The Preparation Of PEG-Chitosan Nanoparticles Containing ACEshRNA And Treatment To Spontaneously Hypertensive Rats

Objective:In this study, firstly chitosan nanoparticles were prepared and PEG surface modification was given, then their physics and biological characteristics were analysised to obtain a long release non-viral vector-mediated systems. Secondly we observed its transfection and cells cytotoxicity efficiency by transfected cultured rat vascular endothelial cell in vitro, and researched the downward effect of ACE gene in the molecular and protein levels. Finally we selected the appropriate dose of transfection in spontaneously hypertensive rats to observe the antihypertensive effect as well as target organ protect effect.Methods:1. Preparation and biological characteristics of PEG-chitosan nanoparticles containing ACEsh RNA1) Using the ACE gene target sequence obtained from the laboratory'pre-experiment screened: 5'-AACCTAACATGTCAGCCTCTG-3 ' (gene sequences loci: 3603-3624). The synthesis of plasmid was assisted by Wuhan Jin Sai Corporation. The substantial extraction and purification of recombinant plasmid were obtained by bacteria alkaline lysis method. The Sequencing was detected by random enzyme digestion method and the concentration and purity of plasmid.were detected.2) To prepare chitosan nanoparticles by ionic crosslinking method and modify them with PEG, then prepare PEG-chitosan nanoparticles containing ACEshRNA by rehabilitation method under the condition of different pH values as well as the volume ratio of chitosan and plamid. The chitosan nanoparticles morphology was observed by Spray gold electron microscopy . At the same time, the particles size,zeta potential value and multi-dispersity were measured by the zeta potential machine. The pDNA / CS complex'poly-formation and charge properties were analysised by Gel retardation method. The supernatant DNA concentration after centrifugation was detected by UV method. Furthermore we calculated embedding rate, got PEG-CS/DNA nanocomposites cumulative release curve and analysised nanoparticles against nuclease digesting.2. The appropriate conditions of transfecting PEG-chitosan nano Containing ACEshRNA on vascular endothelial cells1) Rat vascular endothelial cells were cultured by Organize paste method and identified of cytosolic factorⅧby immunofluorescence assay . Cells survival rate were detected by Trypan blue staining method.2) Vascular endothelial cells were respectively transfected 0-96h at the best allocation ratio (nanoparticle: plasmid 1:1) of CS / DNA nanocomposites combination and PEG-CS/DNA nanocomposites with 60ul, 80ul, 100ul, 120ul, and took the corresponding naked plasmid group and blank group as controls .3) The expression of green fluorescence of transfected cells was observed with fluorescence microscope. Transfection efficiency was detected by flow cytometry and cell activity was determined by MTT method.3. The inhibit of ACE expression after PEG-chitosan nano containing ACEshRNA transfected on rat vascular endothelial cells1) The expression of ACE mRNA was detected with Real-time fluorescence quantitative PCR method before and after transfection in each PEG-CS/DNA nanocomposites group, and naked plasmid and blank group as control.2) ACE protein expression was detected with Western-blot method in collect cells of each group before and after transfection of 24h, 48h, 72h .3) The concentration of ACE and Ang-Ⅱwere detected in 2mL culture medium before and after transfection of 72h with Enzyme-linked immunosorbent assay methods.4. ACEshRNA-PEG-CS nano-vector-mediated RNAi effect in Spontaneously Hypertensive Rats1) 12-week-old adult male spontaneously hypertensive rats (SHR) and high blood pressure model control mice were studied. 25ug DNA mediated diluted to 500ul with sterile saline mediated by ACEshRNA-PEG-chitosan nanocomposites was injected into rats through tail vein , repeated after 10 days. And given daily gavage Lotensin 10mg / day /animal,blank SHR control group, chitosan group normal blood pressure group as control, respectively tail vein injection of 500ul sterile saline or the same concentration of chitosan nano-liquid.2) Rat tail artery systolic blood pressure (SBP) and heart rate were measured at the same point respectively before injection and 1-3 days after injection with tail cuff method in quiet environment and awaken condition.3) The myocardial, aortic, renal ACE mRNA or protein expression were detected by fluorescence quantitative RT-PCR or Western-blot assay. The serum levels of ACE and Ang-Ⅱcontent were detected by enzyme-linked immunosorbent assay . The hepatic and renal function were analyed by automatic biochemical method.4) At the end of the experiment, the whole heart (HW) and left ventricular weight (LW) were weighted to achieve heart / body weight, left ventricular / body weight ratio. And preparation of heart, kidney and aortic Organize frozen section to observe PEG- CS-EGFP-ACE-shRNA distribution under fluorescence microscope, inspect histology change under light microscopy and transmission electron microscopy.Results:1. Preparation and biological characteristics of PEG-chitosan nanoparticles containing ACEsh RNA1.1 The DNA plasmid concentration was 0.5ug/ul, the purity was 1.88 detected by UV.1.2 The impact of physical nature of various factors on chitosan nano-composite: (1) the particle size of chitosan nanoparticles increased with the solution pH value increasing. when the pH value is 5.5 , PEG chitosan nanoparticles average diameter is about 125.8±5.6nm with size uniformity, smallest multi-dispersity, more concentrated distribution and positive zeta potential which is benefited to combine of the negatively charged plasmid. (2) After PEG modified, zeta potential of particle size had no significant effect, but compared to the same conditions, the dispersity were significantly reduced, showing more suitable to prepare uniform nanoparticles and combine plasmids. (3) A smaller average chitosan particle size was obtained with 1:1 volume ratio (mass ratio) of chitosan nanoparticles to plasmid , with the character of the better permeability through the cell membrane, smaller dispersity , distributed more concentrate and positive zeta potential, which all benefit to combine the plasmid with negative charge.1.3 The impact of plasmid-binding capacity of chitosan of various factors: (1) Chitosan nano-and PEG-chitosan nano-plasmid complexes have effective combination of plasmid , and the plasmid negative charge were decreased. When gel electrophoresis, the plasmid stayed in the holes and naked plasmid went out of the hole. (2) When PH <7 most of amino with positively charged of the chitosan molecules can effectively combine plasmid DNA plasmid to make it stay in the hole. (3) At volume ratio of 1:1,1:2,1:3, the chitosan nanoparticles can be effectively combine plasmid.1.4 The impact of embedding rate of various factors on complex: (1) The DNA-embedded rate of chitosan nano-composite was highest (90.43±3.9)% at pH =5.5.. (2) Embedding rate increased significantly in each group after the surface modification by PEG. . (3) the embedding rate were higher at the volume ratio of 1:1,1:2 , respectively (88.87±13.2)% and (80.13±12.9)%, no significant difference between the two groups; And as the ratio increased embedding rate decreased.1.5. the impact of vitro release of the compound with various factors : (1) The release of CS / DNA nanocomposites in PBS solution is basically same at pH = 3.5-7.5. There are the beginning stages of the release burst, the release of the preceding curve near a straight line, about after 4 days (96 hours), the stability release began, the release curve risen slowly, maintaining a smooth release of about eight days. And the nanoparticles quickly released in pH = 9.0 PBS solution, only lasted about 4 days. (2) The release curve of different gene complexes with different volume ratio all had the begining burst release when fixed at PH = 5.5 . The release curve rapid increased at the beginning, about after 4 days (96 hours)the stability release started. The duration of the release shortened with volume ratio increasing. At volume ratio 1:1, the duration of release was longest, maintained smoothly release about eight days; at volume ratio 1:5, the release time was shortest, only maintained about four days. (3) The beginging burst release characteristics of chitosan nanoparticles containing ACEshRNA were not enfluenced by PEG modified when fixed volume ratio 1:1 and pH = 5.5 . The release begun to stabilized after about 4 days (96 hours) , the release curve slowly raise, but the release duration was extended to 10 days.1.6 DNaseⅠdigestion experiments: (1) PEG-CS-ACEshRNA gene complexes can be effective against DNA degradation at the volume ratio 1:1-1:3, and the plasmid was completely blocked into the hole. At the volume ratio of 1:4,1:5 bands can be showed indicating the nuclease protective effect decreased. (2) PEG-chitosan nanoparticles can effectively protect the plasmid from DnaseⅠdigestion. (3) The protection of plasmid DNA nuclease degradation can be observed at PH = 3, 5.5, 7. At pH = 11.0, the obvious bands can be seen, suggesting that the nuclease protective effect decreased. 2. The appropriate conditions of transfecting PEG-chitosan nano Containing ACEshRNA on vascular endothelial cells2.1 Cultivate stable rat vascular endothelial cells were confirmed by immunofluorescence identification with cell survival rate of over 95%.2.2 Determination of transfection efficiency: (1) Using fluorescence microscopy we can see there is the expression of green fluorescence, while the naked plasmid group and blank control group were no fluorescent expression. (2) Flow cytometry transfection efficiency: 1) Without PEG-modified chitosan-based nanoparticles on the endothelial cells of plasmid transfection rate is low, up to (26.0±3.9)%, After PEG chemical modification, the transfection efficiency has significantly increased,up to (59.4±5.2)%. 2) The transfection efficiency was highest at the volume of 100ul, that is, the plasmid DNA content of 25ug. At a certain range, transfection efficiency and the amount transfection complex volume was proportional . Instead, transfection efficiency rates begin to decline with the volume of transfection increase 3) At fixed volume of transfection, as time increased, the transfection efficiency gradually increased and reached the peak at 72h (57.1±7.1)%, (p <0.05); It began to decreased at 96 hours , but still was significantly higher than at 0h (p <0.05).2.3 Determination of cytotoxicity: There was no significant difference compared with blank control group when transfection complexes volume was <100ul ,suggesting there were non-toxic to cells. The cell growth was significantly inhibited at the composite volume was 120 ul after transfected 24h, and with the time prolong inhibition gradually increasing (p <0.05).3. The inhibit of ACE expression after PEG-chitosan nano containing ACEshRNA transfected on rat vascular endothelial cells3.1 ACE gene mRNA expression changes before and after transfection in different groups: ACE mRNA expression levels decreased after 24 hours PEG chitosan nanocomposites plasmid transfection cells (14.7±5.9)%, up to (53.6±5.4)% after 48 hours, up to (60.1±2.1)% after 72-hour compared with the blank control group and the plasmid control group (all P <0.05).3.2 ACE protein expression changes before and after transfection in different groups: protein expression had no significant change in PEG-CS/DNA group at 24 hours after transfection , significantly reduced at 48 hours compared to blank control group and plasmid control group (P <0.05), reduced lower at 72 hours (P <0.01). ACE protein expression was no significant change in the blank control group and the control plasmid group at different time points before and after transfected, according to the RT-PCR results.3.3 The content of ACE and Ang-Ⅱin Vascular Endothelial Cell culture medium; ACE and Ang-Ⅱwere significantly reduced in culture medium in PEG-CS/DNA group after 72 hours transfection compared to the blank control group and the group of naked plasmid.(P <0.05),suggesting that PEG-CS nano-ACEshRNA transfection system can not only significantly reduced ACE gene at the molecular protein level but also on the function of a significant reduction in ACE content, reducing the synthesis of Ang-Ⅱ.4. The RNAi effect mediated by PEG-CS-nano containing ACE-shRNA in spontaneously hypertensive rats4.1 Tail arterial pressure and heart rate changes: The arterial pressure was no significant difference in each SHR group before the intervention (P> 0.05),. arterial pressure in each SHR group were significantly higher than that in normal blood pressure control group before intervention (P <0.01). In SHR gene therapy group at the first 3 days after injection, tail arterial pressure decreased significantly about (22±4) mmHg, there is significant difference compared with before treatment (P <0.05), after that the pressure declined slowly, sustained 8 days, and the biggest pressure drop-down was as 33mmHg. At about 11 days after injection, blood pressure began to rise; after second the injection, tail arterial pressure further significantly dropped about (24±5) mmHg, antihypertensive effect sustained 10 days, then blood pressure began rise, the biggest drop-down rate was about 25mmHg. The largest accumulated drop-down rate after second injection was up to 50mmHg. In Lotensin treatment group the blood pressure started to decrease after 3 days , continued to 13 days up to the maximum drop-down 39 mmHg, significantly lower than the maximum drop-down rate in gene therapy group (p<0.05). Followed by 7 days, no significant drop in blood pressure was observed in Lotensin treatment group. Tail arterial pressure continued to rise in SHR control group and the chitosan control group. In normal blood pressure control group no significant changes in arterial pressure were observed. There was no significant change in heart rate Rats in each group before and after treatment (P> 0.05).4.2 PEG-CS-EGFP-ACE-shRNA distribution in tissues and organs: The expression of green fluorescent substantial can be found in heart, aorta, kidney tissue, these tissures were known as rich in ACE.4. 3 The downward effect of ACE features of heart, kidney, aorta:after PEG - CS - EGFP - ACE - shRNA transfection 72h . 1) RT - PCR results: ACE mRNA expression of myocardium, aorta, renal was significantly lower in SHR gene therapy group than other SHR control groups, (P <. 05), but had no statistical difference compared with normal blood pressure control group (P>. 05)., suggesting with PEG - CS - EGFP - ACE - shRNA treatment can significantly reduce the expression of ACE mRNA. 2) Western blot results: ACE protein expression of myocardium, aorta, renal in SHR gene therapy group was significantly lower than those in other SHR control groups (P <. 05), no statistical difference compared to normal blood pressure control group. (P>. 05), according to RT - PCR results. 3) Serum ACE and Ang -Ⅱcontent: At 72 hours after transfection, in SHR ACE gene therapy group , ACE as well as Ang -Ⅱsignificantly reduced, which was lower than other SHR control groups. (P <. 05), had no statistical difference compared with normal blood pressure control group (P >. 05). In SHR drug treatment group, before and after treatment ACE had no significant change, but Ang -Ⅱsignificantly reduced, confirming ACEI drugs can inhibit Ang -Ⅱsynthesis.4.4 Influence of left ventricular structure and function: 1) Detection of left ventricular hemodynamic function: left ventricular diastolic pressure (LVDP), left ventricular end diastolic pressure (LVDEP) and maximal diastolic velocity (- dp / dtmax) in the each SHR group were higher than normal blood pressure group. Those in the blank control group, chitosan control group were significantly higher than those in the drug treatment group and the gene therapy group (P <. 05), suggesting that in the early stage of hypertension diastolic dysfunction existed. Gene therapy group and drug treatment group can be significantly improved diastolic dysfunction, there is obvious difference between the two groups, the former more significantly reduced LVDP and (- dp / dtmax), suggesting that PEG - CS - ACEshRNA can more significantly improved diastolic dysfunction. 2) The heart / body weight, left ventricular / body weight: the heart / body / weight and left ventricular / body weight was significantly higher in SHR control group and the chitosan control group than those in normal blood pressure blood pressure group (P <. 05), suggesting there is a clear cardiac hypertrophy in untreat SHR groups. The above indexes in SHR Drug treatment group and the gene therapy group were significantly decreased (P <. 05), the latter reduced more obviously, but didn't fell to normal levels in the control group blood pressure, suggesting compared with the ACEI drugs, PEG - CS - EGFP - ACE - shRNA more obviously reduce cardiac hypertrophy. 3) Histological observation: under light microscopy, myocardial cells hypertrophy were observed in SHR control group and SHR Chitosan control group than those in the normal control group. Myocardial hypertrophy were reduced significantly in SHR gene therapy group and drug treatment group. Observed under electron microscopy: myocardial cell membrane integrity, myofibrillar clear, with a more tidy, clear cross striations can be seen in SHR gene therapy group and the drug treatment group compared to those in SHR control group and chitosan control group. And mitochondria without swelling, a small amount of local increase in myocardial interstitial collagen fibers ,without obvious hyperplasia also can be found in both treatment groups,suggesting that both PEG-CS-EGFP-ACE-shRNA and ACEI drugs can improve the SHR myocardial ultrastructure changes.4.5 The liver and kidney function: in each group were no significant difference (P> 0.05).It showed that PEG-CS-nanoparticles containing ACE-shRNA had no effect on the liver and kidney functions.Conclusions1. We have prepared the PEG -chitosan nanoparticles containing ACEshRNA with uniform size, narrower particle size distribution. Under the condition of PH = 5.5 and the volume ratio 1:1, PEG chitosan nanoparticles as a gene vector have good drug encapsulation rate, a prolong release reaction in vitro release characteristics, and can effectively inhibit the nuclease degradation of plasmid DNA,Which provided a foundation for the later in vitro transfection on cultured cells and in vivo transfection experiments.2. After compared transfection efficiency rate under different conditions as well as surface modification with PEG we have gotten the appropriate transfection conditions: DNA25μg, DNA: CS nanoparticles volume ratio of 1:1, transfected for 72 hours.3. The RNA interference mediated by ACEshRNA PEG-CS-nanoparticles played a successful downward biology effect on ACE gene expression ,ACE inhibition and Ang-Ⅱsynthetic in cultured rat vascular endothelial cells, proved ACEshRNA PEG-CS-nanoparticles transfection vector can effectively carry gene into target cell and produce gene downward effect.4. ACE-shRNA-PEG-CS nanoparticles was injected into spontaneously hypertensive rats through the tail vein, which successfully played a role in gene silencing, effectively inhibited ACE mRNA and the corresponding functional protein expression. Compared to the traditional long-acting drugs ACEI , blood pressure was droped even more, one time drug delivery can maintained antihypertensive effect about 10 days, and significantly improved the myocardial remodeling and cardiac diastolic function. Therefore, the ACE-shRNA- PEG-CS-nanoparticles mediated RNA interference technology is a potential new strategy for the treatment of hypertension.