Study On Tumor Targeting Of Methotrexate Loaded Polyamidoamine Dendrimer System

Polyamidoamine dendrimer（PAMAM） has been synthesized for examination in numerous applications due to its unique nanoscopic dendritic architecture.The stepwise synthesis of PAMAM dendrimer affords molecules with a highly regular branching pattern,a unique molecular weight or a low polydispersity index,and a well-defined number of peripheral groups.The high density of exo-presented surface groups allows attachment of targeting groups or functionality while the inner space allows encapsulation ofbioactive agents.Besides,PAMAM dendrimer is not toxic nor immunogenic in a certain dosage range.Methotrexate（MTX） is an antifolate that competitively binds to dihydrofolate reductase which inhibits precursors of DNA and RNA and inhibits cell replication.It is a currently employed antineoplastic agent in the treatment of acute lymphoblastic leukemia（ALL）,choriocarcinoma and trophoblastic tumors.MTX has been in clinical use for more than 35 years and its limitations lie in toxic side effects,such as hepatotoxicity and nephrotoxicity.In addition,MTX has a high occurrence of drug resistance which also limits its effectiveness.The purpose of this study was to evaluate the potential of PAMAM dendrimer as drug delivery vehicle using methotrexate as a model drug.On one hand,the multitude of primary amines in surface sites of PAMAM or PEGylated PAMAM dendrimer are employed to form complex with MTX via electrostatic interaction.On the other hand, MTX is attached to primary amines of PAMAM dendrimer via amide linkage before PAMAM dendrimer be PEGylated.The PEG-PAMAM-MTX conjugate may both prolong MTX half-life and achieve selective accumulation in tumor tissue through enhanced permeability and retention（EPR） effect.The effect of pH and agitation time on PAMAM complexation was studied while preparing PAMAM/MTX complex.On an average,up to 22 or 41 molecules of MTX could be complexed with one molecule of PAMAM_G4 or PAMAM_G5.The in vitro release of MTX from PAMAM/MTX complex is appreciably slower compared to free drug in low ionic strength release medium.However,such sustained release effect was destroyed with the increase of ionic strength.The accumulated release of MTX from PAMAM/MTX complex was less than 65%in 10mM Tris-HC1 buffer solution in 120 hours,in contrast,more than 90%of MTX released from the complex within 6 hours.A series of PEGylated PAMAM dendrimers(PAMAM_G4-PEG₁₁, PAMAM_G4-PEG₂₁,PAMAM_G4-PEG₂₉ and PAMAM_G5-PEG₁₆,PAMAM_G5-PEG₃₀, PAMAM_G5-PEG₃₇) was synthesized.Those PEGylated PAMAM dendrimers exhibited lower hemolytic toxicity and cytotoxicity to KB cell than non-PEGylated PAMAM ones.On an average,up to 34,45 and 49 molecules of MTX could be complexed with one molecule of PAMAM_G4-PEG₁₁,PAMAM_G4-PEG₂₁ and PAMAM_G4-PEG₂₉;up to 53,122 and 140 molecules of MTX could be complexed with one molecule of PAMAM_G5-PEG₁₆,PAMAM_G5-PEG₃₀ and PAMAM_G5-PEG₃₇. PAMAM-PEG/MTX complex demonstrated sustained release character,however, with the increase of the ionic strength in the buffer solution,it was easier for MTX to escape from the complex.Stability test was carried out under the condition of 3-5℃（in refrigerator） and 20-25℃（room temperature）,PAMAM/MTX and PAMAM-PEG/MTX complex showed sound stability after storage for 3 months.A high performance liquid chromatographic（HPLC） analysis was developed to determined MTX plasma concentration.The method was showed to be accurate sensitive and selective.The pharmacokinetic study was carded out in SD rats. Compared to MTX injection,the prolonged drug action of both PAMAM/MTX and PAMAM-PEG/MTX could be seen.The half-life of PAMAM_G4/MTX, PAMAM_G5/MTX complex was 1.86 and 1.94 time as long as that of MTX injection, PAMAM_G4-PEG₁₁/MTX,PAMAM_G4-PEG₂₁/MTX and PAMAM_G4-PEG₂₉/MTX was 2.00,2.07 and 2.06 times as long as that of MTX injection,PAMAM_G5-PEG₁₆/MTX, PAMAM_G5-PEG₃₀/MTX and PAMAM_G5-PEG₃₇/MTX was 2.07,2.20 and 2.52 times as long as that of MTX injection.PAMAM_G4-MTX and PEG-PAMAM_G4-MTX conjugates were synthesized via the formation of an amino bond between primary amines of PAMAMG4 dendrimer and carboxy group of MTX.NMR studies suggested that 36 molecules of MTX and 8 molecules of PEG were conjugated to PAMAM_G4.The pharmacokinetics study in rats plasma showed that the half-life of PAMAM_G4-MTX and PEG-PAMAM_G4-MTX conjugates was 17.4 and 21.3 times longer than that of MTX.The antitumor effects of PAMAM/MTX complexes and PEG-PAMAM_G4-MTX conjugates were studied on S-180 solid tumor beating mice.PEG-PAMAM_G4-MTX conjugate showed significant antitumor effect of all.At the same dose,the inhibitory rate of tumor for PEG-PAMAM_G4-MTX,PAMAM_G4-MTX,PAMAM_G4/MTX and PAMAM_G4-PEG₂₉/MTX were 69.74%,46.41%,44.33%and 52.63%.The above results suggest the potential applications of PAMAM dendrimer in drug delivery systems.