JBChemYear4Readings
Characterization of Stratum Corneum Molecular Dynamics by Natural-Abundance 13C Solid-State NMR (Bjorklund, Nowacka, Bouwstra, Sparr, Topgaard (2013)
Summary
Abstract
Despite potential benefits of trans-cutaneous drug delivery, the mechanisms that speed up drug delivery are poorly known.
They plan on recording the 13C NMR signal given out and recording how it changes upon hydration and heating.
They plan on using polarization transfer solid state NMR using Insensitive nuclei enhanced by polarization transfer (INEPT) pulse sequences and
Introduction
Materials and Methods
Results and Discussion
Conclusions
Ionic Liquids for oral insulin delivery (Banerjee, Ibsen, Brown, Chen, Agatemor, Mitragotri (2018)
Summary
Abstract
The paper will discuss the discovery of choline Geranate or CAGE. It is noted that diabetes is a growing problem and the oral delivery of insulin is not ideal as the stomach and intestine digests and ruins the structure of the enzyme. The injection of insulin is poorly adhered to by patients so there is increased interest in through the skin delivery. Cage seems to do this effectively in rats and the injected insulin lasts considerably longer than injected insulin. Biocompatability and stability both appeared good.
Introduction
Oral delivery of drugs is considered to be the best and easiest route but this is impractical for biological molecules such as proteins as the stomach biodegrades them, annulling any benefits they may give once in the blood stream.
Insulin is currently administered using needles by has a poor adherence rate. Phobia of needles and pain etc.
Orally taken insulin would be the best as it would be the closest mimic to naturally occurring pancreatic insulin.
This is because, the insulin is transported in a normal human to the portal vein where 80% is retained. Orally bioavailable insulin would follow as similar route. The rest reaches systemic circulation. Therefore, there is up to 3 times as much insulin in the portal vein as there is in systemic circulation.
Materials and Methods
Results
Insulin was stable in CAGE for long periods of time
Insulin is an alpha-helical conformation which is essential for its receptor interaction.
Insulin was stored in CAGE at room temperature away from direct sunlight and another set in a fridge at 4°C.
Insulin was biologically still effective in rats after 2 months of sitting at room temperature and 4 months of sitting in a fridge.
Insulin applied to rats caused hypoglycemia and demonstrated good pharmokinetics.
The blood sugar levels and insulin levels were monitored every half an hour for 5 hours in non diabetic rats.
There were 4 studies done. An injection of cage and insulin, a cream rubbed on the rats of insulin and cage, a cream rubbed on the rats of saline CAGE, and a cream rubbed on the rats of saline insulin.
The injection and the cream with both the cage and the insulin showed decreased blood sugar levels within 2.5 hours but whereas the injection showed the sugar levels to be back to normal after 5 hours, the cream still showed decreased blood sugar levels after 5 hours.
Neither the CAGE nor the insulin by itself showed any considerable blood sugar drop except one that would be expected by a fasting animal.
Insulin-Cage administered together showed increased oral bioavailability
An enterically coated capsule of insulin and cage was given to the rats. This showed a drop in blood glucose levels and a longer stay at these reduced levels than either the injected or the cream results of the previous section
A control was set up whereby cage was administered and then 30 mins later insulin. This had no effect on blood glucose levels so they have to be administered together for any effect.
Cage demonstrates good oral bioavailability in vivo
No differences were noted between the intestines of animals treated with cage/insulin and saline insulin. No damage was noted.
Discussion
Insulin is poorly adhered to especially when out in public. This leads to increased hospitalisation and increased costs.
Other methods have been used including nanoparticles, oil in water nano dispersions , directing ligands etc but very few have had any notable success. Most of the reside at less than 5% which is not good enough.
Ionic liquids have demonstrated a good ability at delivery of poorly water soluble molecules such as danazol.
Choline is important in many plant and animal processes and is present in the ampiphillic lecithin that emulsifies things. It has a median lethal dose of 3,400 mg/kg
Geranic acid is commonly used as flavouring and is safe up to a median dose of 3,700 mg/Kg.
The dose of CAGE rarely exceeded 80 mg/kg
CAGE demonstrated concentration dependant insulin transport, with a figure of 10 fold increase at 50 milimolar CAGE.
The route of CAGE is likely to be through the lipids in between the corneocytes that make up the stratum corneum.
Markers were used to prove that it moves through these lipid bilayers. These markers were Lucifer Yellow and fluorescein isothiocynate. Both displayed increase transport through these layers when added to CAGE.
The transepithelial electircal resistance (TEER) was also measured and found to decrease after CAGE had passed through the lipid bilayers. This is similar to other chemical enhancers such as sodium caprate.
However, it was found that CAGE decreases the transcellular uptake of the marker coumarin-6 which predominantly crosses the cell membranes by transcytosis (the means by which the molecule is captured within a vesicle that is able to move through the lipid membrane and is then ejected on the other side).
CAGE was also found to help penetrate through mucus in the intestine by thinning the mucus by up to 5%. CAGE also hinders tryptic digestion when compared to insulin in a buffer solution.
It is believed that CAGE forms a physical barrier between enzymes and the insulin preventing degradation. Furthermore, the CAGE can help with movements across mucus and through the lipid bilayers as CAGE helps open small junctions.
The elimination half life for CAGE was 2 times higher than injected insulin and maintained its efficacy for considerably longer than injected insulin.
Ionic liquids similar to CAGE have been shown to have high drug loading properties - the quantity of drug molecules that are attached to each molecule of the ionic liquid.
There was no toxicity in rate following considerable dosage of CAGE and no damage to the intestine.
Increased efficacy by presenting the insulin to the intestinal cells as monomers - no polymerisation can occur within the CAGE structure.
Conclusions
Mechanism of Antibacterial Activity of Choline-Based Ionic Liquids (CAGE) (Ibsen, Ma, Bannerjee, Tanner et al 2018)
Summary
Abstract
Anti-microbials are in short supply at the moment. This could lead to a wealth of problems but ionic liquids are slowly showing that they have anti-microbial properties and could be used in the future.
The paper reports on a series of geranic acid and choline mixtures that have displayed anti-microbial mixtures. The paper discusses how they interact with the cell wall of bacterial cells and how to kill them. Afterall, the preferential anti-micrbial just targets microbes and is not toxic to humans
Four ratios were used to investigate the effects of CAGE.
1:4/1:2/1:1/2:1 of choline : geranic acid.
The mechanism was found to be related to the fact that choline is attracted to the negatively charged cell membrane and inserts geranic acid into the lipid bilayer.
Introduction
CAGE was one of the best molecules discovered that possessed both antimicrobial properties and had minimal toxicity to human cells.
It has been discovered that there is a minimum concentration below which the ionic liquid has minimal antimicrobial powers. It has also been found that the longer the alkyl chain, the more effective the antimicrobial properties of the ionic liquid are.
The proposed mechanism was that of the alkyl chain inserting itself into the cell membrane. Another mechanism suggests that the cation blocks the acetylcholinesterase.
Materials and Methods
Results and Discussion
Conclusions
Definitions of ionic liquids and deep eutectic solvents by Banerjee, Ibsen, Brown et al 2018
- Cage is seen as an ionic liquid since it is mainly formed of ionic species and has a melting point of below 100 degrees Celsius.
- Cage is not a classical ionic liquid since it contains neutral geranic acid
- Ionic interactions play a key role in cages properties as conductivity was reduced which is indicative of ion pairing
- Hydrogen bonding does exist as indicated by a proton shift in Proton nmr of the OH peak
- Cage also has a reduction in freezing temperature something that is characteristic of a deep eutectic solvent.
- No current term for cage is currently in use as it has similarities to ionic liquids and deep eutectic solvents deep eutectic solvents with a complex anion has been expressed as a possibility for cage.
Simplified stratum corneum model membranes for studying the effects ofpermeation enhancersBarbora AmélieČuříkováa, Kamila Procházkováa, Barbora Filkováa, Petra Diblíkováa,Jan Svobodaa, Andrej Kováčikb, Kateřina Vávrováb, Jarmila Zbytovskáa
Abstract
Skin samples for intro experiments are hard to source and can be highly variable in nature.
The paper prepared skin mimics from a mix of Stearic acid, cholesterol, cholesterol sulphate and ceramides. These ceramides were N-2-hydroxystearoyl phytosphingosine (CER[AP]) and/ or N-stearoyl phytosphingosine (CER[NP]).
Permeation of theophylline (TH) and Idomethacin were compared both through this membrane and porcine skin.
Mixed ceramides gave the optimal result
This was used to test two different known drugs able to permeate through the stratum corneum to compare the results of this mimic against porcine skin results. The agents were theophylline (TH) and indomethacin (IND)
The 1:1 mix of ceramides gave the closest results to the porcine skin results. Following on from this trial, this mixture was used to test the two permeation enhancers L-Pro2 ((S)-N-acetylproline dodecyl ester) and Azone (N-dodecyl azepan-2-one).
Both permeation enhancers were found to increase the rate of TH and IND through the membrane more markedly than the porcine skin
L-Pro2 was found to be a better permeation enhancer than Azone
Introduction
The function of the skin is 2-fold – to prevent entry of foreign chemicals and bacteria and to regulate temperature and water loss
The skin is formed when terminally differentiated keratinocytes (corneocytes) become embedded in a mix of at least 12 different types of ceramides, cholesterol, free fatty acids and a small quantity of cholesterol sulphate and other lipids.
This lipid structure is divided into 2 parts, a long phase of about 13 nm and a short phase of about 6 nm packed in a hexagonal orthorhombic manner.
Usually the permeation enhancers disrupt the lipid membranes, but sometimes they do affect the corneocytes.
A franz diffusion cell was mostly used in the testing of the membranes and the permeation of chemical enhancers.
Testing of skin can be difficult as different species have different skin qualities, some have a greater number of pores than others, so testing for human medicines is tough.
The types of lipids used in a mimic is important; shorter chain ceramides can increase permittivity whereas sphingosine-CER display lower permeabilities.
Materials/Method
The thickness of the porcine skin was 1.02 +/- 0.04 mm
IND/TH are standard permeation markers.
Enhancement Ratio (ER) = where Je is the flux in the presence of an enhancer and J is the flux without an enhancer
Electrical impedance was used to characterise the effects of the permeation enhancers on the integrity of the lipid membranes.
Electrical Impedance Ration (EIR)= EI1/EI2 where EI1 is the impedance before the application of the enhancer and EI2 is the impedance after the application of the enhancer.
Results
TH/IND reached a state of steady flux through the mimic skin quicker than through the porcine skin
None of the mimics had a flux through the membrane that was significantly different to that of the porcine skin
The closest replica was that of the 1:1 mix of ceramides
Electron microscopy showed an even thickness of about 9.5 um. This lies close to the thickness of real stratum corneum lipid membranes.
IR spectroscopy showed all the correct peaks which have been assigned in the paper
Both azone and L-Pro2 increased the flux through the membrane in line with result from the porcine skin
The effect was considerably negated if the L-Pro2 was applied before applying the drug itself.
The electrical impedance of the membrane was affected regardless of when the enhancer was added.
Discussion
IR shows that the mimic had a rigid organisation of lipid structure as there were low carbon wavenumbers.
The mimic only had lamellar spacing of 4.5nm – no replica of the longer spacing.
Predominately hexagonal orthorhombic lateral packing.
TH is a small molecule with balanced lipophilicity that is likely to cross the membrane via free volume diffusion
IND is a larger more lipophilic molecules that prefers lateral diffusion along the lipid bilayers.
Both molecules diffuse better when the head group is less polar.
There was a smaller lag time for the diffusion of the molecules through the lipid membranes than through the porcine skin. This is because corneocytes hinder the process of the molecules through the skin whereas the chemicals can interact with all of the skin of the mimic as there are no impermeable stacks of corneocytes that prevent their diffusion. The membrane was also slightly thinner than that of the porcine skin.
The smaller TH molecule had quicker diffusion times
Azone molecule disrupts the rigid form of the lipids so the membranes become easier to permeate through.
L-Pro2 created phase separation within the lipid membranes resulting in a less ordered phase in the stratum corneum lipids.
Propylene glycol can act as a permeation enhancer by disrupting the keratin but a control test proved that the enhancement seen in these two enhancers was not due to that
Enhancement ratio greater for the mimic than for the porcine skin as it is purely lipid so has a greater surface area over which it can work.
Higher wavenumbers in the aftermath of the application of the enhancers shows the lack of rigidity in the lipid membranes.
Lipids and barrier function of the skin - Wertz 1999
Abstract
This paper aims to review the structure of the skin and the resulting physical and chemical properties.
Lipids accumulate in small organelles within keratinocytes called granular organelles. Once terminally differentiated into corneocytes, the lipids are extruded into the intercellular spaces.
Enzymes process these lipids into a range of ceramides, cholesterol and fatty acids that is then organised into two lamellar bilayers. The barrier nature of the stratum corneum is largely reflective of the phase behaviour of the intercellular lipids.
Lamellar granules are thought to be assembled by acylglucosylceramide
Discussion
The stratum corneum is the outermost layer, and the thinnest layer of the epidermis.
This structure provides the barrier to external bacteria and chemicals entering the body whilst regulating water and heat loss.
A major aspect of differentiation as keratinocytes become dead corneocytes is the accumulation of lipids within internal organelles known as granular organelles.
Upon cell death, these are expelled into the intercellular lipid matrix along with enzymes that process these lipids into those that eventually form the lipid domains that make up the stratum corneum.
Enzymatic processing takes the expelled lipids from phospholipids, glycolipids and cholesterol into ceramides, fatty acids (straight chained) and cholesterol.
Oleic acid which is transferred from phosphoglycerides to cholesterol during the process can be separated at the last moment as oleic acid is a potential permeability enhancer.
Granular organelles containing lipids are not particularly dense and can be isolated by density gradient centrifugation.
Granular organelles are ovoid in shape roughly 200 nm in diameter.
It is filled with flat vesicles containing an unusual glycolipid which is to be found in figure 1 (from the actual paper)
This unusual structure is the primary source of linoleic acid that is then found in the stratum corneum. Linoleic acid is crucial for the barrier properties of the stratum corneum
A linoleate containing acylglucosylceramide has also been suggested to be a principle player in the formation of granular organelles.
The longer tail of the acylglucosylceramide is twice as long as the shorter tail. This longer tail is long enough to span an entire bilayer whilst the other tail inserts into a different but equally close lipid bilayer. Thus it acts as a rivet to hold two bilayers together to promote the flattening of lipid vesicles and to stack them efficiently
This acylglucosylceramide plays a key role in the formation of these lamellar granules as lamellar granules are only present in cells where this acylglucosylceramide is also present. Neither are present in cells in your mouth etc.
The epidermis of fish and reptiles do not contain the acylglucosylceramide and also do not contain the lamellar granules.
Prior to the exocytosis of the lipids into the external lipid medium, the membrane bounding of the lamellar granules fuses to the cell membrane.
Glucosylceramides become deglycosylated at this point and the linoleate is removed and recycled.
The resulting w-hydroxyceramide becomes covalently to the cell envelope and this hydroxyceramide coats the membrane layers and is responsible for the corneocytes becoming impermeable.
Once deglycosylated, the resulting acylceraide is dumped into the intercellular space and plays an important role in organising the lipids in the membrane.
The bilayers that were riveted together in the lamellar granules are still riveted together and the bilayers move around together
The major lipid classes are ceramides (50%), cholesterol (25%) and fatty acids (10%).
There are 6 types of ceramides that one can separate in porcine skin.
Free fatty acids found in human and porcine skin are straight chained, saturated species about 16-30 carbons in length
Cholesterol is the largest SINGLE lipid and the skin is saturated with cholesterol. Cholesterol is thought to be able to make the gel phase more fluid and more pliable than simply having no cholesterol. This is hugely important in skin as it needs to be fairly flexible. Brittle skin would rip and the organism would die.
All the lipids are cylindrical or rod shaped and as such are perfect for forming highly ordered, flat lipid membranes.
It is thought that both liquid phase and gel phases coexist.
This coexistence is though to occur in a mosaic manner - gel phases are separated from one another by a continuous liquid domain.
Polar molecules can diffuse along the polar head groups whilst non polar molecules diffuse through the non polar matrix on the inside of lipid bilayer.
Diffusion is easier in the liquid domain than in the gel domain.
The phase boundary has the greatest number of packing defects and therefore diffusion is easiest here.
Near the junction between corneocytes and the lipid membranes there exists three bands, one broad (5nm), one narrow (3nm) and then another broad (5nm).
A zipper like structure holds together the corneocytes made from sphingosines from the hydroxyacylceramides. Free lipids fill some of the space created
6 band (total width 26 nm) and 9 band arrangements also exist.
Landmann units exist in 12 band formations where the middle 2 are both broad and held together.
The lipid layers are covalently bound to the corneocytes to provide continuous protection
The acylceramide that exists in the granule organelles also exist in the Landmann units
If the acylceramide is removed, then the 13nm repeating band width is no longer seen in x-ray diffraction studies.
Desmosomes are thought to be highly important in producing a cohesive layer. Their degradation is important to cell replacement from below
Mechanism of Antibacterial Activity of Choline Based Ionic Liquids (CAGE) Ibsen, Ma, Banerjee
Abstract
Ionic Liquids (ILS) have shown great promise in our fight against antibiotic resistant microbes
The mechanism is unknown but if we were to know we could devise highly potent but minimally toxic (to humans) reagents.
Previously CAGE has been shown to be highly effective against bacteria, fungi and viruses but begnin to humans
Four variants of CAGE investigated with the minimum concentration required to kill E. Coli found in each case.
Molecular Dynamics simulations to find mechanism of CAGE on E. Coli is found to be related to how the choline is attracted to the negatively charged membrane.
This aids insertion of the geranic acid into the lipid bilayer
This was confirmed with propidium iodide staining via flow cytometry and scanning electron microscopy
FTIR shows the lipid membrane was altered in a similar fashion to a phase transition
No resistance was shown upon multiple exposures to the ionic liquid
Introduction
Antibiotics are becoming increasingly useless and hard to replace.
ILS are a very diverse group of compounds with the potential to mix and match ions to tune their behaviour
The properties of ILS can also be tuned by altering the side chains
It could have a similar mechanism to cationic biocides or surfactants such as benzalkonium chloride
Choline has reported to have displayed anti-microbial properties with a range of counterions
Choline and geranic acid form an ionic liquid with good antimicrobial properties whilst not being toxic to humans
Low quantities of CAGE provided complete bacterial neutralization
Mechanism postulated to be that the aliphatic chain inserts into a similar manner to pesticides/surfactants
Could also be due to the inhibition of acetylcholinesterase by the cation
Molecular dynamics simulations show imidazolium cations interact with the polar headgroup of lipids and insert hydrophobic tails into the membrane
These interactions are highly dependent on the charge and structure of the the cations and counterions and the complexity of the membranes
The secondary effects of chain insertion on signalling etc is unknown
This information could lead to improved tuning and avoidance of resistance
Investigated choline bicarbonate: Geranic Acid ratios of 1:4/1:2/1:1 / 2:1
NMR INCLUDED IN THIS PAPER
Results
NMR results are all good with integrals working out well
Minimum bactericidal concentration is lowest for combinations of choline bicarbonate and geranic acid than the 2 together
This seems that they have to work together to have any effect
13 nM CAGE was bacteriostatic. 6nM allowed slight colony growth which plateaued and no further growth was seen.
Propidiumm Iodide (PI) was used to stain dead bacteria cells. After 2 hours 8mM CAGE showed limiting staining, 26 mM CAGE showed considerable staining.
Quaternary ammoniumm choline cation with a small hydroxyl alkyl chain can penetrate the lipopolysaccharides (LPS) domain and form stable interactions with the negatively charged membrane.
Choline facilitates the penetration of geranate ions into the lipid tails – the negative headgroup is not in the hydrophobic domain and is stabilised by the choline.
Choline without the excess geranic acid (2:1 and 1:1 ratios) easily penetrates the membrane binding to the negatively charged core and the lipid head groups
Higher density of choline results in a higher density within the core LPS headgroups
Geranate penetrates the outer LPS leaflet and reaches the inner leaflet
Equilibrium is reached within 6-7 us.
Bicarbonate ions with short alkyl chains don’t insert into the LPS domain
Compared to choline in the 2:1 and 1:1 ratios, the number of contacts is 6-8 times lower for geranic acid
In 1:2 and 1:4 mixes, uncharged geranic acid molecules penetrate the outer and the inner leaflets of the membrane, especially in 1:4.
Penetration of pure geranic acid without any choline present is dire
Replacing choline with an Na+ ion displays no improvement in the penetration of the geranate ion. The Na+ ion penetrates the LPS core and makes contact with the membrane lipids but Na+ is a hard ion so doesn’t interact with the geranate ion. The geranate ion does not get pulled through. Therefore, choline is an essential ingredient for the penetration of geranate ions.
Scanning Electron microscopy was used to image the cell membrane before and after application of CAGE. The membrane appeared rough and flakey compared to the smooth surface of untreated cells. 2:1 choline : geranic acid gave a bubbly texture whilst only a small number had burst the cell membrane.
FTIR was used to investigate cells grown in a sublethal concentration of CAGE.
This resulted in increased peak height (greater increase for 24 hour growth compared to 2 hour growth) which demonstrates an increased quantity of these bonds. Therefore, the cell has responded by increasing the number of lipids in the membrane.
An upwards shift in the frequencies of these peaks also denotes a conformational change within the membrane from an ordered gel phase to a disordered liquid phase
Cell resistance was investigated but neither method displayed any chance of resistance with lipid formations not changing to prevent penetration
Discussion
Geranic acid and choline are both naturally occuring safe compounds
Outer membrane is LPS rich
Inner membrane is DPPE rich
E.Coli is heavily negatively charged --> The fact they are negatively charged makes them susceptible to CAGE.
LPS negative charge is effectively screened by the choline Cation which facilitate the movement of geranic acid into the inner layer
1:4 CAGE has the highest penetration
Hydrophobic geranic acid is able to dissociate from the cation enabling it to penetrate further
It is structurally similar to fatty acids hence has a good ability to penetrate further in but combining it with choline gives It better solubility in aqueous environments
Simplified stratum corneum model membranes for studying the effects of permeation enhancers - Curikova 2017
Abstract
Skin samples for intro experiments are hard to source and can be highly variable in nature.
The paper prepared skin mimics from a mix of Stearic acid, cholesterol, cholesterol sulphate and ceramides. These ceramides were N-2-hydroxystearoyl phytosphingosine (CER[AP]) and/ or N-stearoyl phytosphingosine (CER[NP]).
Permeation of theophylline (TH) and Idomethacin were compared both through this membrane and porcine skin.
Mixed ceramides gave the optimal result
This was used to test two different known drugs able to permeate through the stratum corneum to compare the results of this mimic against porcine skin results. The agents were theophylline (TH) and indomethacin (IND)
The 1:1 mix of ceramides gave the closest results to the porcine skin results. Following on from this trial, this mixture was used to test the two permeation enhancers L-Pro2 ((S)-N-acetylproline dodecyl ester) and Azone (N-dodecyl azepan-2-one).
Both permeation enhancers were found to increase the rate of TH and IND through the membrane more markedly than the porcine skin
L-Pro2 was found to be a better permeation enhancer than Azone
Introduction
The function of the skin is 2-fold – to prevent entry of foreign chemicals and bacteria and to regulate temperature and water loss
The skin is formed when terminally differentiated keratinocytes (corneocytes) become embedded in a mix of at least 12 different types of ceramides, cholesterol, free fatty acids and a small quantity of cholesterol sulphate and other lipids.
This lipid structure is divided into 2 parts, a long phase of about 13 nm and a short phase of about 6 nm packed in a hexagonal orthorhombic manner.
Usually the permeation enhancers disrupt the lipid membranes, but sometimes they do affect the corneocytes.
A franz diffusion cell was mostly used in the testing of the membranes and the permeation of chemical enhancers.
Testing of skin can be difficult as different species have different skin qualities, some have a greater number of pores than others, so testing for human medicines is tough.
The types of lipids used in a mimic is important; shorter chain ceramides can increase permittivity whereas sphingosine-CER display lower permeabilities.
Materials/Method
The thickness of the porcine skin was 1.02 +/- 0.04 mm
IND/TH are standard permeation markers.
Enhancement Ratio (ER) =
where Je is the flux in the presence of an enhancer and J is the flux without an enhancer
Electrical impedance was used to characterise the effects of the permeation enhancers on the integrity of the lipid membranes.
Electrical Impedance Ration (EIR) = EI1/EI2 where EI1 is the impedance before the application of the enhancer and EI2 is the impedance after the application of the enhancer.
Results
TH/IND reached a state of steady flux through the mimic skin quicker than through the porcine skin
None of the mimics had a flux through the membrane that was significantly different to that of the porcine skin
The closest replica was that of the 1:1 mix of ceramides
Electron microscopy showed an even thickness of about 9.5 um. This lies close to the thickness of real stratum corneum lipid membranes.
IR spectroscopy showed all the correct peaks which have been assigned in the paper
Both azone and L-Pro2 increased the flux through the membrane in line with result from the porcine skin
The effect was considerably negated if the L-Pro2 was applied before applying the drug itself.
The electrical impedance of the membrane was affected regardless of when the enhancer was added.
Discussion
IR shows that the mimic had a rigid organisation of lipid structure as there were low carbon wavenumbers.
The mimic only had lamellar spacing of 4.5nm – no replica of the longer spacing.
Predominately hexagonal orthorhombic lateral packing.
TH is a small molecule with balanced lipophilicity that is likely to cross the membrane via free volume diffusion
IND is a larger more lipophilic molecules that prefers lateral diffusion along the lipid bilayers.
Both molecules diffuse better when the head group is less polar.
There was a smaller lag time for the diffusion of the molecules through the lipid membranes than through the porcine skin. This is because corneocytes hinder the process of the molecules through the skin whereas the chemicals can interact with all of the skin of the mimic as there are no impermeable stacks of corneocytes that prevent their diffusion. The membrane was also slightly thinner than that of the porcine skin.
The smaller TH molecule had quicker diffusion times
Azone molecule disrupts the rigid form of the lipids so the membranes become easier to permeate through.
L-Pro2 created phase separation within the lipid membranes resulting in a less ordered phase in the stratum corneum lipids.
Propylene glycol can act as a permeation enhancer by disrupting the keratin but a control test proved that the enhancement seen in these two enhancers was not due to that
Enhancement ratio greater for the mimic than for the porcine skin as it is purely lipid so has a greater surface area over which it can work.
Higher wavenumbers in the aftermath of the application of the enhancers shows the lack of rigidity in the lipid membranes.
Tanner Ibsen Mitragotri 2018 – transdermal insulin delivery using choline based ionic liquids
Abstract
Transdermal delivery of pharmaceuticals using ionic liquids and deep eutectic solvents is promising They are tuneable and can transport large molecules across the skin Viscosity, miscibility and transport enhancement can be controlled through the choice of ions used Physicochemical properties such as viscosity, miscibility and conductivity was measured Effects of transdermal drug delivery measured on ex Vivo porcine skin
Introduction
Transdermal drug delivery is beneficial because it is non invasive, avoids first pass elimination and improves patient compliance Ionic liquids and deep eutectic solvents have been shown to be biocompatible agents that can improve the permeability of the stratum corneum The ratio between the anion and the cation can significantly alter the physical properties of the ionic liquid and the interactions it has with the stratum corneum
Results and discussion
NMR discussed and presented for cage
The position of the OH peak in choline shifts with changing the ratios of choline and Geranic acid. The shift decreases from 11-10-6 in a linear ish manner as the proton becomes more shielded as more choline is introduced. This suggests that the proton on the choline is hydrogen bonding to the Geranic acid which is in turn deshielding the proton. As the choline moves to excess it cannot hydrogen bond to the Geranic acid any more Degree of hydrogen bonding is likely to change how each variant with biological membrane and the dissolved compound Manipulation of the hydrogen bonding allows poorly soluble drugs to dissolve Ionic liquids with a higher degree of hydrogen bonding shows less skin irritation. NOESY was used to identify through space interactions that occur within 5nm This represents a global average of interactions as instantaneous ones are too short to be seen. NOESY won't show h bonding between the Geranic acid and the choline as the lability of the proton means these bonds are also formed and broken quicker than the timescale of the experiment NOESY is being used to investigate non hydrogen bonding interactions that occur over a long period in solution Each variant displays different inter and intra ionic interactions CAGE 1:2 ; primary interactions are between the sp3 protons (both terminal and internal methyl groups) and the methyl groups attached to the nitrogen on choline CAGE 1:1; Same as 1:2 plus through space interactions between the hydroxyl proton on the internal protons on Geranic acid. (In this case the internal CH3 group and the protons on the two neighbouring carbons to the right (13/14/17/18) For 2:1 CAGE interactions with the nitrogen methyl groups dominate For CAGE 1:4; interactions exist between both the choline and the external and internal protons of Geranic acid. This demonstrates that the bonding in CAGE is probably a complicated form of molecules all rapidly exchanging protons Most likely 4 Geranic acid molecules circling one choline molecule Different microscopic interactions might make the CAGE act differently in biological situations Will also effect which molecules will be soluble More Geranic acid, more hydrophobic solvent. More choline, more hydrophilic solvent. For 1:1 CAGE the proximity and strength of the hydroxyl proton to the alkyl groups of the geranate ion suggests that they may prefer to interact with each other rather than a drug molecule or lipids in the skin An almost linear trend of more choline the higher the viscosity and conductivity appears We might expect a less viscous ionic liquid to be better at conducting but it could be due to the fact that short lived neutral pairings are produced resulting in about 1/2 of all ions not contributing to conductivity This can be interpreted as the Geranic acid content changes the viscosity changes. As there is more Geranic acid, the viscosity decreases and it becomes more fluid. However, it disrupts the natural pairing of cation and anions and increases proton transfers to decrease conductivity 1:1 pairing is the odd one out. Conductivity is very low as it's much more likely to go around as a neutral molecule whilst the viscosity is much greater as ions cannot flow without bumping into an oppositely charged ion and forming a pair Conductivity and viscosity needs to be minimised in a cream based medicine High conductivity leads to high skin irritation Pairing of ions shields the charge and protects the skin Thermal stability not effected by the change in ion ratios All variants had MPs below 100 Short term stability : 1:2>1:4>1:1>2:1
The glass transition temperature was measured for all variants and found to be between –50 and –150 for all variants in the order 2:1<1:2<1:4<1:1 whilst 2:1 was by far the lowest.
Follows a trend of increasing geranic acid content to increasing Tg.
Water miscibility of CAGE can be altered by changing the ratio of choline to geranic acid. A high choline component means it is completely miscible with water. 1:2 ratio of choline to geranic acid was sparingly soluble and 1:4 was completely immiscible
FTIR has been used to interrogate changes in the intracellular lipids in the stratum corneum and the mechanism by which penetration is enhanced
Extraction/fluidization of the lipids of the bilayers that surround the corneocytes in the SC increases the permeability of the skin
CAGE may simply work as a solvent, diffusing into the lipids and partition in them, weakening inner-lipid interactions and extracting the excess CAGE from the surface of the skin into the bilayers
FTIR was used to investigate the effect of CAGE on the lipid structure.
Lipid bond vibrations have peaks between 2800 cm-1 and 3000 cm-1 and the peak height is indicative of the amount of that vibration present.
Peak height of the peaks at 2850 cm-1 (symmetric CH2 stretching) and 2920 cm-1 (Asymmetric CH2 bond stretching) decreases with increasing geranic acid content
Lipid extraction is shown by reduction in the peak height of the peak at 2850 cm-1. Broadneing/positive shift of this peak is representitive of a fluidisation.
Octanol/water partition coefficients show increasing hydrophobicity with increasing geranic acid.
This leads to increased lipid removal from the SC.
CAE 1:2 shows the best detectable transport of insulin through the porcine membrane
1:4 was ok but 2:1 and 1:1 were terrible and just accumulated on the skin
Geranic acid by itself is also useless at transporting things across the membrane despite a similar level of lipid extraction to that shown by the 1:2 and 1:4 CAGE variants
Geranic acid is highly hydrophobic and may not be able to dissolve proteins
Choline by itself will also not work
Ex vivo models can predict in vivo results because the stratum corneum is the greatest barrier to drug permeation but is made up of dead corneocytes. Thus ex vivo procine skin gives very closely aligned results to in vivo studies