Cyclosporine CsA—The Physicochemical Characterization of Liposomal and

Colloidal Systems

Summary
In this paper the respective authors have given a review on the possibilities of testing different
cyclosporine formulations with an importance on different parameters which can improve
biocompability and stability of cyclosporine administration. They have chosen cyclosporine A (CsA)
for their investigation. The compound CsA is an organic compound and has the formula
C62H111N11O12. It is a cyclic polypeptide which has 11 amino acids. CsA is associated with
biocompatibility and bioavailability problems. This is due to its poor or low solubility in physiological
fluids because of having lipophilic structure. It has been reported that there were considerable efforts
made to design a suitable vehicle for cyclosporine A oral administration which can have improved
absorption and reduced side effects (1-9). The aim of the review is to see the potential of CsA
formulations in respect with physiochemical properties of cationic CsA. They have tested the feasibility
of cyclosporine colloidal system for oral administration in various techniques and compared with
investigations which are similar. Dipalmitoylphosphocholine (DPPC) and cholesterol were used as lipid
matrix to develop the cyclosporine A colloidal system. Furthermore the matrix was stabilized using
ethanol and soybean oil or n-tetradecane as oil phase in emulsion via high pressure homogenizer for
obtaining droplet size and narrow size distribution. This was then placed under normal pH and
physiological temperature.
They have used CsA (98.5%), ethanol (96%), cholesterol and dipalmitoylphosphocholine (DPPC) both
99% respectively. They have also reviewed the usage of antibacterial chitosan for the bioavailability of
CsA. The authors have reviewed the different aspects or parameters of formulations of CSA such as
solubility, liposomal system and the use of DLS or BAM analysis in different subtopics. They have
reported that with their findings and comparison with published literature that Cyclosporine A is not a
suitable oral drug formulation due to its poor solubility and bioavailability however the drug can be
easily produced in the size range of 200-500 and can show better absorption and bioavailability in To
improve the bioavailability of CsA in liposomal system.They have suggested the use of Chitosan, lipid
or other oil-water formulation. These formulations can improve the bioavailability of the drug and can
reduce the side effects with low dose regimen. Dynamic light scattering(DLS) technique has been used
to find zeta potential and size distribution for both liposomal system and oil-water emulsion to assess
the physio-chemical characeterteristics of CsA and its stability in the mentioned systems followed by
BAM analysis for morphological study.
Based on assessing the manuscript, I will be discussing the strength and weaknesses of the manuscript
followed by novelty of the review article and finally my recommendation.
Strength and Weaknesses of the Manuscript

The authors have discussed on the CsA solubility in water where they have recorded the data with
increased temperature. The solubility slowed with increased temperature. So the problem in solubility
had been shown to give low bioavailability where the oral bio-availability of the drug can be in the
range of 10 % to 89%.. It should be noted that they have given reasoning for this is because of the
compound having intramolecular H-bonds that has a rigid configuration . They have also explained that
is most likely for the D-alanine amino acid having a conformational change which is the residue of the
drug that loses water with temperature increase. The lipid based liposomal system was developed by
injecting alcohol into the DPPC and homogenized where the phase temperature was 41 ◦C For
measuring particle size distribution they have used Dynamic Light Scattering (DLS) where different
CsA formulations mean particle size was measured in 5 1- minute cycle using Zeta Potential-Bi mass
(Brookhaven, Preston, UK). The zeta potential also determined in the same apparatus. The particle size
distribution was also measured using laser light scattering technique for the liposomal formulations
with and without CsA. The recorded size distribution was CsA, CaCl2 > CsA, H2O > CsA, KCl.
In the subtopic 3.1 they have discussed stability of Liposomal CsA where they have briefly described
the usage of Cyclosporine A as a good candidate for including into liposomes. In this chapter they have
mentioned about different charged liposome and the particle size for good stability. Study of Czogalla
et al. was reported where liposomal formulations were developed less than 200 nm . This size is small
compared with literature reports where existing literature has found liposome in blood frequently to be
more than or equal to 200 nm. The author also recommended that despite there is proven research for
all types (charges) liposome can be developed into powders, only positive and neutral liposomes
showed to have more stability high stability in blood. According to their research depending on the type
of application and location of action cyclosporine A in water or KCl is a better approach for small sized
liposomal system but for liposomal stability as a key indicator CsA in calcium chloride solution is a
good choice.
Brewster Angle microscopy (BAM) was used to look into the pure DPPC monolayer which showed
irregular domains where CsA monolayer had no irregular domain and appeared to be homogenous. In
this BAM analysis and surface-pressure area isotherms images were used to investigate the CsA effect
on model DPPC membrane. The images obtained are helpful to understand the interaction of living cell
membranes with drug CsA.
In the oil-water emulsion subtopic, they reported the possible formation of tetragonal crystalline particle
in one study which was not present in the other. The comparison showed that the differences meant
stability of the emulsion is dependent on the different formation of CsA. The characteristics of the
formulation in soybean oil and cholesterol to look into the zeta potential and size diameter. From the
DLS data it can be noted that size distribution in Cyclosporine A – oil was narrow than the CsA oil and
ethanol. Therefore it can be said that formulation system by addition of phospholipid-ethanol has better
bioavailability.
They have also looked into the alternative such as using Chitosan where they have mentioned a study
of Hermans et al. In this study rabbits were used for in-vivo experiment where concentration of CSA
was found in conjunctiva and cornea and showed to be active against inflammation (11). Wiacek et al
used soybean oil as oil phase where poor concentration and poor penetration was found in tear film and
cornea respectively due to poor solubility of CsA. With the in-vitro reports showing almost no toxic
effect the chitosan-CsA can be helpful delivery system for optical drug delivery system.
The use of CsA in the formulation of oral drug delivery system was also discussed where data and
notable findings were presented from previous literature. It was concluded that the oral bioavailability
data correlated with particle size distribution. The comparison studies showed that in one particular
study CsA administered invasively for a lung patient showed no toxic effects or hepatotoxicity however
oral administration had shown hepatotoxicity.
The author had also described about ocular and skin CSA drug delivery system where different studies
were compared for a better understanding. As mentioned cationic o/w emulsion showed efficiency in
ophthalmic drug.
Overall the different subtopic covers most of the type of delivery from oral to skin and the potential use
of CsA as a drug delivery with existing literature and their comparative research. The topics are well
covered and understandable with clarity given on reasoning behind results. Furthermore existent
literature published and mentioned in this article was cross-matched for accuracy which proved to be
correct. The graphs and tables were explained in a detailed manner especially the DLS data for two
different systems were helpful in making conclusive decision in regards to the size distribution and zeta
potential parameter.

Weakness
In table 2 of the manuscript they have shown the characterization DPPC liposome with and without
CsA with the help of DLS to show the particle size distribution and zeta potential values. The order of
the particle sizes were not less than 100 nm which is rather high for an optimal liposomal drug delivery
system (10). They have also looked into the alternative such as using Chitosan and described a few
studies however the studies were not adequate to state the usage of chitosan. It was mentioned that a
particular chitosan formulation had shown good absorption in an in-vivo system but the chapter needed
more in depth analysis.
The chitosan nanoparticle mentioned didn’t have much information regarding the different types of
method to be used for its efficacy as a drug delivery system where additional information with more
safety and toxicity information would have made the effectiveness of chitosan as a potential
nanoparticle which can be used for better therapeutic vehicle apart from its antimicrobial activity.
Appraisal of the method
To define nanoparticles and characterize it Dynamic light Scattering is an important technique to see
the size distribution and zeta potential for comparison studies. Furthermore BAM analysis which is
commonly used to investigate thin films on liquid surfaces such as Langmuir films. Both of these were
used by Wiacek et al for their review which have given good comparative results for their study.
However for figure 4, the zeta potential at 20 and 37 °C would have given a better understanding if
presented in a graph plot diagram than a box plot diagram.
Novelty of the research
Since the article is a review article I have looked into the aspect of what new findings in terms of
information rather than results is found from this article. In my opinion the studies have been established
before and have given us insights on the use of CsA as a delivery vehicle in respect to both oil in water
and oil in emulsion systems. However it should also be mentioned that the combined technology of
both micro emulsion and liposomal systems can have the possibility to enhance efficacy and absorption
of Cyclosporine A. In all CsA preparations the absolute zeta potential was below 12 mV and showed
positive zeta potential with a higher increased ethanol concentration.
Recommendation
My recommendation as a peer reviewer would be to accept the paper with minor revisions. Minor
revisions are required for the weaknesses that I have mentioned above. The author should find relevant
information for the particle size distribution found in the DLS data. The size was higher than 200 nm.
So the author may find papers on how to improve the nanoparticle size or explain further on what might
be the reason behind the size distribution. Furthermore chitosan can improve the bioavailability of CsA
as mentioned in the review article but adequate safety and toxicity effects are not mentioned which are
important parameters.

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