Synthesis of a supermolecular nanoparticle γ-hy-PC/Ada-Dox and its antitumor activity / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To synthesize a (2-Hydroxypropyl)-γ-cyclodextrin-polyethylenimine/adamantane-conjugated doxorubicin (γ-hy-PC/Ada-Dox) based supramolecular nanoparticle with host-guest interaction and to identify its physicochemical characterizations and antitumor effect.</p><p><b>METHODS</b>A novel non-viral gene delivery vector γ-hy-PC/Ada-Dox was synthesized based on host-guest interaction. 1H-NMR, NOESY, UV-Vis, XRD and TGA were used to confirm the structure of the vector. The DNA condensing ability of complexes was investigated by particle size, zeta potential and gel retardation assay. Cytotoxicity of complexes was determined by MTT assay in BEL-7402 and SMMC-7721 cells. Cell wound healing assay was performed in HEK293 and BEL-7404 cells. The transfection efficiency was investigated in HEK293 cells. H/E staining and cell uptake assay was performed in BEL-7402 cells.</p><p><b>RESULTS</b>The structure of γ-hy-PC/Ada-Dox was characterized by 1H-NMR, NOESY, UV-Vis, XRD, TGA. The drug loading was 0.5% and 5.5%. Gel retardation assay showed that γ-hy-PC was able to completely condense DNA at N/P ratio of 2; 0.5% and 5.5% γ-hy-PC/Ada-Dox was able to completely condense DNA at N/P ratio of 3 and 4,respectively. The cytotoxicity of polymers was lower than that of PEI25KDa. The transfection efficiency of γ-hy-PC was higher than that of γ-hy-PC/Ada-Dox at N/P ratio of 30 in HEK293 cells; and the transfection efficiency was decreasing when Ada-Dox loading was increasing. Cell uptake assay showed that γ-hy-PC/Ada-Dox was able to carry drug and FAM-siRNA into cells.</p><p><b>CONCLUSION</b>The novel vector γ-hy-PC/Ada-Dox has been developed successfully, which has certain transfection efficiency and antitumor activity.</p>

Anticancer effect of triptolide-polyethylenimine-cyclodextrin in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop a drug delivery system triptolide-polyethylenimine-cyclodextrin and to evaluate its anticancer activity in vitro.</p><p><b>METHODS</b>Triptolide was conjugated to polyethylenimine-cyclodextrin by N, N'-carbonyldiimidazole to form triptolide-polyethylenimine-cyclodextrin. (1)H-NMR, FT-IR and XRD were used to confirm its structure. The anticancer effect of the polymer was assessed by MTT assay, erasion trace test and hematoxylin-eosin staining. The potential to condense siRNA and to delivery siRNA into cytoplasm was demonstrated by gel retardation assay, zeta-potential determination and fluorescence staining.</p><p><b>RESULTS</b>Triptolide was successfully conjugated to polyethylenimine-cyclodextrin and the conjugation rate of triptolide was 10% (w/w). siRNA was effectively condensed by the polymer at the N/P ratio of 5, and its particle size was 300 ±15 nm and zeta potential was 8 ±2.5 mV. MTT assay, erasion trace test and hematoxylin-eosin staining revealed that triptolide-polyethylenimine-cyclodextrin had anticancer effect and low cytotoxicity to normal cells. The polymer was able to deliver siRNA to the cytoplasm effectively as demonstrated by fluorescence staining.</p><p><b>CONCLUSION</b>Triptolide-polyethylenimine-cyclodextrin is able to inhibit the growth and migration of cancer cells in vitro and to carry siRNA into cells effectively. It is potential to be used as a novel prodrug for co-delivery of gene and drug in cancer treatment.</p>

Characteristics of cationic polymers PEI-CyD, PEI-PHPA, PEE-PHPA and PEI25kD in vitro and in vivo / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To study the characteristics of cationic polymers polyethylenimine-β-cyclodextrin (PEI-CyD), polyethylenimine-poly-(3-hydroxypropyl)-aspartamide (PEI-PHPA), N,N-Dimethyldipropylenetriamine-Bis(3-aminopropyl)amine-aspartamide (PEE-PHPA) in vitro and in vivo.</p><p><b>METHODS</b>PEI-PHPA, PEI-CyD and PEE-PHPA were synthesized and the chemistry structure of PEI-PHPA, PEI-CyD and PEE-PHPA was confirmed by (1)H-NMR. The particle size and zeta potential of these polymers were measured, and capacity of plasmid DNA condensation was tested. The inhibition of COS-7, A549, HEK293 and C6 cells was measured by MTT assay. The transfection efficiency was determined in HEK293 cell lines. The toxicity, tissue distribution and transfection efficiency of cationic polymers were tested in vivo.</p><p><b>RESULTS</b>When the N/P of polymers/DNA at 30, the particle sizes were close 250 nm and the zeta-potential were near 35 mv. They were able to condense DNA at N/P ratio < 5. The MTT assay showed that the IC(50) of PEE-PHPA was 21.5, 20.2, 7.30 and 37.1 μg/ml, and that of PEI25kD was 15.8, 18.3, 11.4 and 36.7 μg/ml in C6, COS-7, A549 and HEK293cell lines, respectively. The cell viability of PEI-CyD and PEI-PHPA in above cell lines was over 60%. They had high transfection efficiency in HEK293 cell lines. The LD(50) of PEI25Kd, PEI-CyD, PEI-PHPA and PEE-PHPA in vivo was 19.50, 100.4, 521.2 and 630.0, respectively by intraperitoneal (ip) injection. The contractions of these polymers were higher in kidney than in other organs and tissues.PEE-PHPA had slight effect on kidney and liver function.</p><p><b>CONCLUSION</b>PEE and PEI25kD have higher transfection efficiency and higher toxicity; while PC and PHPA-PEI have lower toxicity and higher transfection efficiency to be used as non-viral gene vector.</p>

Modified montmorillonite as multifunction gene and drug delivery system / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop polyethylenimine-Doxorubicin-montmorillonite (PEI-Dox-MTT) as a novel multifunction delivery system.</p><p><b>METHODS</b>Dox was intercalated into montmorillonite, PEI covered to the surface of Dox/MMT to make the nano-particle. XRD, FT-IR and TGA were used to confirm chemical property of the nano-particle. SEM was used to observe the morphology. The capability of drug release was investigated by PBS buffer solution (pH 7.4). The DNA binding ability of nano-particle was detected by gel electrophoresis retardation assay. The cell viability in COS-7 and SKOV3 cell lines was tested using MTT assay. The gastric mucosa protection was evaluated in vitro.</p><p><b>RESULTS</b>XRD image showed that Dox was intercalated into montmorillonite, inter space of which increased to 31.3Å; the FT-IR spectra showed the vibration bands of PEI at 1 560 cm(-1) and 2 850 cm(-1), the vibration band of Dox at 1 350 cm(-1). Size analysis and SEM revealed that the size of nano-particle was 600 nm, and the zeta-potential was 30 mV. Drug release experiment explored that the nano-particle stably released drug in range of 6 X10(-4) ≊ 8 X10(-4) mg/ml within 72 h. MTT assay showed that the cell viability was over 80% in experiment condition in COS-7 and SKOV3 cell lines. 0.3 mg PEI-MMT nano-particle was able to protect gastric mucosa from alcohol.</p><p><b>CONCLUSION</b>Multifunction system of PEI/Dox/MMT has been prepared successfully.</p>

Preparation and characterization of Forms A and B of benazepril hydrochloride / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To prepare Form A and Form B of benazepril hydrochloride and to compare the differences in spectrums, thermodynamics and crystal structure between two polymorphic forms.</p><p><b>METHODS</b>Form A and Form B of benazepril hydrochloride were characterized by Fourier transform infrared spectroscopy (IR), thermal gravimetric analysis (TG), differential scanning calorimetry (DSC), powder x-ray diffraction (PXRD) and single crystal x-ray diffraction (SCXRD).</p><p><b>RESULTS</b>Preparation method, crystal structure and polymorphic stability of Form A and Form B of benazepril hydrochloride were obtained. Based on the analysis of crystal structure of both polymorphs, Form A belonged to monoclone space group P2(1) with a=7.8655(4)Å, b= 11.7700(6)Å, c= 13.5560(7)Å, β= 102.9470(10)°, V=1223.07 (11)Å(3) and Z=2, while Form B belonged to orthorhombic space group P212121, with a=7.9353(8)Å, b=11.6654(11)Å, c=26.6453(16)Å, V=2466.5(4)Å(3) and Z=4. From the DSC and XRD results, Form B of benazepril hydrochloride could be transformed into Form A after heating treatment.</p><p><b>CONCLUSION</b>Form A and Form B of benazepril hydrochloride are both anhydrous and displayed different polymorphs due to different molecular configuration. Furthermore, Form A exhibits more stable than Form B at high temperatures.</p>

Peptide MC10 mediated PEI-beta-CyD as a gene delivery vector targeting to Her-2 receptor / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop a novel non-viral gene delivery vector based on polyethylenimine and beta-cyclodextrin targeting to Her-2 receptor (MC10-PEI-beta-CyD).</p><p><b>METHODS</b>The PEI-beta-CyD was synthesized by low molecular weight polyethylenimine (PEI, Mw 600) cross-linked beta-cyclodextrin (beta-CyD) via N, N-carbonyldiimidazole (CDI). The chemical linker[N-succinimidy-3-(2-pyridyldithio) propionate, SPDP] was used to bind peptide MC10 (MARAKEGGGC) to PEI-beta-CyD to form the vector MC10-PEI-beta-CyD. The (1)H-NMR was used to confirm the structure of vector. The DNA condensing ability,and the particle size of MC10-PEI-beta-CyD/DNA complexes were demonstrated by gel retardation assay and electron microscope observation (TEM). Cell viability was tested by MTT assay. The transfection efficiency was determined on cultured SKOV-3, A549 and MCF-7 cells.</p><p><b>RESULT</b>MC10 was linked onto PEI-beta-CyD successfully. The vector was able to condense DNA at N/P ratio of 5 and particle size was about (170 +/-35)nm. The vector showed low cytotoxicity and high transfection efficiency in cultured SKOV-3, A549 and MCF-7 cells.</p><p><b>CONCLUSION</b>A novel non-viral vector MC10-PEI-beta-CyD with low cytotoxicity and high transfection efficiency has been successfully synthesized.</p>

Peptide TAT modified polyethylenimine-beta-cyclodextrin for gene delivery / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop a novel gene delivery vector TAT-PEI-beta-CyD.</p><p><b>METHODS</b>beta-cyclodextrin (beta-CyD) was linked by low molecular weight (PEI 600) via 1, 1-carbonyldiimidazole (CDI), and TAT peptide (RRRQRRKKRC) was coupled to PEI 600 by [N-succinimidy-3-(2-pyridyldithio) propionate, SPDP]. The copolymer was characterized by (1)H-NMR and FT-IR. Physiochemical characteristics of TAT-PEI-beta-CyD/DNA complexes were tested by agarose gel electrophoresis and particle size measurements. Cell viability and transfection efficiency were evaluated in A293 and B16 cells using PEI 25 kDa as a control.</p><p><b>RESULT</b>TAT peptide was successfully coupled to PEI-beta-CyD. The result of gel electrophoresis showed that the TAT-PEI-beta-CyD was able to condense DNA efficiently at N/P ratio of 4. The particle size of TAT-PEI-beta-CyD/DNA complexes was around 100 nm. The cytotoxicity of TAT-PEI-beta-CyD was lower than that of PEI 25 kDa. The transfection efficiency of TAT-PEI-beta-CyD was higher than that of PEI 25 kDa in A293 and B16 cells at N/P ratio of 30.</p><p><b>CONCLUSION</b>The novel vector TAT-PEI-beta-CyD has been developed successfully with low cytotoxicity and high transfection efficiency.</p>

Peptide CY11 conjugated polyethylenimine-beta-cyclodextrin for gene delivery / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop a novel non-viral gene delivery vector CY11-PEI-beta-CyD and to test its gene transfection efficiency.</p><p><b>METHODS</b>CY11 (CGMQLPLATWY) was conjugated to polyethylenimine-beta-cyclodextrin to form CY11-PEI-beta-CyD with a cross-linker [N-succinimidy-3-(2-pyridyldithio) propionate, SPDP]. (1)H-NMR and TGA were used to confirm the structure of vector. The DNA condensing ability of CY11-PEI-beta-CyD was investigated by gel retardation assay. Cytotoxicity of CY11-PEI-beta-CyD was determined by MTT assay and transfection efficiency was investigated in COS-7, Hela and B16 cells.</p><p><b>RESULT</b>CY11 was conjugated onto PEI-beta-CyD successfully, confirmed by(1)H NMR and TGA. The novel vector effectively condensed DNA at N/P ratio of 4îIt showed low cytotoxicity up to the concentration was 160 Mgr;g/ml. The transfection efficiency was 17-fold higher than that of PEI 25 kDa at N/P ratio of 20.</p><p><b>CONCLUSION</b>The novel vector CY11 -PEI-beta-CyD with low cytotoxic and high transfection efficiency may be used as a potential carrier for gene delivery.</p>

Poly-aspartamide-glutamic acid grafted low molecular weight polyethylenimine as a novel non-viral gene vector / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop a novel gene delivery vector with poly-aspartamide-glutamic acid and polyethylenimine as the backbone.</p><p><b>METHODS</b>alpha, beta-poly-(N-2-hydroxypropyl)-D, L-aspartamide-glutamic acid (PHPAG) was synthesized and low molecular weight polyethylenimine (PEI 1.8 kDa) was grafted to form PHPAG-PEI 1800. Chemical and biological characterization of the polymer was identified.</p><p><b>RESULT</b>The polymer was confirmed by (1)H-NMR, and the molecular weight was about 1.2 x 10(4). The ability of DNA binding was showed by gel retardation assay at N/P ratio of 3. 5. MTT assay showed that the polymer was non toxic in COS-7 and A293 cell lines. In vitro test demonstrated that it had high transfection efficiency in B16 and Hela cell lines.</p><p><b>CONCLUSION</b>PHPAG-PEI 1800 was successfully synthesized,which might be a potential vector for gene delivery.</p>

Lentinan-graft-polyethylenimine-a novel vector for gene delivery / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop a novel vector for gene delivery with low molecular weight polyethylenimine grafted to the natural polysaccharide and conjugated to folic acid (LNT-PEI-FA).</p><p><b>METHODS</b>The properties of LNT-PEI-FA were characterized by (1)H-NMR, FT-IR and TGA, respectively. The particle size of LNT-PEI-FA/DNA complex was measured. The DNA binding ability of LNT-PEI-FA was detected by gel electrophoresis retardation assay.</p><p><b>RESULT</b>The particle size of LNT-PEI-FA/DNA complex was about 200 nm. Gel electrophoresis showed that at N/P ratio of 1.8 (W/W) the polymer was able to completely condense DNA. In vitro experiments showed a high efficiency of gene transfection in A293 and B16 cell lines.</p><p><b>CONCLUSION</b>A novel non-viral vector LNT-PEI-FA was successfully synthesized and characterized, which may be applied in gene transfection research in the future.</p>

Aspirin-PEI-beta-CyD as a novel non-viral vector for gene transfer / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop a novel non-viral gene delivery vector based on PEI-beta-CyD as backbone modified with aspirin, and to identify its physicochemical characters.</p><p><b>METHODS</b>1, 1-carbonyldiimidazole (CDI) was used to bind aspirin onto PEI-beta-CyD to form PEI-beta-CyD-ASP. (1)H-NMR, FT-IR, UV and XRD were used to confirm the polymer structure. The ability of condensation was demonstrated by gel retardation assay. MTT assay was used to test the cell viability in B16, Hela and A293 cell lines. Transfection efficiency of the polymer was tested in B16 cells.</p><p><b>RESULT</b>The structure of PEI-beta-CyD-ASP was confirmed by (1)H-NMR, FT-IR, UV and XRD, which efficiently condensed plasmid DNA at the N/P ratio of 4. The copolymer showed low cytotoxicity and high transfection efficiency in B16 cells.</p><p><b>CONCLUSION</b>The synthesized aspirin-PEI-beta-CyD might be a potential gene delivery vector.</p>

Preparation of floxuridine loaded polycation and its antitumor activity / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop a new prodrug of 5-fluorouracil-polyethylenimine-beta-cyclodextrin-floxuridine (PEI-beta-CyD-Fd) and to test its antitumor activity.</p><p><b>METHODS</b>Floxuridine was conjugated to polyethylenimine-beta-cyclodextrin to form prodrug PEI-beta-CyD-Fd. The structure of synthesized PEI-beta-CyD-Fd was confirmed by (1)H-NMR, FT-IR and UV. MTT assay and cell wound healing assay were performed on human hepatic carcinoma cell line HepG2.</p><p><b>RESULT</b>The drug loading was 2 %. The MTT assay and cell wound healing assay indicated that PEI-beta-CyD-Fd significantly inhibited proliferation and migration of HepG2 cells.</p><p><b>CONCLUSION</b>The synthesized prodrug PEI-CyD-Fd has a significant antitumor activity on HepG2 cells.</p>

Platinum tetrachloride coupled PEG-PEI as drug carrier and gene delivery vector / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct a drug carrier and gene vector PEG-PEI-Pt.</p><p><b>METHODS</b>Polyethyleneglycol (PEG) was coupled to polyethylenimine (PEI 600) and platinum tetrachloride; PEG-PEI-Pt complex was formed in ethanol. The complex was characterized by XRD, UV-VIS and FT-IR and the DNA condensation was tested by electrophoretic mobility shift assay. The cell viability was evaluated by MTT assay in Hela, B16, A293 and COS-7 cells and in vitro transfection efficiency was measured in A293 and B16 cells.</p><p><b>RESULT</b>The structure of PEG-PEI-Pt was characterized by XRD, UV-VIS and FT-IR. PEG-PEI-Pt complex was able to bind DNA at N/P weight ratio of 0.4:1; the complex showed cytotoxicity on Hela and B16 cells. The complex had higher transfection efficiency in A293 and B16 cells than PEI 600.</p><p><b>CONCLUSION</b>A novel drug carrier and gene vector PEG-PEI-Pt was constructed successfully.</p>

Physicochemical characters and gene delivery function of polyethylenimine modified with peptide CP9 containing RGD sequence / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct a novel gene delivery vector using polyethylenimine (PEI) as backbone modified with the peptide CP9 containing Arginine-Glycine-Aspartic acid (RGD) sequence and to verify its physicochemical characters and the gene delivery function.</p><p><b>METHODS</b>The chemical linker [N-Succinimidyl-3- (2-pyridyldithio) ] propionate (SPDP) was employed to bind CP9 onto PEI to form a novel gene delivery vector CP9-PEI. The (1)H-NMR and FT-IR were used to verify the linkage of CP9. The plasmid DNA condensing ability of CP9-PEI,the shape and the particle size of the polyplexes formed with CP9-PEI-plasmid DNA were demonstrated by gel retardation assay, electron microscope observation and particle size assay,respectively. The enhanced transfection efficiency and the integrin targeting capacity were detected by the transfection experiments in HepG2 cells and free RGD peptide inhibition test.</p><p><b>RESULT</b>CP9 was linked onto PEI successfully. The new synthesized vector CP9-PEI could efficiently condense plasmid DNA at N/P ratio of 4 and when N/P ratio was equal to 10, the shape of polyplexes formed with CP9-PEI-plasmid DNA was round or round-alike with particle size of about 200 nm. The transfection efficiency of CP9-PEI was nearly 2 times of PEI in HepG2 cells and the free RGD peptide had the inhibition effect on the efficiency of CP9-PEI.</p><p><b>CONCLUSION</b>The modification of CP9 on PEI can improve the transfection efficiency of PEI and has the integrin targeting ability.</p>

Receptor-mediated gene delivery using polyethylenimine (PEI) coupled with polypeptides targeting FGF receptors on cells surface / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To construct a novel kind of nonviral gene delivery vector based on polyethylenimine (PEI) conjugated with polypeptides derived from ligand FGF with high transfection efficiency and according to tumor targeting ability.</p><p><b>METHODS</b>The synthetic polypeptides CR16 for binding FGF receptors was conjugated to PEI and the characters of the polypeptides including DNA condensing and particle size were determined. Enhanced efficiency and the targeting specificity of the synthesized vector were investigated in vitro and in vivo.</p><p><b>RESULTS</b>The polypeptides were successfully coupled to PEI. The new vectors PEI-CR16 could efficiently condense pDNA into particles with around 200 nm diameter. The PEI-CR16/pDNA polyplexes showed significantly greater transgene activity than PEI/pDNA in FGF receptors positive tumor cells in vitro and in vivo gene transfer, while no difference was observed in FGF receptors negative tumor cells. The enhanced transfection efficiency of PEI-CR16 could be blocked by excess free polypeptides.</p><p><b>CONCLUSION</b>The synthesized vector could improve the efficiency of gene transfer and targeting specificity in FGF receptors positive cells. The vector had good prospect for use in cancer gene therapy.</p>