Negatively And Positively Charged Liposomes Biology Essay

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Several types of liposomes can be prepared, including different sizes and membrane charges. In particular, by applying a saline solution in their preparation, negatively charged liposomes are obtained, while the use of chitosan yields positively charged ones.

In this study our aim was to encapsulate curcumin and resveratrol inside of liposomes. in order to reach to the best liposome size, drug loading, in vitro release and stability we are going to test the effect of different polymer composition and charges, on the size and stability of liposome. in addition we were going to investigate different parameters for formulation of curcumin and Resveratrol liposomes. Particle size, polydispersity and charge were the measured parameters.

We intended to study the effect of liposomes on stability of curcumin and resveratrol by using phosphate buffer assay.

In addition we are going to measure amount of encapsulated curcumin and resveratrol inside of liposomes by using HPLC chromatography technique (quantification and Identification). AUD, retention time and correlation coefficients were the measured parameters.

Basis of techniques used:


Chromatography is a dynamic physicochemical method of separation in which the components to be separated are distributed between two phases, one of which is stationary [the stationary phase] while the other [the mobile phase] moves relative to the stationary phase. High performance liquid chromatography is one of the most powerful means in analytical chemistry. It has the ability to separate, identify, and quantifies the compounds that are present in any sample that can be dissolved in a liquid. (1)


There are two common devices for ultrasound application, namely bath and probe units. There are 2 techniques of sonication(8). Either the tip of sonicator is immersed into the liposome dispersion or a sample in tube or beaker is placed in o the bath sonicator. Ultrasonic probes have the advantage over ultrasonic baths in that they focus their energy on a localized sample zone, thereby providing more efficient cavitation in the liquid(8). sonication is still the most widely used method for preparation of SUVs on a small scale. This method applies high energy input into lipid dispersions and can be applied directly to MLVs. The dissipation of energy at the tip results the local overheating, consequently the vial of sample must be immersed into an ice-water bath. (7)

Sonicated vesicles have diameters of <50 nm, depending on such experimental variables as the type and concentration of the surfactant, age of the vesicles, and the concentration of added electrolyte (Kaler et al., 1982).

Size analysing and Z-potential:

This technique is based on the non-invasive and highly sensitive dynamic light scattering concept to measure the size, zeta-potential and poly-dispersity of nano-particles such as liposomes.

Rotary Evaporation:

Heidolph Rotary Evaporators are especially used for standard distillations, crystallization, product concentration, powder drying and separation of one or several solvents. High distillation rates and available vacuum/temperature programmability guarantee the fastest and most economical results.

Ethical and Safety Considerations:

During all the experiments carried out in laboratory, lab coat and safety glasses were worn at all times. The solutions were carried with care. For protection hands were kept out of the dangerous zone of rotating flasks, steam and slashing bath fluids.


All materials and reagents were supplied by sigma-aldrich

Equepments: Rotary evaporator Heidolph , Probe Sonicator (Soniprep 150), HPLC, Needle, vials, Syringe, Filter (0.45 μm)

Chemical substances: methanol, chitosan (chitooligosaccharide powder, pure water), distearoylphosphatidylethanolamine [DSPE]-PEG, curcumin, Resveratrol, Lecithin (lipoid SPC-3), saline (NaOH, pure water)




Rotary evaporator heidolph


Syringe 0.45 µm filter


In pharmaceutical laboratory

All of the preparation is done just by curcumin. The liposomal resveratrol has not been prepared and examined yet!!

Preparation of hand shaken multi lamellar lipid vesicle(MLV)

Week 1: we were going to prepare negatively and positively charged samples of liposomes. First we weighed 20mg curcumin and 200 mg Lecithin in a weighing boat. We mixed these 2 powders in a round-bottom flask with 5 ml methanol. The flask was placed and rotated at 100 rpm in a thermostatically controlled water bath at 60°C and pressure of 20 mbar for 10-15 min in Heidolph Rotary Evaporator. The sample dried in a rotary evaporator.

We mixed chitooligosaccharide powder (polymer coating) with 10ml pure water to prepare chitosan solution.

After drying of flask by rotary we added 10 ml of chitosan solution to the flask and shaking flask for about 5min. Then we tranfered the solution from flask to vial and put it in a beaker filled by ice and placed it in the sonicator to obtain a dispersion of uniform size for only 1min. Sonicator reduces the size liposomes . After sonication we filtered it by 0.45µm filter for sterilization and also to see are the liposomes size less than 0.45µm or bigger. The sample did not pass through filter. This sample is liposomal curcumin but due to addition of chitosan it is positively charged. We did the same procedure for another pereparation but after evaporation we just added water and not chitosan. So we got a negatively charged liposome which didn't pass through 0.45µm either means both of sample's sizes are bigger than 0.45µm.

After all we took the 2 samples to the HPLC laboratory to measure the amount of curcumin inside of liposomes.

Week 2:

Preparation of liposomal formulations:

Liposomes were prepared essentially according to the lipid film hydration technique reported by Bangham et al. (1965). Curcumin, DSPE PEG and lecithin were accurately weighed and dissolved in a 15ml methanol round bottomed flask. The solvent mixture was evaporated in a rotary flask evaporator under a vacuum pressure of 20 mbar and temperature of 60C and the flask rotated at 100rpm until a smooth, dry lipid film was obtained on the surface of the flask. The film was hydrated with 10ml of saline and chitosan solution at room temperature (25 C) with gentle shaking. The liposomal suspension was then sonicated for 5 min periods using an ice bath with a Probe Sonicator (Soniprep 150). The formulation technique was optimized as regards the following parameters: vacuum, hydration medium, speed of rotation of the flask and size reduction of liposomes.(6)

This week we made -vely and +vely charged liposomes but we used, hydrogenated soya lecithin (HSL or Lecithin), saline, DSPE-PEG and chitosan.

We prepared 4 samples. 2 of them -ve and 2 +ve charged liposomes.

The organic solvent methanol was removed under reduced pressure in a rotary evaporator, and the lipid-curcumin film was dispersed in 0.9% saline or 0.2% chitosan. Multilamellar vesicles (MLV) thus formed were converted to small unilamellar vesicles (SUV) by sonicating in a probe-type sonicator (Soniprep 150). The particle size of liposomes was controlled by controlling the sonication time.

In general charged lipids yield smaller and less lamellar liposomes. Lipid composition, the organic solvent, the rapidity of evaporation, the size of flask, the composition of aqueous phase as well as the power of agitation influence the size distribution and the lipid hemogeneity of the prepared MLVs. (7)

Protocol for the preparation of liposomes

Sample 1:(-vely charged liposome without DSP PEG)

Lecithin (HSL) =100mg

Curcumin= 10mg

Methanol= 15mg

0.9% Saline= 10ml

Sample 2:(-vely charged liposome with DSP PEG)

Lecithin (HSL) =80mg DSPE PEG = 20mg

Curcumin= 10mg

Methanol= 15mg

0.9% Saline= 10ml

Sample 3 :( +vely charged liposome without DSP PEG)

Lecithin (HSL) =100mg

Curcumin= 10mg

Methanol= 15mg

0.2% chitosan= 10ml

Sample 4 :( +vely charged liposome with DSPE PEG)

Lecithin (HSL) =80mg DSPE PEG = 20mg

Curcumin= 10mg

Methanol= 15mg

0.2% chitosan = 10ml

As like the previous sample procedure we mixed the curcumin and lecithin in the flask but this time we added DSPE PEG to 2 of the samples and examine its effect on liposome size and stability. (We should know that polyethyleneglycol (PEG) usage in the formulation of liposome prevent particle-particle interactions which enhance physical stability of nanoparticles to disperse evenly and do not coagulate or accumulate.)

After evaporation and mixture of samples with saline or chitosan we placed the samples in sonicator but this time for 5min then we filtered them through 0.45µm filter.

The physical observation was the samples with DSPE PEG had lighter colour and passed through filter while the samples without couldn't pass and had darker colour.

Week 3:

We prepared 3 sets of 4 samples. 2 +vely charged liposomes and 2 -vely charged all like the week 2 but we used pure curcumin this time. Then like before we passed them through 0.45µm filter. The results are:

Set 1:

Sample 1(saline)= didn't passed

Sample 2(saline+DSPE PEG) = passed

Sample 3 (chitosan) = passed!!!

Sample 4 ( chitosan + DSPE PEG) = passed

Set 2:

Sample 1(saline) = didn't passed

Sample 2(saline+DSPE PEG) = passed

Sample 3 (chitosan) = didn't passed

Sample 4 ( chitosan + DSPE PEG) = passed

Set 3:

Sample 1(saline)= didn't passed

Sample 2(saline+DSPE PEG) = passed

Sample 3 (chitosan) = didn't passed

Sample 4 ( chitosan + DSPE PEG) = passed

Size analysing

In week 3 we took the samples which passed through the filter to the size analyser machine to analyse their size and measuring the Z potential.

Set 1:

Sample 2 (saline+ DSPE PEG)

Size analysing:

Count rate= 259.9 kcps

Z-average size = 218.7 dnm

PDI (poly disperse index) = 0.226

2 peaks on the size analysing graph were seen! Peak 1=70% and peak 2= 30%

Z potential = - 1.44 mV

Sample 3 (chitosan)

Size analysing:

Count rate= 203.3 kcps

Z average size = 583.1 dnm

PDI (poly disperse index) = 0.748

2 peaks on the size analysing graph were seen! Peak 1=46% and peak 2= 54%

Z potential = + 8.36 mV

Sample 4 (chitosan + DSPE PEG)

Size analysing:

Count rate= 75.1 kcps

Z average size = 132.2 dnm

PDI (poly disperse index) = 0.210

Z potential = + 1.58 mV

Set 2:

Sample 2 (saline+ DSPE PEG)

Size analysing:

Count rate= 311.0 kcps

Z-average size = 218.4 dnm

PDI (poly disperse index) = 0.269

3 peaks on the size analysing graph were seen! Peak 1=30%, peak 2= 30% and peak 3 = 40%

Z potential = - 0.746 mV

Sample 4 (chitosan+ DSPE PEG)

Size analysing:

Count rate= 65.5 kcps

Z-average size = 141.6 dnm

PDI (poly disperse index) = 0.235

Z potential = + 1.8 mV

Set 3:

Sample 2 (saline+ DSPE PEG)

Size analysing:

Count rate= 67.8 kcps

Z-average size = 186.4 dnm

PDI (poly disperse index) = 0.280

2 peaks on the size analysing graph were seen! Peak 1=52%, peak 2= 48%

Z potential = - 0.885 mV

Sample 4 (chitosan+ DSPE PEG)

Size analysing:

Count rate= 76.7 kcps

Z-average size = 123.6 dnm

PDI (poly disperse index) = 0.216

Z potential = + 1.96 mV

In pharmacognosy laboratory:

As before all the preparation is done just by curcumin. The liposomal resveratrol has not been prepared and examined yet!

To Measure amount of curcumin inside of liposome samples

HPLC (high performance liquid chromatography): the instrument has 3 parts. Detector (waters 996, photodiode array detector), injector (waters 2695 DC119, separation model LC7) and computer.

HPLC method is used for dentification and quantification of liposomal curcumin and resveratrol by giving different UV-vis spectra and retention times. A Phenomenex Luna 5µm C18 (2) 100Å column (5 μm, 4.6 x 150 mm) was chosen and the mobile phase was formed by methanol and water. in subsequent tests a gradient was tried. The elution mode used is called gradient elution where the mobile phase composition changes during the separation, starting with 90% water (weaker solvent) and 10% methanol (stronger solvent) at time 0 until 10% water and 90% methanol at 40 minutes. In order to shorten the run time and analyzing more samples, time was reduced from 40 to 20. Calibration was performed by using curcumin as control. (I don't have the graphs yet!)

Chromatographic Conditions: The elution was carried out with gradient solvent systems with a flow rate of 1.0 mL/min at ambient temperature.

Curcumin concentrations were calculated on the basis of calibration functions and with regard to the dilution factor. 1 mg/ml of curcumin solution was prepared as mother solution and from this vial we prepared different concentration of curcumin by dilution.

To prepare 0.05mg/ml curcumin solution we should dilute the mother solution (1mg/ml) by 20 times we prepared 4ml of 0.05 mg/ml by taking 200µl from the mother solution and 3800µl methanol in to a glass vial.

Validation of HPLC Method:

Calibration and Linearity: The linearity of the method was evaluated by analyzing a series of standard curcumin. The elution was carried out as described and standard calibration curves were obtained by plotting the concentration of standard curcumin versus peak area. (3)

Rang: The calibration range was chosen to reflect normal curcumin concentrations in liposomal curcumin samples. This range included concentrations from the lower limit of quantification (LLOQ) to the upper limit of quantification (ULOQ). (3)

Determination of the Limit of Quantification (LOQ): The LOQ was defined as the lowest standard curcumin concentration, which can be determined with an accuracy and precision of <20%. (3)

Preparation of the diluted solutions (standard curcumin) for calibration curve:

0.025mg/ml curcumin = 500µl of methanol +500µl from the 4mlvial of 0.05mg/ml curcumin solution

0.0166mg/ml curcumin= 1000µl of methanol +500µl from the 4mlvial of 0.05mg/ml curcumin solution

0.025mg/ml curcumin = 1500µl of methanol +500µl from the 4mlvial of 0.05mg/ml curcumin solution

0.025mg/ml curcumin = 2000µl of methanol +500µl from the 4mlvial of 0.05mg/ml curcumin solution

4ml of 0.05 mg/ml curcumin solution


÷3 ÷5


0.025 mg/ml 0.0166 mg/ml 0.0125 mg/ml 0.01 mg/ml

When we put these preparation in to HPLC they give us calibration curves. Calibration graph was prepared to determine the curcumin content and gives us the linear relationship between peak area response and concentration of curcumin. Calibration curves were derived from injections of these five different concentrations of curcumin versus the peak area.

Preparation of the diluted solutions for sample curve:

The curcumin and resveratrol content of the tested liposomes was determined using the HPLC method above mentioned. For this purpose, 200 μL of liposome solution was added to 800 μL of methanol in the vial. This process causes de disruption the liposomes's membranes, thus releasing the encapsulated drug which could then be quantified.

1)the area under the curve for the first run samples (unpure curcumin, water and chitosan) was measured and it was out of the range so we had to dilute the samples first time by 4 times but it was still out of range and we diluted further by 20 times.

200µl liposomal curcumin+800µl methanol = out of range! Too much

50 µl liposomal curcumin + 950 µl methanol = out of range! Too much

10 µl liposomal curcumin + 990 µl methanol = out of range! Too low

2) the area under the curve for the second run samples (unpure curcumin, saline and chitosan)was measured and it was out of range!

200µl liposomal curcumin+800µl methanol = out of range! Too much

3) the third run is done by 3 sets of 4 samples means 12 samples ( pure curcumin, saline and chitosan) is placed into the HPLC inorder to measure AUC. Each liposome sample is diluted by dilution factor of 2 and 4.

200µl liposomal curcumin+800µl methanol

100 µl liposomal curcumin + 900 µl methanol

50 µl liposomal curcumin + 950 µl methanol

(have not got the results yet!)

Curcumin loading efficiency:

Loading efficiency (%) = C1 [amount of Curcumin in liposomes (mg)] -100

C [initial added Curcumin (mg)


Based on the results of investigation the average diameters of liposomes prepared decreased when DSPE-PEG was added. It has been reported that the Incorporation of PEG bounded covalently to polar head group of phospholipids e.g. DSPE increase the time of circulation of liposomes in blood.

PEG polymerization can inhibit opsonization of the liposomes by plasma proteins also it can enhance the half-life of liposomal drugs. Prolonged circulation of liposomes has been linked to better therapeutic efficacy. (2)

Problems: Methanol forms hydrogen bonds with polar head groups and is definitely more difficult to remove.

The interaction between liposomes and was studied by means of zeta potential measurements.

Results from size analysing confirm the Effect of Composition on the Stability of Liposomal curcumin.

Regression analysis of the experimental data points showed a linear relationship with excellent correlation coefficients (r2) of R² = 0.9867.The linear regression equation for the curve of curcumin is y = 6E+07x - 170653.

The area under the peak is proportional to the amount of compound which has passed the detector.