Protective Effect Of PEGylation Against Poly (Amidoamine) Dendrimers Induced Toxicity

Dendrimers are highly branched macromolecules which have specific size,shape andchemical functions.Poly (amidoamine) (PAMAM) dendrimers are a specific family ofdendritic polymers,which are based on an ethylene diamine core and an amidoaminerepeat branching structure.Dendrimers have recently been successfully used in the field ofbiomedicine,such as drug delivery,gene delivery,cancer diagnosis,etc.Despite theextensive interests in pharmaceutical applications of dendrimers,PAMAM dendrimersbearing amino terminals show certain cytotoxicity.In order to improve theirbiocompatibility,generation 5 (G5) PAMAM dendrimers were modified by conjugation ofpoly (ethylene glycol) (PEG) of two different molecular weights and different number ofchains.To investigate the influence of PEGylation on PAMAM-induced cytotoxicty,theintracellular responses including reactive oxygen species (ROS) content,mitochondrialmembrane potential (△ψm) and apoptosis were examined.Meanwhile,the protectiveeffect of PEGylation against PAMAM dendrimers induced hemolysis of human red bloodcells (RBCs) was studied.Furthermore,the uptake rate of PEGylated dendrimers wasquantified by using FITC labeled PAMAM and PEG-PAMAM.Moreover,the ability oftransfection for PEGylated dendrimers was investigated.The main results are asfollowings:(1) G5 PAMAM were conjugated with PEG-2k or PEG-5k of 13 or 51 PEG chains,which meant 10% and 40% of the total amino terminals of G5 PAMAM were PEGylated,respectively.Thus,four conjugates 13PEG-2k-PAMAM,51PEG-2k-PAMAM,13PEG-5k-PAMAM and 51PEG-5k-PAMAM were prepared.The conjugates werecharacterized by ¹H NMR and FT-IR spectra.(2) The IC₅₀ values (concentration at which 50% of mitochondrial dehydrogenaseactivity was inhibited) were determined to compare the cytotoxicity of differentdendrimers.The cytotoxicity of PAMAM was concentration- and time-dependent.Theorder of IC₅₀ of those conjugates on NIH 3T3 cells was 51PEG-5k-PAMAM,51PEG-2k-PAMAM,13PEG-5k-PAMAM,13PEG-2k-PAMAM,PAMAM.The IC₅₀ valueof 13PEG-2k-PAMAM dendrimers was 12 fold more than that of PAMAM.Moreover,the value of 51PEG-5k-PAMAM was about 100 fold more than that of PAMAM.As a result,dendrimers conjugated with more PEG chains of higher molecular weight were much lesscytotoxic.(3) The cellular internalization of PAMAM and PEG-PAMAM grew steadily over theincubation time.The total rates of cell accumulation of PAMAM,13PEG-2k-PAMAM and51PEG-2k-PAMAM were almost 100%.The rates of the dendrimers modified by PEG-5kwere little less,about 80%.The uptake rate of PAMAM and PEG-PAMAM were alsotemperature dependent,which was inhibited at 4℃.PAMAM and PEG-PAMAM couldenter the nucleus in 15 min.Moreover,the PEGylated dendrimers maintained the ability oftransfection.The transfection effects of 13PEG-2k-PAMAM and 51PEG-2k-PAMAMwere similar to that of PAMAM at appropriate charge ratio.(4) The intracellular responses including ROS contents,mitochondrial membranepotential and apoptosis were examined to elucidate the mechanism of decreasingPAMAM-induced cytotoxicity by PEGylation.The results showed PAMAM inducedapoptosis in a concentration dependent manner.The PEGylation protected remarkablyagainst the PAMAM induced apoptosis.DCFH-DA and Rhodamine-123 were used todetect the levels of intracellular ROS and△ψm,respectively.The results showed PAMAMinduced the burst of intracellular ROS and the dissipation of△ψm,then triggered the cellsa start-up of apoptosis program.These results suggested that cell apoptosis induced byPAMAM was mediated by mitochondrial pathway.PEGylation could effectively reducethe PAMAM-induced cell apoptosis by attenuating ROS production and inhibitingPAMAM-induced△ψm collapse.PAMAM conjugated with more PEG chains of highermolecular weight were much less cytotoxic.(5) The protective effect of PEGylation against PAMAM induced hemolysis of RBCswas studied.PAMAM were PEGylated with three molecular weights (2k,5k and 20k).The RBCs morphology and surface structure were analyzed by optical microscopy (OM)and atomic force microscopy (AFM).PAMAM dendrimers induced obvious hemolysiswhile PEGylated ones exhibited good haemocompatibility.The hemolysis resulting fromPEGylated dendrimers was much lower compared to the parent dendrimers.Moreover,RBCs treated with PEG-5k and PEG-20k modified dendrimers kept their normalmorphology.This study demonstrated that haemocompatibility of PAMAM dendrimers could be greatly enhanced by PEGylation.Dendrimers conjugated with higher molecularweight PEG showed much lower haemotoxicity.The results of hemolysis test andmorphology investigation for PEG-5k and PEG-20k modified groups were closer to thoseof control RBCs.In conclusion,PEGylated dendrimers were more suitable and valuable for practicalapplication in which they presented a much lower cytotoxicity and higherhaemocompatibility.We suggested that PAMAM dendrimers would stimulateoverproduction of ROS in cells,which could disturb the function of mitochondrial andeventually lead to cell death.However,modified with PEG was able to inhibitPAMAM-induced cell death.Meanwhile,dendrimers conjugated with fewer PEG of lowermolecular weight did not significantly change the endocytic properties.Moreover,thePEGylated dendrimers maintained the ability of transfection.The transfection effect ofPEGylated dendrimers would be enhanced by heating activation or conjugation totargeting ligands.