characteristics of micro-circulation distribution and reflects the changes of blood perfusion in brain tissues and lesions according to the time-signal intensity curve(TIC).MRI diffusion-weighted imaging(DWI)is a noninvasive functional imaging technique developed in recent years.The diffusion capacity of water molecules is quantitatively analyzed according to the apparent diffusion coefficient(ADC).However,ADC cannot fully reflect the diffusion of water molecules in living tissues.Intra-voxel incoherent motion(IVIM)is an extension on the basis of DWI,avoiding the confusion of water molecule diffusion and perfusion related diffusion in ADC.The theoretical basis of single exponential model ADC and double exponential model IVIM is that the diffusion of water molecules conforms to gaussion distribution.However,when the b value is higher,the diffusion of water molecules is non-gaussian distribution.Diffusion kurtosis imaging(DKI)is an MRI technique based on the non-gaussian distribution model of water molecule diffusion,which is used to evaluate the heterogeneity and limitation in diffusion of water molecule.At present,MRI and QDs fluorescence imaging have become one of the imaging examination methods for early diagnosis and therapeutic effect monitoring of tumor.From the microscopic level(water molecules diffusion,micro-circulation perfusion and heterogeneity of tumor,etc.),it can comprehensively reflect the texture of GBM cells and histopathological classification;from the macroscopic level(tumor volume change,strengthening degree and mode,accumulation quantity and area in fluorescence tracer,etc.),and it can evaluate specific tracer and drug-targeted therapy in rat C6 glioma.Objective:Combining with MRI multi-sequence examination,fluorescent tracing technique and histo-pathological results,to evaluate the feasibility of using ultrasound-targeted microbubbles destruction technique(UTMD)combined with L19-(Q-D-lip)for accurate diagnosis and treatment of rat C6 Glioma.Materials and methods:(1)QDs-DTX-lip preparation with FN active targeted modification:FN was selected as the target site,and the FN targeted peptide CA was modified on PEG-DSPE of the pharmaceutical necessities.The liposome with independent intellectual property rights was used to encapsulate water-soluble QDs,and anti-tumor drugs DTX were loaded,and CA-(Q-D-lip)was obtained by ultrasound-dispersing liposome.The liposomes without FN targeted peptide CA or water-soluble QDs were prepared by the same method.(2)Evaluation of preparation characterization:CA-(Q-D-lip)particle size and δpotential,fluorescence features and encapsulation rate of QDs were tested.The encapsulation rate of DTX and liposome stability of chemotherapy drugs was detected;The infrared spectrometer confirmed whether the CA was successfully modified to the liposome.(3)Modeling of C6 glioma in rats:①fixation and incision selection;②drilling skull;③innoculation of C6 glioma cells.(4)Grouping and drug administration:80 males SD rats with C6 Glioma models were randomly divided into five groups:control group(n=16),QDs solution integrated with UTMD(Q-D+UTMD,n=16),Blank liposome integrated with UTMD(Blank lip+UTMD,n=16),QDs liposomes integrated with UTMD(Q-D-lip++UTMD,n=16)and QDs liposomes modified by FN targeted peptide and integrated with UTMD[CA-(Q-D-lip)+UTMD,n=16].Every group was intervened at fixed time,and then was scanned with routine,dynamic enhancement and multiple b-value diffusion sequences on day 7,day 14 and day 28 of rat modeling.(5)Imaging examination and evaluation:1.Calculate the volume of GBM after MRI enhancement,V=(W×L×H×π×4/3)×1/8;2.DCE-MRI was performed using Ax LAVA-xvDYN protocol,with a slice thickness of 2mm,120 circles/slice slab,Flip Angle 12°,NEX= 1,FOV 80mmx 80mm,Matrix 160x160,No.4.5 pediatric scalp needle through the rat tail vein for manual injection of gadolinium pentamine(GD-DTPA)within 4 seconds,with a dose of 0.4mmol/kg body weight.MRI scan was performed immediately after contrast injection and the total scan time was 6 minutes and 2 seconds.Depending on TIC characteristics,the early relative signal enhancement rate(ARSER)and the time to peak enhancement(TTP)was calculated.3.Multiple b-value diffusion-weighted imaging:Adopting the protocol of OAx eDWI MB=7 Head scheme,we underwent diffusion-weithed imaging(DWI).After the second orientation sequence,conventional whole brain spin-echo echo planar imaging(SE-EPI)sequences were used to perform diffusion weighted imaging of whole brain transection in SD rats.The imaging parameters were as follows:TE minimum,TR 2000ms,slice thickness 3mm,slice space 0mm,matrix 160x192,FOV 80mmx 100mm,scanning time 4 minutes and 16 seconds,NEX=6;Diffusion sensitive factor(b)values were 0,30,100,200,400,1000,1500s/mm2,respectively.The value of different mathematical diffusion model based on Matlab and C language was performed on image post-processing.B value--signal intensity parameter graphs and results were obtained through Mitalytics software.The images were fitted with different mathematical diffusion models,and the ADC values of the single exponential model,D,D*and f values of IVIM model,Dapp and Kapp values of DKI model were obtained.① ADC-single exponential model:ADC = ln(S0/S1)/(b1-b0),with b values of 30,100,200,400 and 1000s/mm2.② IVIM double exponential model:Sb=S0[(1-f）)×e(-bD)+f×e[-b(D+D*)],where f is perfusion fraction,D is diffusion coefficient(ADC-slow)and D*is pseudodiffusion coefficient(ADC-fast),with b values of 30,100,200,400 and 1000s/mm2.③ DKI model:Si/S0=exp{-bi×Dapp+bi2×Dapp2xKapp/6｝.Kapp is average kurtosis,no unit.Dapp value refers to the ADC value corrected by non-gaussian distribution,and b value is 30,100,200,400,1000 and 1500s/mm2.The diagnostic efficacy of different mathematical diffusion models(ADC-single exponential model,IVIM-double exponential model and DKI model)was evaluated,the best and most stable diffusion model of the three models was preliminarily determined.(6)Fluorescence evaluation:Fluorescence images were collected by the optical imaging system of small animals to observe fluorescence aggregation degree,targeting property of targeted peptide liposome,and fluorescence area and tumor volume were measured.(7)General observation and evaluation of median survival time:In the same period,another 40 healthy adult male SD rats were randomly divided into 5 groups for observation,8 for each group.The modeling was conducted according to the above methods,and the drug was regularly given,which was only used for the survival evaluation during the treatment period,and was not involved in imaging examination and histopathological experiments.(8)Histopathological and drug toxicity evaluation:Twenty-eight days after treatment,two rats were randomly selected from each group after MRI and fluorescence imaging,and brain tissue and other major organs were removed.HE staining was used to observe the toxicity and histopathological changes of the main organs(heart,liver,spleen,lung and kidney)and tumor tissue,and immunohistochemistry was used to observe the expression levels of VEGF,CD31 and Caspase-3 of GBM.(9)Statistical analysis:Statistical analysis was performed using IBM SPSS(statistical product and service solutions,SPSS 19.0)software package.All parameters were first detected by using the Kolmogorov-Smirnov test for gaussian distribution.The data according with gaussion distribution is used x ±SD,not according with gaussion distribution is used the median value.The statistical methods of the data visualization part such as histogram and line graph with error line and the difference between groups was implemented through R language.Two independent samples t test was used in the volume changes,TIC difference,ARSER and TTP comparison,and difference analysis of multiple parameters(ADC,D,D*,f Dapp and Kapp)in multiple b values diffusion-weighted imaging in C6 glioma between the Control group and the different treatment groups.When variance is not equal,approximate t test of Welch method is adopted(considering sample size and variance factors).P values<0.5 were considered to indicate statistical significance.The signifinance level is as follows:*p<0.05,**P<0.01,***p<0.001.Results:(1)CA-(Q-D-lip)was successfully prepared,CA was successfully modified on the liposome by the sulfhydryl bond.The particles and the electric potential of the Q-D-lip were appropriate,the encapsulation rate was in accordance with the standard,and the fluorescence properties of QDs-lip were stable.(2)The ARSER was over 60%.After treatment,the size in the group of CA-(Q-D-lip)+UTMD was apparently decreased,and the slope of ARSER was the smallest,the effect was better than the other four groups.(3)The values of ADC,D and Dapp were going up with increasing time in the group of CA-(Q-D-lip)+UTMD,which were opposite in the other four groups on the whole;The values of Kapp,D*and f were going down with increasing time,which were opposite in the other four groups on the whole;Among the parameters,the diagnostic efficacy of ADC value was much higher and the value was relatively stable.(4)The QDs quantity distribution(day 7)in the group of CA-(Q-D-lip)+UTMD was the highest,the survival time was the longest,and there was good bio-safety in the body.The expression of VEGF and CD31 in each group was compared as follows:Control group>B+lip+UTMD group>Q-D +UTMD group>Q-D-lip +UTMD group>CA-(Q-D-lip)+UTMD group,but the expression of Caspase-3 was opposite.Conclusion:This study can effectively integrate the tracer technology of QDs of CA-(Q-D-lip)+UTMD,the preparation technology of tumor-targeted nanoparticles and with the mediated technology of BBB reversibly opened by UTMD,making the most advantages of MRI multi-sequences and QDs fluorescence imaging,cooperation and co-compensation.It can provide more comprehensive and accurate imaging information for the precise location and effective treatment of GBM.