Study Of The Polyamidoamine Dendrimer Conjugating With Alkali Blue For The Lymphatic Tracing And Targeting

Since lymphatic circulation is the main pathway of tumor metastasis,lymph tissues(including lymphangiogenesis and lymph nodes)became an important target in cancer chemotherapy,and nanoparticles as lymphatic tracer carriers for sentinel lymph nodes during surgery became a hot topic in the field of molecular imaging.Combining fields of research described above,the purpose of this project is to develop an Alkali blue modified dendrimer carrier to achieve the purpose of targeting to tumor metastasis lymphatic system.This new dark blue carrier,entrapped chemotherapy drugs in the unique three-dimensional structure,were delivered to the lymphatic system and tumor cells to clearly sketch out the location of tumor and lymph nodes,and ultimately express the dual role of lymphatic tracing and lymphatic targeting.In this study,amide-linked AB conjugates of G3.5-COOH dendrimer were synthesized by bonding the carboxyl ending groups of PAMAM and amino groups of AB in DMSO using DCC as a condensation agent and DMAP as a catalyst.The water-soluble,dark-blue PAMAM-AB was obtained.UV-vis,FT-IR,1H-NMR,and HPLC characterization were performed to prove the successful synthesis of PAMAM-AB.The calculated AB payload of PAMAM-AB conjugate was 7 per dendrimer molecule(27.16%by weight).Hydrolysis stability of PAMAM-AB in vitro was evaluated.Results indicated that PAMAM-AB conjugates with amide bonds were quite stable against hydrolytic cleavage for the 7-day time period study at pH 5.0,pH 5.8 and pH 7.4 PBS,and no free AB was detected in reaction solutions.In plasma,only 0.55%of AB was released from PAMAM-AB for 6 days,and increased to 1.28%for 7 days.An HPLC method was developed for the determination of PAMAM-AB in the plasma and biological samples.MB solution(MB-S),MB water-in-oil microemulsion(MB-ME)and MB multiple microemulsion(MB-MME)were selected as the control,the lymphatic tracing of PAMAM-AB with different dose were studied to determine the blue-stained intensity of PAMAM-AB in right popliteral lymph nodes(PLNs),iliac lymph nodes(ILNs)and paraaortic lymph nodes(PALNs)after s.c.administration.Results indicated that PLNs,ILNs and PALNs could be obviously blue-stained within 10 min after PAMAM-AB administration at a dose of 10 mg/kg.MB and PAMAM-AB concentrations in plasma,PLNs,ILNs,PALNs,the injection site and other tissues were quantitatively analyzed using HPLC to evaluate the intralymphatic targeting ability and lymphatic tropism of PAMAM-AB.PAMAM-AB displayed a more rapid lymphatic absorption,a higher AUC value in lymph nodes and a longer lymph nodes residence time compared with MB-S,MB-ME and MB-MME.The higher distribution percent and the targeting efficiency of PAMAM-AB in PLNs,ILNs and PALNs proved the better lymphatic targeting ability of PAMAM-AB.With the anti-cancer drug paclitaxel(PTX)as the model drug,PTX-loaded polyamidoamin-alkali blue(PTX-P-AB)was prepared.The method for calculate the encapsulation efficiency(EE)and drug loading(DL)was established.With the criteria of EE and DL,factors,such as the speed of the centrifuge,the centrifugation time,the reaction time,and the ratios of PAMAM-AB and PTX were studied to optimize the formation and the technology.The physicochemical properties and in vitro drug release were evaluated.The EE and DL of PAMAM-AB was(35.57±2.61)%and(26.35±1.51)%,respectively.The zeta potential and drug content of PTX-P-AB essentially showed no apparent change after 12 h comparing,indicating that drug in a PAMAM-AB dendrimer delivery system was stable.The in vitro release behaviors of PTX-P-AB were respectively investigated in mimic subcutaneous and lymphatic system.Results showed that PTX release from PTX-P-AB dendrimer system in mimic subcutaneous release condition was significantly slower than in mimic lymphatic system.The release rate was in descending order of pH 5.8 PBS,pH 6.8 PBS,pH 5.0 PBS,and pH 7.4 PBS,and PTX release from PTX-P-AB gradually speeded up with the increase of the osmotic pressure of PTX-P-AB.An HPLC method was developed for the determination of PTX in the plasma and biological samples.The lymphatic targeting ability of PTX-P-AB with the osmotic pressure of 300 mmol/kg,500 mmol/kg and 700 mmol/kg(OSM-300,OSM-500 and OSM-700)was evaluated in healthy mice and in tumor-bearing mice with the Taxol(?)as control.PTX-P-AB displayed the more rapid lymphatic absorption,the higher AUC value in LNs and the longer LNs residence time compared with Taxol(?).The higher distribution percent,relative tissue exposures,targeting efficiency and relative targeting efficiency of PAMAM-AB in PLNs,ILNs and PALNs proved the better lymphatic targeting ability of PTX-P-AB.With the increase of the osmotic pressure of PTX-P-AB,the lymphatic drainage from the injection site enhanced,and the uptake into lymph increased.The lymphatic uptake in tumor-bearing mice was higher than that in healthy mice.After subcutaneous administration of S180 tumor to Kunming mice every other day for 14 days,the pharmacodynamic model was successively established.The metastatic tumor in PLNs was selected to investigate the anti-cancer effect of PTX-P-AB.Results indicated that the PAMAM-AB dendrimer alone had no toxicity,and PTX inhibited the mice growth due to the toxicity.The metastasis-inhibiting rate of PTX-P-AB group was higher than Taxol(?)group,and the anticancer activity was increased by increasing the osmotic pressure of PTX-P-AB,indicating the better anti cancer activity of PTX-P-AB than Taxol(?).