Dear Dr Saleha Hafeez

In response to your question, we must keep in mind that liposomes and nanoliposomes (made of phospholipids without incorporating stabilizing agents or without any coating, as is the case with your experiment) are generally highly dynamic and soft structures. Particularly vesicles with low phase transition temperature (Tc) are in the liquid crystalline phase at temperatures above their Tc (for more details on this aspect of lipid vesicles see our attached manuscript).

You mentioned that you have used "serum" as a diluent (would you please provide more details regarding the exact nature of your serum and at what stage you incorporated it to your formulation / sample). Also you mentioned that the suspension medium of your liposomes is water (again, pls specify what type of H2O you have used and what was its pH?).

We must specify what types of proteins / ions / other solutes are present in your formulation.

Also, pls give details of the DLS technique you use (does it require you to perform dilutions prior to analysis or not? Do you use correct Refractive Index and Medium Viscosity in your DLS assay?).

Attached Paper:

Danaei, M., M. Kalantari, M. Raji, H. Samareh Fekri, R. Saber, G. P. Asnani, S. M. Mortazavi, M. R. Mozafari, B. Rasti, and A. Taheriazam. "Probing nanoliposomes using single particle analytical techniques: effect of excipients, solvents, phase transition and zeta potential." Heliyon 4, no. 12 (2018): e01088.

I totally agree with Dr. Mozafari

manufacture of liposomes without stabilizing agents or coating leads to a weak liposome structure in terms of homogeneity of liposome molecules, differences in the size of liposome molecules or possibly clusters and aggregations in the liposome and an increase in volume. I think your formulation need more improvement and optimization

In addition, there are several things to consider at hydration step (solvent pH, ionic strength,temperature....etc)

Regards..

Thank you sir Mozafari and Hanan,

We used distilled water maintained at pH 7.4.

Normal serum was used (no dilutions were made) at pH 7.4. Serum has serum albumin and immunoglobulins.

We diluted the samples before dls analysis.

We made liposomes through thin film hydration method where we hydrated with distilled water and then centrifuged and then replaced the supernatant water with serum.

Dear Saleha Hafeez

The initial pH of your formulation is fine.

Two other parameters to consider with respect of your above question:

1. changing the isotonicity / osmotic pressure / electrolyte structure of your liposome suspension medium. This will cause burst of the vesicles and as a result decrease in the mean hydrodynamic particle size (as you observed). The proteins in the serum will interact with the ingredient molecules of your liposomes. Smaller size aggregates will form and contribute to the decrease in particle size.

2. Performing dilution (I assume you used serum for this) prior to DLS analysis.

Any dilution done with your liposomal formulation will change its nature and physicochemical characteristics (including size and PDI).

Modern particle size apparatus do not require dilutions for accurate analysis. I try to find some literature supporting this.

Have a look at this literature on the effect of dilution on particle sizing data:

Zu, L. (2014, March). Effects of dilution on particle size measurement in dynamic light scattering. In ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY (Vol. 247). 1155 16TH ST, NW, WASHINGTON, DC 20036 USA: AMER CHEMICAL SOC.

Abstract

The purpose of this paper is to articulate the effects of sample dilutionin particle size measurement using DLS technology, and how these effects may be minimized by using Photon Cross Correlation Spectroscopy (PCCS). Dynamic Light Scattering (DLS) is a non-invasive, fast and well established technique for characterizing submicron particle size and distribution. Generally, it requires significant sample dilution in order to achieve a correct result by avoiding multiple scattering phenomena. Sample dilution may change the chemical environment of the suspended particles in the sample. As a result, the measured size and distribution may deviate from its original value. PCCS is a subset of DLS, extending the range of sample concentrations that can be measured with DLS up to 106. A set of measurements for a series diluted NIST traceable monodispersed polystyrene latex nanoparticles gives comparative results for using PCCS vs PCS. It enables the clarification and verification of concentration dependency in particle size measurements and eliminates many of the assumptions in DLS measurements which are inherent with sample dilutionsthat may lead to skewed conclusions in a real application. Graph1 shows two repetitive PCCS measurements give a correct peak diameter of 101nm (green and blue curve), and PCS measurements give an artificially smaller peak diameter of 55nm (red and purple curve) in the concentration level.

Thank you so much sir

checking the bonds of your lipossom composition