Preparation of tetracycline-loaded chitosan-alginate polyelectrolyte complex (Tet/CS-AG PEC)

nanoparticles
Reagents Materials/Equipment
Sodium Alginate Volumetric Flask, 100-mL Magnetic Stirrer
Chitosan Volumetric Flask, 25-mL Stir Bar
Distilled Water Dropper pH Meter
Acetic Acid, 2% Syringe, 25-mL Syringe Pump
Sodium Hydroxide, 1M Needle, Gauge 27 Centrifuge
Tetracycline Beaker, 250-mL Centrifuge Tube
Pipet

Preparation of 2% (w/v) Acetic Acid

Preparation of 1M Sodium Hydroxide
Preparation of 0.4% (w/v) Chitosan
Preparation of 0.2% (w/v) Sodium Alginate
Preparation of Nanoparticles
Add 25 mL of the 0.4% (w/v) chitosan solution dropwise over 90 minutes using a syringe
pump into the alginate solution through a 27-gauge needle while 50 mL of the 0.2% (w/v) alginate
solution is concurrently magnetically stirred at a speed of 300 rpm.
The resulting mixture will be stirred magnetically for 1 hour to improve curing and
separation of nanoparticles then allowed to settle over 24 hours.

Solutions of sodium alginate and chitosan with final concentrations of 0.2% and 0.8% (w/v)
respectively will be prepared by dissolving each polymer in distilled water. Because chitosan is
insoluble in neutral solutions, the pH of the chitosan and alginate working solutions will be
adjusted to 5.4 and 5.2, respectively with 2% acetic acid (w/v) and 1M sodium hydroxide. The
solutions will be magnetically stirred overnight at a speed of 300 rpm at room temperature to
ensure complete dissolution. Solutions will be filtered through 0.22-um syringe filter prior to use.
For preparing the blank nanoparticles, This will result into a solution with a mass ratio of
1:1 chitosan to alginate.
The solution with precipitated nanoparticles will be centrifuged at 4,800 rpm for 5 minutes
in accurately weighed Eppendorf vials to separate the free polymers from the nanoparticles. The
supernatant will be removed cautiously with a pipet and saved for later analysis of its tetracycline
content. The precise weight of the recovered nanoparticles will be calculated by accurately
weighing the Eppendorf vials after the removal of the supernatant. The particles in the centrifuge
tube will be washed by adding 5 mL ultrapure water and the tube will be again centrifuged at 4,800
rpm for 5 minutes. The second supernatant will be removed and discarded if preliminary tests
shows that the second supernatant contains no tetracycline residue. Finally, the particles will be
resuspended in 5 mL ultrapure water before further experimentation.
 NANOPARTICLE PREPARATION DATA SHEET
Chitosan, Sodium Alginate,
Tetracycline
Preliminary Trial 0.8% (w/v) 0.02% (w/v) Remarks
(mg)
(mL, pH) (mL, pH)
Trial 1
Trial 2
Trial 3
Chitosan, Sodium Alginate,
Tetracycline
Blank 0.8% (w/v) 0.02% (w/v) Remarks
(mg)
(mL, pH) (mL, pH)
NP-B-1
NP-B-2
NP-B-3
NP-B-4
NP-B-5
NP-B-6
NP-B-7
NP-B-8
NP-B-9
NP-B-10
NP-B-11
NP-B-12
Chitosan, Sodium Alginate,
1 Polymer : Tetracycline
0.8% (w/v) 0.02% (w/v) Remarks
0.5 Tetracycline (mg)
(mL, pH) (mL, pH)
NP-0.5-1
NP-0.5-2
NP-0.5-3
NP-0.5-4
NP-0.5-5
NP-0.5-6
NP-0.5-7
NP-0.5-8
NP-0.5-9
Chitosan, Sodium Alginate,
1 Polymer : Tetracycline
0.8% (w/v) 0.02% (w/v) Remarks
1 Tetracycline (mg)
(mL, pH) (mL, pH)
NP-1-1
NP-1-2
NP-1-3
NP-1-4
NP-1-5
NP-1-6
NP-1-7
NP-1-8
NP-1-9
NP-1-10
NP-1-11
NP-1-12
NP-1-13
NP-1-14
 NP-1-15
NP-1-16
NP-1-17
NP-1-18
NP-1-19
NP-1-20
NP-1-21
NP-1-22
NP-1-23
NP-1-24
NP-1-25
NP-1-26
NP-1-27
NP-1-28
NP-1-29
NP-1-30
NP-1-31
NP-1-32
NP-1-33
NP-1-34
Chitosan, Sodium Alginate,
Tetracycline
za 0.8% (w/v) 0.02% (w/v) Remarks
(mg)
(mL, pH) (mL, pH)
NP-2-1
NP-2-2
NP-2-3
NP-2-4
NP-2-5
NP-2-6
NP-2-7
NP-2-8
NP-2-9

Films Total Preparations

Blank 13
NPC 16
Tet 16

Outsourced Test Total Preparations

Blank 12
1:0.5 9
1:1 34
1:2 9
Films Total Preparations
Blank 13
NPC 16
Tet 16
Preparation of Tet/CS-AG PEC nanocomposite poly(vinyl alcohol) films and tetracycline-loaded
poly(vinyl alcohol) films

Reagents Materials/Equipment
Poly(vinyl alcohol) Beaker Magnetic Stirrer
Distilled Water Volumetric Flask, 25 mL Stir bar
*Nanoparticle Suspension Polypropylene Petri Dish Sonicator
Tetracycline Pipet oven

This procedure is adapted from the methods employed by Niamlang et al. (2017), Pei et al.
(2008) and Giovino et al. (2012). Poly(vinyl alcohol) will be dissolved in 20 mL of distilled water at
90 ºC for 4 hours and then cooled down to room temperature to obtain a concentration of 5%
(w/v). After that, the nanoparticle suspension (5 mL) with 1:1 tetracycline-polymer mass ratio will
be added in the poly(vinyl alcohol) solution and then sonicated for 20 min in order to uniformly
disperse the nanoparticles. The homogeneous solution will be cast on a Teflon film-coated glass
plate measuring 9.5 by 9.5 cm and then dried at a temperature of 35 ºC for 48 hours in a dark
temperature-controlled incubator to avoid photodecomposition of tetracycline.
For drug release comparison, tetracycline-loaded poly(vinyl alcohol) films will be prepared
by adding tetracycline of equal amount to the encapsulated drug in 20 mL of 5% (w/v) poly(vinyl
alcohol) solution and then fabricated by the same casting procedure.
Encapsulation Efficiency

This procedure is adapted from the methods employed by Niamlang et al. (2017) and Cover
et al. (2012). Incorporation of tetracycline into the particles will be characterized by measuring the
tetracycline contained in the centrifugation supernatant. Since the total amount of drug in each
formulation batch was known (100 mg of tetracycline), any tetracycline not found in the
supernatant could be assigned to the particles.
The encapsulation efficiency of tetracycline-loaded chitosan-alginate polyelectrolyte
complex nanoparticles will be determined by measuring the absorbance of the collected
supernatant. The amount of tetracycline will be measured at 275 nm using UV-Vis
spectrophotometry. The results will then be calculated to obtain the amount of free tetracycline
in the supernatant. The encapsulation efficiency will be calculated by the following equation:
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑇𝑒𝑡𝑟𝑎𝑐𝑦𝑐𝑙𝑖𝑛𝑒 𝑎𝑑𝑑𝑒𝑑 − 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐹𝑟𝑒𝑒 𝑇𝑒𝑡𝑟𝑎𝑐𝑦𝑐𝑙𝑖𝑛𝑒 𝑖𝑛 𝑆𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡
𝐸𝐸(%) = 𝑥100%
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑇𝑒𝑡𝑟𝑎𝑐𝑦𝑐𝑙𝑖𝑛𝑒 𝑎𝑑𝑑𝑒𝑑
Moisture Content
This procedure is adapted from the method employed by Soradech et al. (2012). For this,
each film sample will be cut into 3 by 3 cm and dried in an oven at 100 ºC for 24 h. Before and
after drying, the weight loss was measured as water content and expressed as a percentage, based
on the initial weight of film calculated from the equation:
𝑊𝑒𝑖𝑔ℎ𝑡 𝑏𝑒𝑓𝑜𝑟𝑒 − 𝑊𝑒𝑖𝑔ℎ𝑡 𝑎𝑓𝑡𝑒𝑟
𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝐶𝑜𝑛𝑡𝑒𝑛𝑡 (%) = 𝑥100%
𝑊𝑒𝑖𝑔ℎ𝑡 𝑎𝑓𝑡𝑒𝑟
Water Uptake
This procedure is adapted from the method employed by Pei et al. (2008) and Sharma et
al. (2012). Film samples 3 by 3 cm will be stored in desiccators to constant weight before testing.
The water uptake capacity of the films will be determined gravimetrically by swelling the films in
pH 7.4 phosphate-buffered saline solution at room temperature for 24 hours. The swollen films
will be removed, carefully blotted with filter paper to remove excess surface water, and
immediately weighed. The procedure will be repeated until the membrane attains constant
weight. The water uptake will be calculated from the following formula:
𝑊𝑒𝑖𝑔ℎ𝑡 𝑎𝑓𝑡𝑒𝑟 − 𝑊𝑒𝑖𝑔ℎ𝑡 𝑏𝑒𝑓𝑜𝑟𝑒
𝑊𝑎𝑡𝑒𝑟 𝑈𝑝𝑡𝑎𝑘𝑒 (%) = 𝑥100%
𝑊𝑒𝑖𝑔ℎ𝑡 𝑏𝑒𝑓𝑜𝑟𝑒
Water Vapor Permeability
This procedure is adapted from the method employed by Hosseini et al. (2016). The water
vapor permeability of the films will be performed according to the ASTM E96-05 method, as
modified by McHugh, Avena-Bustillos, and Krochta (1993). Circular glass cups with a diameter of
49 mm and a depth of 1.1 cm will be used. The film samples will be sealed onto the cup mouth
containing 6 ml distilled water (100% RH; 2.337 x 103 Pa vapor pressure at 20 ºC), then placed in
a desiccator containing silica gel at 20 ºC and 0% RH (0 Pa vapor pressure). The water transported
through the film and adsorbed by the desiccant will be the weight loss of the glass permeation cell
(measured every 2 hours for 10 hours). Weight loss over time will be plotted to obtain the slope
(r2>0.99). The measured water vapor permeability of the films will be calculated using the
following equation:
𝑊𝑉𝑇𝑅 𝑥 𝐿
𝑊𝑉𝑃 =
𝛥𝑃
where WVTR is the water vapor transmission rate (g mm/kPa h m2) calculated from the slope of
the straight line divided by the exposed film area (m2), L is the film thickness (mm), and ΔP is the
water vapor pressure difference (kPa) between the two sides of the film.
In Vitro Release of Tetracycline
This procedure is adapted from the method employed by Niamlang et al. (2017). Whole
Tet/CS-AG PEC nanocomposite poly(vinyl alcohol) films, tetracycline-loaded poly(vinyl alcohol)
films, and Tet/CS-AG PEC nanoparticle suspensions (5 mL) will be immersed for 1, 2, 3, 4, 24, 48,
72, 96,120, and 144 hours in both 25 mL pH 7.4 phosphate-buffered saline solution and pH 5.5
acetate buffer. The tetracycline released in the phosphate-buffered saline solution and acetate
buffer will be measured by UV-Vis spectrophotometry at 270 nm and 276 nm, respectively. Based
on the equation obtained for tetracycline from the standard curves, the concentrations of
tetracycline released at different times will be calculated. The percentage of tetracycline released
will be calculated based on the following equation:
𝑅𝑒𝑙𝑒𝑎𝑠𝑒𝑑 𝑇𝑒𝑡𝑟𝑎𝑐𝑦𝑐𝑙𝑖𝑛𝑒 𝑎𝑡 𝑇𝑖𝑚𝑒𝑝𝑜𝑖𝑛𝑡
𝐷𝑟𝑢𝑔 𝑅𝑒𝑙𝑒𝑎𝑠𝑒 (%) = 𝑥100%
𝑇𝑜𝑡𝑎𝑙 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐿𝑜𝑎𝑑𝑒𝑑 𝑇𝑒𝑡𝑟𝑎𝑐𝑦𝑐𝑙𝑖𝑛𝑒