| FR (folate receptor), which is a well-known tumor associated antigen, exhibits limited expression in normal tissues, but is greatly over-expressed in a variety of carcinomas. Due to its high binding affinity for cell surface FR, folate conjugation allows delivery of non-specific drugs selectively into pathologic cells without causing harm to normal tissues. Liposomes are versatile tools used in drugs and gene delivery. Therefore, folate-targeted liposomes were also prepared for tumor diagnosis and treatment.MRI is capable of producing three-dimensional images of tissues containing water with a high degree of spatial resolution. However, it suffers from a relatively low sensitivity. Optical imaging has higher sensitivity, but it has problems associated with tissue auto-fluorescence and tissue absorption/scattering of light. Combined the two technique together can complement each other. Tumor can be visualized by MRI and verified by fluorescence microscopy of histology samples. Moreover, the idea of delineate tumors both by preoperative MRI and by intraoperative optical imaging is attractive. Therefore, we design a multifunctional folate receptor targeted, paramagnetic and fluorescent liposomes for targeting and imaging cancer cells.1. Synthesis of Gd-DTPA-bis(SA)DTPA-bis(SA) was prepared from DTPA and SA, and then chelated by Gd3+ to form Gd-DTPA-bis(SA). Gd-DTPA was also prepared. The products were verified by IR and UV, and the concentrations of Gd were detected by ICP-AES. Gd-DTPA-bis(SA) contains Gd of 13.3%, which was similar to the theoretical value (14.9%). Gd-DTPA contains Gd of 26.1%, which was also similar to the theoretical value (27.4%). The phase-transition temperature of Gd-DTPA-bis(SA) was 65.32℃, which can be shown from the DSC spectrum. Phase-transition temperature was an important character of lipids, that is why Gd-DTPA-bis(SA) could form liposomes with other lipids. 2. Preparation of multifunctional liposomeLiposome was composed of HSPC, mPEG2000-DSPE, Cholesterol, Gd-DTPA-bis(SA), Folate-PEG3350-CHEMS. For MRI better imaging, 25% lipid amount of Gd-DTPA-bis(SA) that incorporated in the liposomal bilayer was chosen. To show the efficiency of targeting, the amount of Folate-PEG3350-CHEMS was found by cell uptake experiment, and confirmed that 1% total lipids of the amount of Folate-PEG3350-CHEMS was the best choice. Other factors influencing the stability of liposomes, like the total amount of lipids and the volume of Calcein solution were all analyzed.The liposomes were approximately 125-150nm with low polydispersity index, and could be stable at least for three months. The fluorescent character of Calcein did not change when encapsulating in liposome. The incorpotation rate of Calcein was pretty low, ranging from 1.2~4.7%, detected by UV. While the incorpotation rate of Gd was much higher, after being analyzed by ICP-AES. Liposomes were also imaged by MRI.3. Cell uptake of liposomes in vitro3.1 optical imaging of cells3.1.1 KB cells were incubated with fluorescent liposomes (containing Calcein but without Gd), analyzed by FCMThe average fluorescence intensity of KB cells after uptake of F-Calcein-L was 163 times more than the intensity of cells after uptake of Calcein-L. And it was 6.8 times more than the intensity of cells after uptake the mixture of F-Calcein-L and free FA.3.1.2 KB cells were incubated with biomodal liposomes (containing both Gd and Calcein), observed by fluorescence microscopeAfter 1h, cells incubated with F-Gd-Calcein-L showed greater green fluorescence than Gd-Calcein-L. In contrast, the fluorescence of cell reduced after adding free FA to F-Gd-Calcein-L. Additionally, it can be noticed that liposomes were primarily located on the surface of cell membrane, due to their preferential binding to FR on the membrane. This phenomenon demonstrated that liposomes were taken up by cells through an endocytic pathway.3.1.3 Cell lines of KB, Hela, A549 were incubated with biomodal liposomes (containg both Calcein and Gd), analyzed by FCMThe average fluorescence intensity of KB cells and Hela cells after uptake of F-Gd-Calcein-L was much higher than the intensity of cells after uptake of Gd-Calcein-L. And the uptake could be blocked by free FA.However, the average fluoresce intensity of A549 cells were nearly the same after incubation with different liposomes.Form the fluorescent experiments, it can be seen that folate-decorated liposomes could be targetd delived to folate receptor positive cells but not the negative ones. And this targeted effect can be blocked by free folic acid.3.2 MRI imaging of cells3.2.1 Hela cells were incubated with liposomes containing Gd without Calcein, imaged by MRIHela cells were incubated with Gd-L, F-Gd-L and F-Gd-L+FA for 2.5h. Then the drugs was washed and cells were imaged by MRI. F-Gd-L showed the highest signal intensity and the action could be blocked by free FA.3.2.2 Hela cells and KB cells were incubated with liposomes containing both Gd and Calcein.Hela cells were incubated with L, Gd-Calcein-L, F-Gd-Calcein-L, F-Gd-Calcein-L+FA and Gd-DTPA for 1h. Imaging from MRI showed that cells incubated with F-Gd-Calcein-L obtain highest signal intensity and the action could be blocked by free FA. Comparing with Gd-DTPA, which enter cells by diffusion and can be removed soon, liposomes prolong the circulation time and enhance the signal.KB cells were incubated with L, Gd-Calcein-L, F-Gd-Calcein-L, F-Gd-Calcein-L+FA and Gd-DTPA for 1h. Imaging from MRI showed that cells incubated with F-Gd-Calcein-L obtain highest signal intensity and the action could be blocked by free FA.Form the MRI imaging, it also can be demonstrated that folate-decorated liposomes could be targetd delived to folate receptor positive cells but not the negative ones. And this targeted effect can be blocked by free folic acid.In conclusion, a folate-decorated, MR- and fluorescence- detectable liposome was prepared with narrow size distribution and can be imaged by MRI imaging and fluorescent imaging. It could be targeted deliverd to folate receptor positive cells (KB cells and Hela cells). In contrast, for the folate receptor negative cells, it did not show any preferential uptake of folate-decorated liposomes. This multifunctional liposome may serve as a useful diagnostic tool to co-localize tumors both by MRI and fluorescence. |
