Liposomal characterisation

Our liposomes form a protective shield around nutrients, helping to prevent degradation as they travel through the harsh environments of your digestive system. In practice, this is about shielding the nutrients from severely acidic conditions.

To demonstrate the shielding ability of our liposomes with respect to nutrient degradation through digestion, experts analysed under simulated gastric and intestinal conditions to measure how the Vitamin C encapsulated in our liquid liposomal delivery system degraded over time compared to an unencapsulated Vitamin C.

The in vitro simulated digestion stability of the Zooki Vitamin C was analysed under simulated gastric, and intestinal conditions to assess its degradation process.

Simulated gastric fluid (SGF) was produced with modifications according to Frenzel et al., (2015) and Lee et al. (2012) by mixing 25 ml of distilled water with a pH value set to 2.0 with hydro chloric acid and 1 ml of 0.4 % pepsin solution (800–2000 U/mg of protein); 5 ml of liposome solution were added and the solution was incubated at 37 °C at 150 rpm on an orbital shaker; 500 μl samples were taken after 0.5, 10, 20, 30, 60, 90 and 120 min and evaluated for liposomal size and content of vitamin C.

Simulated intestinal fluid (SIF) was prepared with modifications according to Troncoso et al. (2012) and Parmentier et al. (2012) by dissolving 27.74 mg of calcium chloride and 100 mg of bile acid extract in 20 ml of phosphate buffered saline (PBS) at a pH value of 6.8. 250 mg of pancreatin were dissolved in 30 ml of PBS. Both solutions were incubated over night at room temperature and 150 rpm on an orbital shaker and then warmed to 37 °C. The pancreatin solution was centrifuged to remove particulate matter and the supernatant was combined with the bile acid solution, followed by the addition of 5 ml of liposome solution. The mixture was incubated at 37 °C and 150 rpm on an orbital shaker and 100 μl of a 15 mM calcium chloride solution were added after 0, 30, 60, 120, 180 min. Samples of 1 ml were taken after 0, 10, 20, 30, 60, 120, 180, 240 min and analysed in terms of liposomal size and vitamin C content in the dissolution media.

Particle size is a significant parameter for the liposomal system. The mean particle size (PS) for Zooki vitamin C was measured by using the dynamic light scattering technique (Litesizer 500, Anton Paar, USA) at 25 °C. Every measurement was executed with at least three sets of six runs.

The in vitro simulated digestion stability of the Zooki Vitamin C and vitamin C were analysed under simulated gastric, and intestinal conditions over 120 min to assess their degradation process and the results are shown in Fig. 1. A burst degradation of more than 50% in SGF and more than 60% in SIF conditions in the first 30 min was observed for vitamin C (control) whereas, Zooki Vitamin C registered slow degradation (less than 85%) both the SGF and SIF conditions, thereby confirming the protecting function of the encapsulation of vitamin C in the Zooki vitamin C formulation.

Before you can start utilising their benefits, the nutrients that survive the acidic environment of the digestive system need to be absorbed through the gut wall and into the bloodstream.

Most ordinary, non-encapsulated nutrients are absorbed into the bloodstream through “active transport” - your body expends energy to absorb nutrients into the bloodstream through “protein channels”. The body doesn’t have enough of these protein channels in the gut wall to absorb the high concentrations of nutrients found in supplements, which leads to the majority of the nutrients in regular supplements passing straight through the body without making it into the bloodstream.

By encapsulating nutrients in liposomes, nutrients in Zooki products are absorbed directly into the bloodstream through M-cells in the gut lining, by-passing the need for the protein channels that act as bottlenecks for nutrient absorption.

Zooki Liquid Vitamin C – In Vivo Absorption Study Summary

In this study, the oral bioavailability of (Zooki Vitamin C liquid with 1000 mg Vitamin C (Test product), and reference product (Vitamin C, conventional Vitamin C with 1000mg vitamin C) was evaluated in an open label, randomized, single dose, two- treatment, two-way crossover, oral bioavailability study in healthy, adult, human participants under fasting conditions.

The sample size of the study was 24, with 12 participants randomized to each of the two study arms at a 1:1 ratio and received dosing as per randomization. During the second period of study, the participants were interchanged for the crossover study after the washout period of 7 days. The subjects were fasted for at least 10.00 hours before dosing. In morning, a single oral dose, 1000 mg ascorbic acid per sachet (15ml) of either test product (T) or reference product (R) administered orally, (as per randomization schedule) along with 250 mL water, to the subjects in sitting posture, under the supervision of Investigator and trained study personnel including Quality Assurance auditor(s).

In this study period, 8 blood samples were collected including the pre-dose sampling in each period. For the pre-dose blood sample (00.00 hours) 05 ml will be collected within 60 minutes prior to dosing. Post dose blood samples 05 ml collected at 00.50, 01.00, 02.00, 06.00, 12.00, 24.00 and 48.00 hours.

After collection of blood samples were placed in a thermo-insulated box containing wet ice and transferred the box to the sample processing room where the blood samples were centrifuged within 30 minutes at 4000 ± 50 rpm for 10 minutes at 02 °C to 08 °C to separate the plasma, which were transferred into suitably labeled polypropylene tubes.

Analysis of ascorbic acid (vitamin C)

One millimolar citric acid buffer is added to the plasma samples to precipitate out the proteins and centrifuged and filtered through 0.22 micron and the aliquot inject to the UPLC system. Vitamin C content in the plasma was measured by ultra performance liquid chromatography (UPLC) (Agilent 1200 Infinity Series, Germany) using an ZORBAX Eclipse Plus C18 column, 5 μm, with a volume of 4.6 x 250 mm. The mobile phase was 0.1 % acetic acid and methanol in the ratio of (90:10). The flow was isocratic at a rate of 1 ml/min. at 32 °C. The eluate was detected using an Agilent VWD detector set at 254 nm.

Results and discussion

The absorption of the liposomal vitamin C were compared with conventional vitamin C. The total average pharmacokinetic variables for liposomal vitamin C and conventional vitamin C calculated from plasma total vitamin C are given in Table 1.

The maximum plasma vitamin C (Cmax) and the AUC for the liposomal vitamin C were approximately 1.16 and 37.91 mg/dL, respectively. In comparison, these values for the conventional vitamin C were 0.32 and 9.69 ng/dL, respectively (Table 1 and Fig. 1). The time to reach the maximum plasma concentration (Tmax) is 2 h for both liposomal vitamin C and vitamin C, which indicates that the release patterns of vitamin C in both the groups are the same. The absorption of the liposomal vitamin C was 3.91-fold greater than the vitamin C in terms of absorption (AUC0–t) and 3.61- fold greater in terms of the rate of absorption (Cmax) (Table 1).

Zooki Dry Glutathione – In Vivo Absorption Study Summary

In this study, the oral bioavailability of investigational product – Liposomal Glutathione (600-650 mg capsule containing NLT 250 mg of L-Glutathione/ Capsule) of Your Zooki Holdings, and the reference product (600-650 mg capsules which contains 250 mg of conventional Glutathione) of Molecules Food Solutions Private Limited was evaluated in an open label, randomized, single dose, two-treatment, two-way crossover, oral bioavailability study in healthy, adult, human participants under fasting conditions.

The sample size of the study was 24, with 12 participants randomized to each of the two study arms at a 1:1 ratio and received dosing as per randomization. During the second period of study, the participants were interchanged for the crossover study after the washout period of 7 days. The subjects were fasted for at least 10.00 hours before dosing. In morning, a single oral dose, 2 capsules, (1 capsule 600-650 mg) of either test product (T) or reference product (R), which contain NLT 500 mg of glutathione administered orally, (as per randomization schedule) along with water, to the subjects in sitting posture, under the supervision of Investigator and trained study personnel.

In this study period, 8 blood samples were collected including the pre-dose sampling in each period. For the pre-dose blood sample (00.00 hours) 5 ml will be collected within 60 minutes prior to dosing. Post dose blood samples 5 ml collected at 00.50, 01.00, 02.00, 06.00, 12.00, 24.00 and 48.00 hours.

After collection of blood samples were placed in a thermo-insulated box containing wet ice and transferred the box to the sample processing room where the blood samples were centrifuged within 30 minutes at 4000 ± 50 rpm for 10 minutes at 02 °C to 08 °C to separate the plasma, which were transferred into suitably labeled polypropylene tubes.

Results and discussion

The absorption of the liposomal Glutathione were compared with conventional Glutathione. The total average pharmacokinetic variables for liposomal Glutathione and conventional Glutathione calculated from plasma total Glutathione are given in Table 1.

The maximum plasma Glutathione (Cmax) and the AUC for the liposomal Glutathione were approximately 56.63 mg/dL and 1087.59 mg.h/dL, respectively. In comparison, these values for the conventional Glutathione were 15.16 mg/dL and 302.7 mg.h/dL, respectively (Table 1 and Fig. 1). The time to reach the maximum plasma concentration (Tmax) is 2 h for both liposomal Glutathione and Glutathione, which indicates that the release patterns of Glutathione in both the groups are the same. The absorption of the liposomal Glutathione was 3.59-fold greater than the Glutathione in terms of absorption (AUC0–t) and 3.74-fold greater in terms of the rate of absorption (Cmax) (Table 1).

Conclusion

The data indicate that the liposomal Glutathione exhibits greater absorption as compared with conventional Glutathione.

Zeta potential is a measurement of charge on the surface of particles (such as liposomes) suspended in a solution. We’re specifically trying to measure the difference in charge between the surface of the liposome and the solution itself. Zeta potential tells us about the stability of liposomes. For example, a higher zeta potential implies the liposomes are more stable and less likely to aggregate. 

• Think of Zeta Potential as the force that keeps two magnets apart. When you push magnetic forces with the same negative charge together, they repel each other. 
• Now imagine that you have a football, but the entire surface of that football is magnetised with a negative charge. You throw that football into a bath and watch it float around with other footballs that have also been negatively charged.
• That repelling force at the surface of the football prevents it from colliding (or fusing) with other footballs. The negative force that stops a football from fusing with another is spread across its entire spherical surface and holds the football together, stopping it from falling apart.

In general, a higher Zeta Potential, either positive or negative, will indicate a more stable encapsulation. This is because the electrostatic repulsion between the particles will be stronger, making it more difficult for them to aggregate or fuse together over time.

Some general guidelines for interpreting Zeta Potential results:

• Zeta Potentials greater than 30 mV are considered to be stable.
• Zeta Potentials between 10 and 30 mV are considered to be borderline stable.
• Zeta Potentials less than 10 mV are considered to be unstable.

Examples of Zeta Potential values for both our liquid and dry delivery systems. Note the results show a negative charge with a greater value than 30mV; a good indication of the stability and integrity of the liposomal structures present.

Dynamic light scattering (DLS) is a technique that is used to measure the size and distribution of particles in a solution. 

It’s called “dynamic light scattering” because it’s based on the principle that the Brownian motion of particles (how particles naturally move) causes light that is fired through it to fluctuate in intensity. 

This is to say, if we fire light through a solution, we can gauge the size of the particles inside by looking at how the light varies as it comes out the other side. Essentially, the fluctuations in the scattered light can be used to calculate the size and distribution of particles.

To measure DLS, a laser beam is shone onto the solution containing the liposomes. The scattered light is then recorded and analysed. 

DLS results are typically presented as a distribution of particle sizes. The distribution is plotted as a histogram, with the particle size on the x-axis and the number of particles in each size range on the y-axis. 

With liposomes, a narrow distribution indicates stability and a small particle will lend itself well to bioavailability. So, we’re looking for small liposomes that don’t vary too much in size.

Cryogenic Transmission Electron Microscopy (Cryo-TEM for short) is a technique for capturing images of particles at the nano scale after they’ve been frozen using liquid nitrogen. It’s a complex but intuitive technique that won a Nobel prize in 2017. The CRYO (cryogenic) part references the freezing stage and the TEM (transmission electron microscopy) references the technique for capturing the images.

Cryo-TEM is important because it allows us to identify the nature of the lipid structures, which with respect to liposomes can often be extremely challenging. Many of the examples you’ll see in textbooks or online will be of empty liposomes as part of a simple solution. Using Cryo-TEM to identify liposomes as part of a more viscous solution that contains other ingredients is much more challenging and there are very few companies that have successfully captured images of liposomes with real finished product designed for human consumption.

You can think of Cryo-TEM like taking a photo of a butterfly mid-flight with a flash camera. The butterfly is the biological sample and the ‘flash’ freezes the butterfly in place so the electrons fired at the butterfly can produce an image unblurred by movement. The images can be used to identify the different components of liposomes, such as the lipid bilayer, the aqueous core, and any encapsulated molecules.

Sample preparation

Sample fixation by freezing is employed in the cryo-TEM method (JEOL-2100) which is nowadays considered one of the best methods for colloidal drug carrier characterization. The preparation of a good-quality sample is a multi-phase procedure. To make sample analysis by means of the cryo-TEM method possible, the sample must demonstrate the following characteristics:

• The size of the particles should be in the submicron range, since larger particles will be removed during the drying phase by quickly blotting with filter paper along with the extra sample.
• The sample shouldn't have a viscosity that is too high, because it could be hard to make the mixture into a uniform layer.
• Highly concentrated samples should be diluted (20x) prior to analysis to make sure that particles seen in the TEM image are not densely concentrated.

First, samples with a concentration of 1 mg/mL is made. Carbon-coated hollow grids were used for the cryo-TEM technique. To hydrophilize the grid for optimal aqueous sample distribution, a glow discharge is carried out right before the sample application. A sample droplet (about 2-5µl) was put into the grid using a pipette after it has been fixed in the preparation chamber (under the UV light having the shortest wavelength of 254 nm) for 10 minutes. The sample was then instantly frozen in liquid nitrogen. After freezing, the sample was kept at a very low temperature (-175°C). The grid containing the vitrified sample (which has been transformed into a glassy state) was taken out of the liquid nitrogen container while it is cooling, and any extra nitrogen is blotted using filter paper. Using precooled tools, the sample was quickly transported into liquid nitrogen and put into the cold cryo container. The cooled holder is then swiftly moved and put inside the electron microscope.

Examples of Cryo-TEM images of Liposomes for both our liquid and dry delivery systems above. We can see the dark cores which indicates good encapsulation efficiency, as well as the iconic dual lipid outer layer

Field Emission Scanning Electron Microscopy (FESEM) is a type of electron microscopy that uses a high-powered electron beam to image the surface of objects. It is a powerful tool for studying the morphology of liposomes.

FESEM is important for studying liposomes because it can provide high-resolution images of their surface features, such as their size, shape, and surface roughness. 

FESEM works by focusing a beam of electrons onto the surface of the sample. The electrons interact with the atoms in the sample, and the resulting interactions produce secondary electrons, backscattered electrons, and other signals. These signals are then used to create an image of the sample surface.

It’s important to realise that the images generated by FESEM aren’t photos in the typical sense. You wouldn’t be able to take a detailed photograph with a camera of a grain of sand because there’s only so far you can zoom in. Instead of using a traditional camera or microscope, we shoot a beam of electrons at the grain of sand. The electrons interact with the atoms in the object, and the resulting interactions produce signals that can be used to generate an image of the object's surface.

With FESEM images, we’re looking specifically at the surfaces and their textures. Liposomes should be spherical with smooth surfaces and hopefully not aggregated or clumped together. 

FESEM is a different type of imaging to CRYO-TEM and whilst in some ways not quite as detailed, it still provides valuable insight into the surface morphology of the liposomal structures present.

Sample preparation

• The prepared formulation samples were spread on glass with a micropipette (0.5 microliters) cover slip and stuck to the grid with double-sided adhesive tapes.
• Then, a JEOL JFC 1600 (JEOL, Tokyo, Japan) autofine coater was used to coat the stubs with platinum in a vacuum.
• Finally, the platinum-coated samples were observed and examined with the help of FESEM (JEOL JSM-820 Model), and photographs were taken.

Examples of FESEM images of Liposomes for both our liquid and dry delivery systems. The smooth surfaces and spherical shapes give us another glimpse into the nature of these micro sized nutrient couriers

For both our liquid and dry liposomal delivery systems, we’ve presented independent test results that substantiate the following:

• The Zeta Potential values have demonstrated the stability and integrity of the liposomes present, with a low likelihood of aggregation or fusing over time.
• The DLS values show narrow distribution and small particle size, an indication of the stability and bioavailability of the liposomes present.
• The Cryo-TEM images show spherical structures with dark cores and a dual lipid layer: hallmark characteristics of liposomes with a filled core of active ingredient.
• The FESEM images give us insight into the morphology of the liposomes, illuminating the smooth surfaces and further demonstrating the spherical structures.

As well as the physical characteristics, we’ve demonstrated the properties you would expect to see of liposomal encapsulation of nutrients. Through in vitro and in vivo clinical trials, we’ve demonstrated:

• Up to 10 x greater nutrient protection through the digestive system
• Up to 4 x higher absorption into the bloodstream

Ultimately, using a wide range of methods, for both our liquid and dry delivery systems, we have evidenced the successful creation and presence of liposomes.