Experimental design (HCI):
Objective:
To find out the optimum size of BSA nanosphere for drug delivery. The optimum BSA nanosphere will be
compared with the optimum nanofibers to see which type of nanoparticles is most effective as drugs
against Agrobacterium tumefaciens.
Justification for BSA nanoparticles: (Why protein nanoparticles?)
- Biodegradable polymers are sustainable and well controlled for targeted drug delivery. (Sustainable:
Antibiotics can be stored for long periods of time unlike liposomes, which cause antibiotics to leak out easily)
- Non-toxic, non-antigenic (No antigens on the protein; due to defined primary structure and high content of charged
amino acid, lysine) and biodegradable so that it does not accumulate indefinitely in tissues; no effect on
plant health
- Improve the therapeutic effects and reduce the side effects of the formulated drugs
- Ease of preparation by coacervation process
- Current techniques involve simply spraying plants with antibiotic. It is washed off, resulting in pollution
- Protein nanoparticles can pass through cell membranes, decreasing runoff and benefiting the
environment
- Nanoparticles act as vectors for drug delivery. It releases drugs slowly, reducing the frequency of
treatment. Lower frequency of treating a plant, allowing for a more economically efficient treatment. At
the same time, a reduction in dosage allows for lower resistance of bacteria against tetracycline and
ampicillin.
Hypothesis:
1. If antibiotics can be incorporated into BSA nanosphere, then the antibacterial properties of the
antibiotics will be present in the fibers that are formed.
2. If the nanosphere are prepared into an aerosol spray, then it will eradicate (suitable?) Agrobacterium
tumefaciens grown for in-vitro samples.
Constants:
1)
2)
3)
4)
5)
6)
Formation of nanoparticles: 30mg/ml of BSA, 3ml of BSA per ml of ethanol and 7.5 pH is used
Types of antibiotics: Ampicillin and Tetracycline
Type of bacteria (A. tumefaciens)
Volume of Agrobacterium (Refer to varun’s question on wikispace)
Size of potato strips and agar disk for in vitro testing
The temperature and humidity that the method of treatment is exposed to (To be standardize with AOS)
Independent Variable:
1) Temperature (4oC-35oC; 5 oC, 10 oC, 15 oC, 20 oC, 25 oC, 30 oC, 35 oC)
Dependent Variable:
The size of the nanosphere, which would affect the efficacy of the nanosphere against A. tumefaciens grown in invitro samples. There are two samples: one that measures the zone of inhibition of the treatment in agar discs, and
another that measures the efficacy of the treatment by growing A. tumefaciens on potato discs and using a
spectrophotometer.
Control:
-
Control solution (LB broth with no bacteria) to establish baseline for spectrophotometer
Water (negative control) and ethanol (positive control) will be added in the agar wells to see its
impact on the bacteria colony
Repeated Trials:
1) Nanosphere will be formed in 7 different temperatures to obtain different sized nanosphere. The
antibiotic (Ampicillin and Tetracycline) will be loaded into the different nanosphere formed at
different temperatures. For each temperature range, the antibiotic loaded nanosphere are then
divided into 4 equal parts. One of them will be sent for characterization. The other three will be
used for in-vitro testing.
Plate 1: Water, BSA, antibiotic loaded nanosphere and nanosphere
Plate 2: Ethanol, BSA, antibiotic loaded nanosphere and nanosphere
Plate 3: Antibiotics, BSA, antibiotic loaded nanosphere and nanosphere
Since each plate can hold a maximum of four wells, we will follow this method. It allows us to perform
triplicates to show the effect of antibiotic loaded nanosphere. The effect of water, ethanol, and antibiotics
can be seen too.
In total, there will be 28 samples required, of which 7 will be sent for characterization and 21 would be
used for experimentation.
Materials:
Chemicals: (to be obtained)
-
Bovine Serum Albumin: (98% concentration product number A6003 from Sigma Aldrich)
Ampicillin: (?)
Tetracycline : (98% concentration dihydrate product number 75965 from Sigma Aldrich)
Anhydrous ethyl alcohol
glutaraldehyde
Ethanolamine
Tween-20 (Polysorbate 20)
acetate membrane
polyvinylchloride copolymer membrane
Coomassie Blue reagent
Equipment:
-
Scanning Electron Microscope (at NUS)
Spectrophotometer (at Lab)
SpectaPor cellulose dialysis tube (where?)
Camera
Microbes:
-
Agrobacterium tumefaciens grown in LB broth agar. Methodology of growing bacteria?
Methodology:
Formation of Nanosphere (Coacervation technique)
Coacervation technique was implemented for preparation of BSA nanoparticles.
1) Anhydrous ethyl alcohol was added to 150 ml BSA (5 mg/l in 10 mM Tris/HCl contained 0.02%
sodium azide, pH 7.5) What does it mean?) till the solution became turbid, then 150 μl of 25%
glutaraldehyde was added for cross linking. The reaction was continued at different temperatures
each repeat (5 - 35°C). E.g: 5oC, 10oC, 15oC, 20C, 25oC, 30oC, 35oC.
2) Ethanolamine was added to block the non-reacted aldehyde functional group.
3) Tween-20 (Polysorbate 20) was added at a final concentration of 0.01% v/v to stabilize the
preparation.
4) Large aggregates were eliminated by centrifuge (50, 000 g, 30 min, 4°C).
5) Remove the supernatant (assuming that the nanospheres are inside the solution)
6) Nanoparticles were dried in vacuum oven at 25 ◦C for 1 day.
7) The supernatant was dialyzed and subsequently micro and ultra-filtrated through an acetate
membrane and polyvinylchloride copolymer membrane with cut off of 0.2 μm and 300 kDa,
respectively. The concentration of BSA determined with Coomassie Blue reagent. ???
(What equipments are used? (SDS-page; 200rpm); )
8) Repeat steps 2-6 for different temperatures of the reaction. Obtain 4 samples for each temperature,
providing 28 samples of nanoparticles.
9) 60mg of nanoparticles (PALA or BSA) were introduced in a SpectaPor cellulose dialysis tube (cut-off
of 12,000–14,000Da) and kept in contact with a 0.04M antibiotic solution for 24 h at room
temperature.
10) Nanoparticles were washed with deionized water for 2 hours to remove the un-bonded antibiotic.
11) The amount of adsorbed antibiotics was evaluated by UV–vis spectroscopy by checking the
absorbance of the antibiotic solution at 270 nm before and after the contact with the nanoparticles
and by comparing it with a standard curve (absorbance vs. concentration).
12) The amount of drug in supernatant was then subtracted from the total amount of drug added
during the formulation (W). Effectively, (W - w) will give the amount of drug entrapped in the pellet.
Characterization of nanosphere
Determination of particle size by photon correlation spectroscopy
The size distribution of BSA nanoparticle was analyzed by photon correlation spectroscopy (PCS). PCS
determined the average particle size and Polydispersity Index (PI) which is the measurement for the range
of particle sizes within the measured sample particles. The sample analyzed in PCS device should consist of
well dispersed particles in liquid medium.
In the specified condition, the particles possess are in constant random motion, which is refer to Brownian motion using
Einstein-diffusion equation stated above:
Determination of particle morphology
The morphologies of the BSA nanoparticles were observed by Scanning Electron Microscope (SEM)
provided by NUS.
Determination of Particle Purity
The purity of BSA nanoparticles was investigated by Fourier Transform Infra-Red Spectroscopy (FTIR).
Testing efficiency of BSA-antibiotic nanosphere:
Spectrophotometry and turbidity
1.
2.
3.
4.
5.
6.
7.
8.
9.
Using a inoculating loop, taken sterile from the package, inoculate the A. tumefaciens onto a potato
in vitro culture contained within a Petri dish
Place Petri dish into a incubator machine at maximal growing temperature and leave for 2-4 days
Prepare one solid colony from which additional Petri dishes can be easily and readily cultured
Place a 1 cm by 1 cm square of BSA-antibiotic fiber onto the potato in vitro disk
Leave disks for 2-4 days
Scrape off the surface of the potato strips, trying to minimize the amount of potato and maximizing
the amount of bacteria, and place in a test tube
Place test tube into the spectrophotometer, and determine the optimum wavelength for testing
Establish a frame of reference by finding the absorbances of known concentrations to predict and
compare the concentrations of bacteria in a specific solution
Perform step 4 for each varying concentration of the BSA-antibiotic nanosphere
Zone of inhibition
1.
2.
3.
4.
5.
Prepare Petri dish with agar (gel form)
Streak surface with A. tumefaciens through the use of an inoculating loop
Leave dish in incubator for 2-4 days
Place 1cm by 1cm squares of mesh in designated areas of the dish, approximately four per dish
Leave treatment for two days, measuring the zone of inhibition that is formed around each square
(if at all any zone is formed)
Notes/Queries:
1) Graphs required:
- Size of nanoparticle vs antibiotics loading
- Loading efficiency vs amount of antibiotics incubated
- Size of nanoparticles vs temperature
2) Bacteria can either consume the nanoparticle; nanoparticle can diffuse into the bacteria;
Thus, in the case that bacteria consumes the nanoparticle, what is the enzyme that may digest it?
3) We may be unable to include glucose for our nanosphere since glucose may or may not attach to the
nanoparticles.
5) Formation of bacterium: (Do we have our own methods?; After growing bacteria, we have to send them for the
spectrophotometry as a basis for comparison, we will be using the same method to determine cell turbidity
for the potato strips)
“Making competent cells of Agrobacterium tumefaciens
Inoculate 5 ml LB broth amended with Rif 100/Tet 5* with a colony of A. tumefaciens or 15 μl of A tumefaciens
glycerol stock. We have used two strains, AGL-1 and C58C1. AGL-1 works much better. Incubate overnight at
28°C shaking 250 RPM. Next day inoculate 50 ml LB broth amended with Rif 100/Tet 5 in a sterile 250 ml
flask with 2 ml of the above overnight culture. Make glycerol stock of the remainder (850 μl of culture and
150 μl of sterile glycerol). Incubate flask at 28°C shaking 250 RPM for ~8 hours to OD 600 nm of 0.6. Chill the
flask on ice for 5 minutes. Pellet cells in 50 ml Falcon tubes at 3000 RPM at 4°C for 5 min. Resuspend cells in
1 ml of 20 mM CaCl2. Make 100 μl aliquots and store at –80°C (use 50 μl of cells per transformation)
*Antibiotic Stock Solutions
These antibiotics are used to reduce bacterial contamination since A. tumefaciens grows slowly. However, the AGL-1
strain does not grow with these antibiotics, so they should not be used if strain AGL-1 is being used.
Rifamipicin 25 mg/ml in 100% methanol (add 4 ml/L LB = Rif 100) Tetracycline 5 mg/ml in ethanol (add 1
ml/L LB = Tet 5
Store the antibiotic solutions at –20°C
Add all antibiotics after autoclaving LB
6) Questions by AOS:
1) What is the agar made of, and what MSDS's/potentially hazardous chemicals should we be aware of?
2) What form of the antibiotics are we using (the MSDS's for the different forms are different, so we need to know)
7) What about Mdm Mary Ng Mah Lee?