Polylysine copolymers for gene delivery
Polylysine copolymers for gene delivery
Polylysine and its copolymers have gained wide application as non-viral gene vectors. Although polylysine itself is toxic to cells, the covalent attachment of polyethylene glycol to the polylysine chain segments greatly reduces cytotoxicity and increases the efficiency of gene transfection. We have also synthesized a degradable analog of polylysine, polyaminobutyl glycolic acid, and an octadecanoylated stearylpolylysine for DNA delivery, the latter being strongly hydrophobic to low-density proteins. This ternary "terplex" system was shown to have significant levels of transfection both in vivo and in vitro.
Author: T. Friedman et al, Translated by W. Jing et al. This experiment is from "Gene Transfer".
Operation method
In vitro transfection of plasmid D N A Move In vitro transfection of plasmid D N A Material reagents A 7R 5 cell, murine smooth muscle cell line (American T y p e Cell Culture [A T C C ]) The cells were cultured in DMEM (Dulbecco minimal essential medium) medium containing 10 % fetal bovine serum at 37°C. 0.5 % of the total volume of the cell culture was used. CO2 incubator. Growth medium and fetal bovine serum (F B S ) (HyClone Laboratories) o-nitrophenyl-卩-D ■ galactose pyranoside (ONPG) Phosphate Buffer (P B S) Plasmid DNA, p S V -p-glactosidase control vector (6821b p , Promega) terplex complex Different complexes were prepared by varying P L L or L D L . Octadecylated PLL [synthesized by stearate bromo AT-alkylated PLL (M.W. 50 0 0 0 )] and varying amounts of LDL were mixed in P B S solution at room temperature (see Kim et al. 1998b for more details). Instrumentation. Cell culture tanks, controlled at 5 % C02 (e.g., Napco, Precision Scientific). Spectrophotometer Methods 1 . One day prior to transfection, A 7R 5 cells were inoculated in a 24-well plate at a concentration of 2 X IO4 cells/m l at 37°C at 5 %. 2. To determine the amount of solid plasmid DNA, mix DNA with terplex containing various components in a PBS solution and use lug of DNA per well in a 24-well plate for a single transfection. It is best to prepare it fresh before transfection! 3- Add 40ul of DNA solution (as control) or terplex containing PLL, LDL and plasmid DNA to each well. 4- Incubate the cells at 37°C, 5 % . The cells were incubated at 37°C, 5 % C02 for 48 h, washed three times with PBS, and collected for the detection of β-galactosidase activity. 5- The β-galactosidase activity of the transfected cells was measured by spectrophotometer at 420 nm using ONPG as substrate. reagents A 7R 5 cells, murine smooth muscle cell line (A m e r i c a n T y p e Cell Culture [A T C C ]) Cells were cultured in DMEM (Dulbecco minimal essential medium) medium containing 10 % fetal bovine serum at 37°C in a 5 % C02 incubator. CCD-32 L u , human lung fibrogenic cell line (A T C C ) The cells were cultured in 25 mm2 polystyrene culture flasks (T-25 culture flasks, Falcon) with EMEM (Eagle's minimal essential medium) medium containing 10 % fetal bovine serum. Trypsin digested and resuspended for experiments. Growth medium and fetal bovine serum (FBS) (HyClone Laboratories) M T T (3-[4, 5-dimethylthiazol-2-yl3-2, 5-diphenyltetrasodium bromide) CSigmaAldrich) Oligonucleotides Antisense oligonucleotides were synthesized by Midland Certified Reagent Co- by iminochemical method and purified by gel filtration terplex complex Different complexes were prepared by varying the amount of oligonucleotides. Stabilized terplex complexes were prepared by mixing octadecylated PLL [synthesized by stearobromo N -alkylated PLL OVL W. 50 000] and a fixed amount of LDL, along with varying amounts of oligonucleotides, in a PBS solution at room temperature (see Kim et al. 1998b for more details). Instrumentation Cell culture tanks, controlled at 5 % C02 (e.g., Napco, PrecisionScientific). Methods 1. One day prior to transfection, inoculate 9 6-well plates with A 7R 5 or C C I >32 L u cells at a density of 5 X 104 cells/well and incubate overnight at 37°C, 5 % C O 2 concentration. 2. Prepare different terplex complexes by varying the amount of oligonucleotide D N A . It is best to prepare them fresh before transfection! 3. Add the terplex complex to cultured cells and incubate the cells at 37°C, 5 % C0 2 for 4h, both with and without serum. 4- Cell proliferation was analyzed using the M T T method (M o s m a n n 1 9 83). reagents Buprenorphine Gsoflurane (isoflurane) Ketamine (50m g/kg, intramuscular injection) Phosphate Buffer (PBS, p H 7.4) Plasmid DNA, p CM V - V E G F Plasmid D N A was purified by a double CsCl gradient and confirmed by A 26qM 28 and 0.8 % agarose gel electrophoresis. 8 % agarose gel electrophoresis. Rabbit, N e w Z e a l a n d W h i t e (2. 5 ~ 3. 5k g ) (W e s t e m O r e g o n ) Color solution Hematoxylin and eosin (H & E) trichrome xylazine (8.8m g/kg, intramuscular injection) Instrumentation Blood tube insertion (angiocatheter) Electrocardiogram scanner Needle (30 gauge) Respirator (rodent ventilator, Harvard Apparatus) Standard planimetry software (NIHIMAGE) Suture (5-0 Prolene) Echocardiography development system (General Electric Vivid Five or Hewlett-Packard 5500) Methods I. Sedation of New Zealand White Rabbits. White rabbits of 2.5-3.5kg mass were selected and given intramuscular injections of Ketamine (50mg/kg) and Methylphenidate (8.8mg/kg). The rabbits were shaved and tubes were inserted into their windpipes. The number of animals to be prepared was determined depending on the purpose of the study. Randomly assign these animals to experimental or control groups (see Step 6). 2 . Surgical anesthesia with isoflurane was administered to the left chest of each animal under sterile washboard. The animals were immobilized on a ventilator with respiratory rates of 6 0 % and 4 0 % F i O 2 3.The left thorax was incised, the lungs indented and the pericardium opened. 4- A 5-0 suture is placed around the first branch of the spinothalamic artery, and an IO s occlusion test is performed to determine the ventricular area. 5 . Prepare the injection solution. For each experimental group of animals, prepare a 500ul injection containing 50ugLDL, 50 ug octadecylated PLL and 50 ugpCMV-VEGF, dissolved in PBS (PH 7 . 4 ). For each control animal, the injection was allowed to be used as a control solution. For each control animal, prepare 500ul of PBS injection containing 50ug LDL, 50 ug octadecylated PLL. Preferably, the injection solution should be freshly prepared. 6 . A 30-gauge needle is inserted into the myocardium and the prepared injection is injected into each experimental animal in 4 separate injections (12.5 ul each). 7. Immediately after injection, myocardial infarction was induced as described by Affleck et al. (2001). Myocardial atrophy was determined by visual inspection and electrocardiographic changes. 8. After a period of observation, temporary closed gas was exhaled to inflate the lungs. Evacuate the air remaining in the pleura with a cannula. 9. suture the ribs and muscle layers and skin. 10. Remove the ventilator tube when the animal can breathe naturally. Buprenorphine (0.05m g /k g , intramuscular) was injected for pain relief every 12 h for 48 h after surgery. 11- Rabbits were lightly anesthetized with ketamine (20m g /k g ) and mephenothiazine (5m g /k g ), and the electrocardiograms of the experimental animals were recorded on days 1 and 21 after surgery. In our study . In our study, cardiac echo maps were used as a blinded control for the treatment group. 12- Images were collected with a Hewlett-Packard 5500 or General Electric Vivid Five using a 12M H z probe In our experiments all presentations were acquired by an experienced ultrasonic cardiac tracing analyst and verified by another experienced ultrasonic cardiac tracing analyst. verification to ensure inter-observer and inter-imaging reliability by the same observer. The long-axis orientation of the right parietal sternum was used for 2D measurements of the expired volume fraction. Two-dimensional measurements of the excretion 13. On postoperative day 21, experimental animals were anesthetized by ketamine and isoflurane inhalation and bled to death. 1 4 . The heart was perfused with IOOml of regular saline from the aortic root and fixed by perfusion of the heart via neutral buffered formalin pressurization [IOOmmHg (Im m H g=I. 33 322X102P a ) ]. 1 5 . Sections were paraffin-encapsulated every 5m m from the short axis direction and stained with hematoxylin and eosin. 16- Measurement of normal and infarcted left ventricular myocardium using Standard planimetry software ( N I H I a m g e) For more product details, please visit Aladdin Scientific website.
The plates were incubated at 37°C with 5 % CO2 .
r m y b antisense oligonucleotide (I8m e r , 5^ G T G T C G G G G G G T C T C C C G G G C -3') mismatch (m i s m a t c h e d) antisense oligonucleotide (18m e r , 5 '-G T C T C C C C G G C T C C AC C C C C C C G G G ○ 3,)
Discharge fraction was obtained by manually recording the change in ventricular area at the end of diastole and at the end of systole. The measurements were repeated 3 to 5 times for each set of data, and the average values were used as the experimental results. The short-axis orientation of the right parietal of the sternum was used to characterize the annulus at the base, the medium-sized papillary structures, and the apex. M-mode measurements were derived from the partial contraction changes at each level so that the expulsion fraction was calculated. The reported expired volume fractions, partial systolic changes, and LV systolic and diastolic areas represent the mean of these values.
area.
■ Percent LV myocardial infarction is equal to the LV myocardial infarct area (all slices) divided by the total LV myocardial area and multiplied by 100.