| PAMAM-OH was conjugated with S-Methyl-L-cysteine(SMLC)via a?-thiopropionate bond to obtain PAMAM-SMLC(PAMS).1H-NMR result showed that the degree of attachment of SMLC was 83.9%.PAMS was further modified by PEG and folic acid(FA)and the attachment degree of PEG-FA was 11.8%.This study concluded two sections.In the first section,based on the PAMSPF,a novel biodegradable gene delivery system(PAMSPF/DNA)was prepared.After being uptaked into cells,the cleavage of ?-thiopropionate linkage in the endosome facilitated the degradation of PAMSPF/DNA and release of DNA,which was necessary for transcription.In the second section,another gene delivery system(PAMS/DNA/10NLS)was prepared based on PAMS,DNA and nucleus location signal peptide(NLS).The synergistic effect of NLS and GTPase-activating protein 1(RanGAP1)on the nucleus import of PAMS/DNA/10NLS was investigated.SECTION 1:The construction and evaluation of a pH-sensitive degradable polyplex(PAMSPF/DNA).The agarose gel electrophoresis result showed that PAMSPF was able to retard DNA mobility completely and protect DNA in the presence of heparin and nuclease at an N/P(molar ratio of PAMS-nitrogen atoms to DNA-phosphates)ratio of 2.Hoechst 33258 binding assay showed that the encapsulation efficiency of PAMSPF/DNA was above 90%at an N/P ratio of 2.The morphology of PAMSPF/DNA was confirmed visually by transmission electron microscopy(TEM).PAMSPF/DNA was present in the form of spherical nanoparticles with well-defined shapes.The diameter and Zeta potential of PAMSPF/DNA were measured by dynamic light scattering(DLS).As the N/P ratio increased,the particle size decreased and the Zeta potential increased.The diameter of PAMSPF/DNA was about 200 nm and Zeta potential was more than+10 mV when the N/P ratio reached 8.The degradation of PAMSPF/DNA was analyzed by 1H-NMR and agarose gel electrophoresis.The results showed that the PAMSPF/DNA degraded completely and free DNA was released after a 3 h incubation at pH 5.5.The in vitro expressions of green fluorescence protein(GFP)and luciferase were determined to investigate the transfection efficiency of the PAMSPF/DNA.The higher the N/P ratio,the higher the transfection efficiency of PAMSPF/DNA exhibited in HepG2 cells.For KB cells,the transfection efficiency of PAMSPF/DNA reached a maximum at an N/P ratio of 16.However,when the N/P ratio increased above 20,the transfection efficiency began to decrease.MTT results showed that the cytotoxicity of PAMSPF was N/P ratio-dependent generally for both cell lines.However,the viability of KB cells was lower than that of HepG2 cells.The different cytotoxicity and transfection efficiency of PAMSPF/DNA in KB and HepG2 cells was due to the different FRs level in these two cells.Folate receptor targeted delivery of PAMSPF/DNA and competitive inhibition of free folic acid(FA)were assayed by flow cytometry.The results showed that the uptake of PAMSPF/DNA into high-FRs KB cells was higher than that into low-FRs HepG2 cells.When free folic acid was added,the uptake of PAMSPF/DNA into these two cells was same.The endosomal escapse of PAMSPF/DNA in KB cells was studied using laser scanning confocal microscopy(LSCM).Almost all the PAMSPF/DNA escaped from the endosomes and entered into the cytoplasm after a 2 h incubation.The tumor targeting ability of polyplexes was assayed by small animal imaging technology.The result showed that most of PAMSPF/DNA was accumulated in the tumor site after 0.5 h injection.At 24 h post-administration,PAMSPF/DNA exhibited more fluorescence than other reference polyplexes in tumor tissues,showing that PAMSPF/DNA has high tumor targeting ability and displayed a long circulation time in vivo.SECTION 2:The synergistic effect of NLS and RanGAP1 on the nucleus import of PAMS/DNA/10NLS.The obtained obove results showed that the release of DNA can increase the transfection efficiency.However,the free DNA has to enter into the nucleus as soon as possible to avoid being damaged in the cytoplasm and be transcribed.As a result,PAMS/DNA/10NLS was prepared.The synergistic effect of NLS and RanGAP1 on the nucleus import of PAMS/DNA/10NLS was investigated.The agarose gel electrophoresis results showed that PAMS/DNA/10NLS was able to retard DNA mobility completely at an N/P ratio of 2.The diameter and Zeta potential of PAMSPF/DNA were measured by dynamic light scattering(DLS).As the N/P ratio increased,the particle size decreased and the Zeta potential increased.After incubation in pH 5.5 for 0.5 h,PAMS/DNA/10NLS began to degrade and displayed a time-dependent degradation.After incubation for 3 h,the degradation rate was 92.9%.Transmission electron microscopy(TEM)result showed that PAMS/DNA/10NLS degraded into smaller partices after after incubation for 3 h.Forster resonance energy transfer(FRET)technology confirmed the degradation of PAMS/DNA/10NLS inside cells.MTT assay was used to assess the cytotoxicity of PAMS/DNA/10NLS.PAMS/DNA/10NLS didn't exhibit any significant cytotoxicity when N/P ratio was below 12.When N/P ratio reached 16,the cytotoxicity of PAMS/DNA/10NLS was higher than control.As a result,N/P=12 was used to compare the cytotoxicity of different polyplexes.The cytotoxicity of PAMS/DNA/10NLS was same as that of PAMS/DNA,showing that NLS didn't cause any extra cytotoxicity.The result obtained in HEK-293 cells was same to in KB cells.RanGAP1 overexpressed cells(RanGAP1 cells)were produced by transfecting cells with RanGAP1 plasmid.RanGAP1 cells had higher GTPase activities than normal KB cells(normal cells).The nucleus import of PAMS/DNA/10NLS was studied visually using a laser scanning confocal microscope(LSCM).With the help of NLS,PAMS/DNA/10NLS showed a significantly higher colocalization within the nucleus region than PAMS/DNA in normal cells.The colocalized amount of PAMS/DNA/10NLS within the nucleus region in RanGAP1 cells was higher than that in normal cells after incubation.On the contrary,PAMS/DNA had a similar nucleus importing ability between in normal and RanGAP1 cells.This result indicated that RanGAP1 didn't work on the nucleus import without NLS.As a result,we could conclude that the synergistic effect of NLS and RanGAP1 has the ability to increase the nucleus import.The in vitro expression of luciferase was evaluated to investigate the transfection efficiency of PAMS/DNA/10NLS.PAMS/DNA/10NLS has a higher transfection efficiency than PAMAM/DNA and PEI/DNA at a N/P ratio of 12.The G1 and G2/M cells were produced.The percentage of cells in the Gi phase and G2/M phase were 71.1%and 21.4%,respectively,for normal cells.After synchronization,the percentage of cells in the G1 phase was 85.4%for G1 cells and the percentage of cells in th G2/M phase was 40.1%for G2/M cells,showing that the cells had been synchronized successfully.Both G1 and G2/M cells were transfected with PAMS/DNA or PAMS/DNA/10NLS.For PAMS/DNA,the luciferase activity of G2/M cells was 58.2-fold higher than that of G1 cells.For PAMS/DNA/10NLS,it was clear that the difference became smaller(5.2-fold).With the assistance of NLS,the transfection of PAMS/DNA/10NLS was less dependent on the breakdown of the nucleus envelope than that of PAMS/DNA.For PAMS/DNA,there was no marked difference in reporter gene expression between normal KB cells(normal cells)and RanGAP1 cells.However,for PAMS/DNA/10NLS,RanGAP1 cells exhibited about a significantly higher(11.4-fold)luciferase activity than normal cells,which indicated that NLS and RanGAP1 synergistically increased the transfection efficiency of polyplex.To evaluate the expression efficiency in vivo,polyplexes containing pDs-Red-M-N1 were injected into KB tumor-bearing nude mice.Fluorescence images showed that the PAMS/DNA/10NLS group exhibited a significantly higher fluorescence intensity than the PAMS/DNA group due to the increased nucleus import of PAMS/DNA/10NLS.Moreover,if mice were pre-treated with polyplex containing RanGAP1 gene in advance,the expression efficiency of PAMS/DNA/10NLS was further increased(PAMS/DNA/10NLS+RanGAP1 group).Being similar to the in vitro transfection assay,this might be because NLS and RanGAP1 were able to increase the transfection ability of PAMS/DNA/10NLS synergistically. |
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