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Products for this protocol

• Acetylated BSA (Ac-BSA; Sigma-Aldrich)
   
  BSA is used to facilitate the release of encapsulated DNA from NPs. Specifically, acetylated BSA is used because it is nuclease-free and thus will help prevent DNA from degrading.
   
  
• Cell culture lysis reagent (CCLR; Promega)
   
  Triton X-100 can be used as an alternative.
   
  
• Cells to be transfected
   
  
• Chloroform
   
  
• Isopropyl alcohol
   
  
• Luciferase assay substrate (if using luciferase as a marker gene; see Step 25) (Promega)
   
  
• Media for cells:
   
  
  • Complete growth medium (RPMI 1640 or other medium, depending on the cell line) containing 10% (v/v) FBS
     
    
  • RPMI 1640 or any appropriate serum-free medium, depending on the cell line
     
    
• MicroBCA Kit (Pierce) (see Step 25)
   
  
• Phosphate-buffered saline (PBS, pH 7.4), cold
   
  
• Plasmid DNA (10 mg/mL)
   
  
• PLGA polymer (50:50 polylactide:glycolide ratio, inherent viscosity 1.32 dL/g in hexafluoroisopropanol; Birmingham Polymers, Inc.)
   
  The polymers PLGA and PLA are available in different molecular weights and compositions; each composition has a different degradation rate, which can be selected to achieve the desired release rate and duration of gene expression.
   
  
• Polyvinyl alcohol (PVA) (m.w. 30,000-70,000; 87%-89% hydrolyzed; Sigma-Aldrich)
   
  PVA is a commonly used emulsifier in the formulation of PLGA NPs because the particles formed are relatively uniform and small in size. They can also be redispersed easily in aqueous media because of surface-associated PVA (Sahoo et al. 2002). Furthermore, the type (molecular weight and degree of hydrolysis) of PVA used for emulsification influences NP surface properties, DNA loading, and particle size, and thus gene transfection. PVA with a molecular weight (m.w.) of 30,000-70,000 and 87%-89% hydrolyzed was found to be optimal for the NP formulation used for gene transfection (Prabha and Labhasetwar 2004a).
   
  
• Tris-EDTA buffer (pH 8.0, autoclaved)
   
  

• Aspirator
   
  
• Centrifuge at 4°C
   
  
• Centrifuge tubes (50 mL)
   
  
• Chemical fume hood
   
  
• Equipment for lyophilization
   
  
• Filter (0.22 µm; Millipore)
   
  
• Glass vial (5 mL)
   
  
• Ice bath
   
  
• Incubator preset to 37ºC, 5% CO₂
   
  
• Magnetic stir plate
   
  
• Microcentrifuge tubes
   
  
• Plate (24 well)
   
  
• Shaker-incubator, preset to 37ºC
   
  
• Sonicator, microtip (staged-type) probe (XL 2015 sonicator ultrasonic processor; Misonix Inc.)
   
  
• Ultracentrifuge at 4°C
   
  
• UV spectrophotometer
   
  
• Vacuum desiccator
   
  
• Vortex
   
  

Preparation of Solutions
 

1. Prepare the polymer solution: Dissolve 30 mg of PLGA in 1 mL of chloroform in a 5-mL glass vial with magnetic stirring.
    
   It takes 3-4 h to dissolve the polymer completely.
    
   
2. Prepare the plasmid DNA solution as follows:
    
   
   1. Mix 1 mg of plasmid DNA solution (10 mg/mL) and 200 µL of Tris-EDTA buffer.
       
      
   2. Add 2 mg of nuclease-free Ac-BSA without vortexing the tube. Gentle intermittent tapping of the tube helps dissolve the BSA.
       
      It takes ~3 h to dissolve the BSA completely. Alternatively, the DNA solution can be kept overnight with BSA at 4°C to allow the BSA to dissolve completely.
       
      
3. Prepare a 2.0% (w/v) PVA solution as follows:
    
   
   1. Sprinkle 0.2 g of PVA (m.w. 30,000-70,000) slowly over 10 mL of cold Tris-EDTA buffer while stirring on a magnetic stirrer. It takes ~30 min to dissolve the PVA.
       
      
   2. Centrifuge the PVA solution at 1000 rpm for 10 min at 4ºC.
       
      
   3. Filter through a sterile 0.22-µm filter to remove any undissolved PVA from the solution.
       
      
   4. Add 10 µL of chloroform to saturate the PVA solution.
       
      
Nanoencapsulation of DNA
 

4. Add the plasmid DNA solution (as prepared in Step 2) to the polymer solution in two aliquots of 100 µL each, vortexing for 1 min after each addition.
    
   This forms a water-in-oil emulsion.
    
   
5. Sonicate the emulsification as follows:
    
   
   1. Clean and rinse the sonicator probe with isopropyl alcohol.
       
      
   2. Using a microtip (staged-type) probe sonicator set at 55-W energy output, sonicate the emulsion for 2 min over an ice bath. During sonication, place the probe approximately in the middle of the emulsion and avoid contact with the wall of the tube.
       
      This process reduces the droplet size of the emulsion (primary emulsion).
       
      
6. Add this primary emulsion in two portions to 6 mL of PVA solution in a 50-mL centrifuge tube, vortexing for 1 min after each addition.
    
   This forms a water-in-oil-in-water double emulsion.
    
   
7. Sonicate this double emulsion as in Step 5, but for 5 min.
    
   
8. Stir the resulting emulsion overnight (~18 h) in the same tube at room temperature inside a chemical hood with gentle magnetic stirring to allow chloroform to evaporate. Avoid excessive turbulence.
    
   
9. To ensure complete removal of chloroform, stir the resulting suspension of NPs in a vacuum desiccator placed on a magnetic stir plate for an additional hour.
    
   
10. Recover NPs by ultracentrifugation at 110,000g for 20 min at 4°C. Remove and collect the supernatant.
     
    
11. Resuspend the pellet in 5 mL of Tris-EDTA buffer, first by flushing the buffer over the pellet repeatedly until the complete pellet is suspended and then by sonicating the suspension for 30 sec over an ice bath as described in Step 5.
     
    
12. Repeat Steps 10 and 11 to remove unencapsulated DNA and PVA.
     
    
13. Use the supernatant in Step 10 and the washing in Step 12 to determine the amount of DNA that is not entrapped, by measuring the absorbance at 260 nm in a UV spectrophotometer. Use the supernatant and washing from the NPs, but without DNA, to zero the instrument.
     
    
14. Resuspend the pellet in ~1-2 mL of sterile H₂O and sonicate the suspension for 1 min.
     
    
15. Centrifuge the NP suspension at 1000 rpm for 10 min at 4°C to remove any large aggregates of NPs if present.
     
    
16. Collect the supernatant in a preweighed sterile microcentrifuge tube(s) (make aliquots if necessary), freeze for 45 min at -80°C, and then lyophilize for 2 d in a vial/container to obtain a solid dry powder. Store the lyophilized NPs at 4°C.
     
    The difference in the weight of the tube in the absence and presence of NPs gives the yield of NPs. These NPs are negatively charged at physiological pH, do not undergo aggregation in the presence of serum in media, and are nontoxic when tested at up to 1000 µg/mL concentration in different cell lines.
     
    
Transfection
 
In this procedure, the encapsulated DNA is released slowly inside cells due to degradation of the polymeric matrix of NPs, thus resulting in sustained gene delivery and hence gene transfection (Prabha and Labhasetwar 2004b).
 

17. Seed cells to be transfected in a 24-well plate at a density of 3.5 x 104 cells/well/mL in complete growth medium (containing 10% [v/v] serum). Keep n = 6 wells for each sample of NPs. Seed cells at the same density for a control lane, to which no NPs will be added.
     
    
18. Allow the cells to attach and grow in the plate for 24 h.
     
    
19. Use sterile conditions to prepare a stock suspension of DNA-loaded NPs (4 mg in 0.5 mL of RPMI 1640 or in any appropriate serum-free medium, depending on the cell line). Sonicate in a water bath sonicator for 10 min.
     
    
20. Dilute the NP suspension to 9 mL with RPMI 1640 (or suitable medium for cell growth) containing 10% FBS.
     
    
21. Aspirate the medium from the wells and add 1 mL of NP suspension to each well.
     
    
22. Incubate the plate for 24 h at 37ºC in 5% CO₂.
     
    
23. Aspirate the NP suspension from the wells and replace with fresh medium. Thereafter, replace medium on alternate days, with no further addition of NPs.
     
    
24. Prepare cell lysates at appropriate time intervals to use for measurement of gene expression levels:
     
    
    1. Wash the cells twice with cold PBS (pH 7.4).
        
       
    2. Add 0.1 mL of 1X CCLR or 1.0% (w/v) Triton X-100 solution per well.
        
       
    3. Shake the plates in a shaker-incubator for 30 min at 37ºC to allow for cell lysis.
        
       
25. Determine the gene expression levels in the cell lysates.
     
    If luciferase is used as a marker gene, proceed as follows:
     
    
    1. Measure the expression levels using chemiluminescence intensity with a luciferase assay substrate.
        
       
    2. Determine the protein concentration using the microBCA kit.
        
       
    3. Normalize the levels of luciferase gene expression per milligram of cell protein.