MRI Detection Of Glioma Angiogenesis With Sterically Stabilized Gd-DTPA Liposomes Targeted To Endoglin

Purpose: (1) To synthesize the sterically stabilized Gd-DTPA liposomes targeted to Endoglin and to evaluate its rudimental characteristics. (2) To detect glioma angiogenesis in vivo using MRI and sterically stabilized Gd-DTPA liposomes targeted to Endoglin and to establish a method in vivo to evaluate tumor angiogenesis.Materials and Methods: (1) Three steps were applied to synthesize thePDP-PEG-HEPE, which attaches antibody to the surface of liposome. Firstly,Boc-NH-PEG-HEPE was synthesized with Boc-NH-PEG-HEPE and HEPE. Thenselective deprotection of the tert-butoxycarbonyl (Boc) of Boc-NH-PEG-HEPEwas achieved by using hydrogen chloride (4M) in anhydrous dioxane solutionfor 30 min. Finally, PDP-PEG-HEPE was prepared after Boc-NH-PEG-HEPEreacted with SPDP. During the whole synthesise, TLC was done to prove theproduct.(2)Magneist solution with different concentration was confectedand its fluorescence was measured with HITACHI F-3000. The correlationbetween concentration and fluorescence was analyzed. And the stability offluorescence of magnevist was also observed. (3)IgG-SLs or Endoglin-SLswere prepared following the steps as following: Firstly, liposomes (PDP-SLs)containing HEPC, Choi, mPEG-DSPE, PDP-PEG-HEPE were parepared. Then thedithiol-bond of PDP in the PDP-SLs was reduced to - SH by dithiothreitol(DTT) and HS-SLs was thus obtained. Finally, the derivative anti-Endoglinantibody or IgG (malemidophenylbutyrate-Endoglin or IgG, MPB-Endoglin orMPB-IgG) was attached to the surface of PDP-SLs. The liposome diameter andsize distribution were determined by Nicomp 380 Particle Sizing System.Concentration of phospholipids was deterimined with molybdenum blue method.I25I radiolabelling tests was carried out to determined the coupling ratioof antibody to liposomes and the antibody density on the surface ofliposomes. ELISA studies was performed to confirmed theimmunoreacitivities of IgG-SLs and Endoglin-SLs. (4)Sterically stabilizedGd-labelled immunoliposomes targeted to Endoglin (Endoglin-Gd-SLs) wasprepared by the thin film hydration method and the freeze-thawing method.The shape of Endoglin-Gd-SLs was observed by transmission electron microscope. The liposome diameter and size distribution were determined by Nicomp 380 Particle Sizing System. Gd-DTPA trapping efficency was determined by fluorescent method and the Gd-DTPA concentration in the liposome was also calculated. The effect of concentration, value of pH of Gd-DTPA solution, ratio of HEPC/Chol/mPEG-DSPE/PDP-PEG-HEPE, diameter of liposome on the Gd-DTPA trapping efficency and the Gd-DTPA concentration in the liposome were analyzed. The physical stability of liposome and the leakage ratio of Gd-DTPA were also observed. (5)Forty Fischer 344 rats implanted with F98 gliomas were randomized into four treatment groups and received either: (a)sterically stabilized Gd-labelled liposomes targeted to Endoglin (Endoglin-Gd-SLs);(b)sterically stabilized Gd-labelled liposomes covalently coupled with IgG (IgG-Gd-SLs) ;(c)sterically stabilized Gd-labelled liposomes (Gd-SLs); or (d)Gd-DTPA. MRI scan were performed before, immediately after, and 5, 30, 60, 120min after intravenous injection. MRI enhancement features of each group were observed. To quantify image enhancement over time, CNR at each time point and the relative intensity at time point of maximal enhancement were calculated. After imaging, tumors were resected for a -SMA, VEGF immunohistochemical staining and Endoglin immunofluorescence. E-MVD(microvessel density based on Endoglin immunofluorescence), S-MVD(microvessel density based on a -SMA immunofluorescence), T-MVD (T-MVD= E-MVD+S-MVD) were calculated. VEGF labeling index (VEGF LI) were measured to reflect VEGF expression. The correlation analysis between the relative signal intensity at time point of maximal enhancement and E-MVD, S-MVD, T-MVD and VEGF LI were performed. Results: (1)The results of TLC suggested that PDP-PEG-HEPE was successfully synthesized. (2) The optional conditions for quantitative determination of magnevist were as following: The excitated wavelength was 275nm, emission wavelength was 312nm. The fluorescence of magnevist was stable when pH value was 6.0-8.0, and it positively correlated well with concentration of magnevist when the concentration was 5× 10^-5 mol/L-1mol/L. (3) Mean diameter of IgG-SLs and Endoglin-SLs was 143nm, the size distribution was homogeneous. Mean diameter of IgG-SLs and Endoglin-SLs was slightly larger than that of SLs and PDP-SLs. The concentration of phospholipids was 1. 5725mmol/L. The results of 125I radiolabeling tests showed the the couplingratio of antibody to liposomes was 54. 28%, the antibody density on the surface of liposomes was 78.13ug Ab/umol lipid, which met the needs of experiment. ELISA studies showed the immunoreacitivities of IgG-SLs and Endoglin-SLs was about 44.3% and 51%,respectively. (4)The liposome was round in shape, lipid-membrane and fingerprint-like structure were found. The trapping efficiency and concentration of Gd-DTPA was the highest when the pH value of Gd-DTPA was 6. 0-8. 0, but there was no difference between group pH6. 0 and group pH8.0, and there was also no difference between group pH6.0, pH8. 0 and group pH10.0. While there was significant difference between group pH6. 0, pH8. 0, pHlO. 0 and group 2.0, 4.0 (p<0. 05, 0.01) . The trapping efficiency and concentration of Gd-DTPA in the liposomes was 21. 8% , 0. 067umol/umol respectively, when the concentration of Gd-DTPA solution was 0.67mol/L. While these was 13.4 % , 0.022 umol/umol respectively, when concentration of Gd-DTPA solution was 0. 25mol/L. There was statistical significance between group 0. 5mol/L, 0. 67mol/L, 0. 72mol/L and group 0. 25mol/L (p<0. 05> 0.01) .As for the effect of the size of liposomes on the trapping efficiency and concentration of Gd-DTPA was concerned. The larger the size was, the more The trapping efficiency and concentration of Gd-DTPA was. Statistical significance was found between group lOOnm, 150nm, 200nm and group 50nm (p<0. 05> 0.01) , but there is no difference among the former three groups. Four groups (group A, B, C, D) with different ratio of HEPC/Chol/mPEG-DSPE/PDP-PEG-HEPE were analyzed. The ratio was 3:2:0.1:0.02,4:1:0.3:0.05,2:1:0.1:0.01 , 11:8:0.1:0.01 respectively. The trapping efficiency was statistical different between the former two groups and the latter two groups.As for the concentration of Gd-DTPA in the liposomes was concerned, Statistical difference was found between group A and group C, D (p<0.05), There was also difference between group B and group D (p<0. 05) . While there was no difference between group B and C, between group C and D. The stability of IgG-Gd-SLs stay between Gd-CLs and Gd-SLs during restoration. When IgG -Gd-SLs were suspended in either PBS or 1% human plasma for 10 hours, the leakage ratio of Gd-DTPA was 19. 8%, 11.2%, respectively. (5) Enhancement differences were observed among the four groups. Slight enhancement was found after 5 min and singnal intensity of enhancement region increased steadily within 1h in group Endoglin-Gd-SLs, group IgG-Gd-SLs and group Gd-SLs. Difference of the threegroups lay in enhancement at 2h point. A more significant signal intensity increase was detected after 2h in group Endoglin-Gd-SLs. In group IgG-Gd-SLs, signal intensity of enhancement after 2h was less than that after 1h, but was more than that at baseline. While in group Gd-SLs, signal intensity of tumor almost return to baseline after 2h. In group Gd-DTPA, contrast enhancement was detected soon after Gd-DTPA injection and maximal enhancement was found after 5min. While singnal intensity decreased after 30min and returned to baseline after lh. In addition, the size of enhancement region over time was difference among the four groups. In group Endoglin-Gd-SLs, enhancement region was confined and did not expanded within 2h. While distended enhancement region from periphery to central within lh was found in group IgG-Gd-SLs and group Gd-SLs. In group Gd-DTPA, homogeneous enhancement was found soon after injection.Correlation analysis showed the relative intensity at time point of maximal enhancement correlated well with E-MVD, VEGF LI in the four groups (p<0.05, 0.01), it did not have correlation with S-MVD, T-MVD in group Endoglin-Gd-SLs(p>0.05) , while in other three groups, it correlated well with S-MVD, T-MVD (p<0. 05, 0.01).Conclusions:(1)The optional conditions for quantitative determination of magnevist were as following: The excitated wavelength was 275nm, emission wavelength was 312nm. The fluorescence of magnevist was stable when pH value was 6.0-8.0, and it positively correlated well with concentration of magnevist when the concentration was 5×10^-5mol/L-lmol/L. Fluorescent method is a simple and efficient method to quantitative determination of magnevist concentration. (2)The linkage method of antibodies to the terminal of PEG of liposome surface via thioether bond could retain their immunoreacitivities. The decrease of immunoreacitivities corrlate with two factors as following: the SMPB derivation of antibody, the steric hinderance effect of liposome. (3)Protocol of trapping Gd-DTPA was as following: 0. 67mol/L Gd-DTPA solution with pH 6. 0-8. 0, the 3/3/0. 1/0. 02 (mole ratio) blend of HEPC, Choline, mPEG-DSPE and PDP-PEG-HEPE, the mean diameter should be 100nm-150nm in order to meet the experiment. The stability ofIgG-Gd-SLs stay between Gd-CLs and Gd-SLs during restoration. (4) Enhancement of Endoglin-Gd-SL correlate well with glioma neovasculature.It is feasible and efficient to detect tumor angiogenesis in vivo using...