Nanomedicine

Question 1

Describe the structure of liposomes.

Answer 1

They are spherical vesicles which consist of a singular or multiple phospholipid bilayers.
The structure of a phospholipid consists of a polar head group with two non-polar tails.
In a liposome the outer circular head group of the phospholipid faces outwards to the external aqueous environment and the internal circular head group faces inwards to the internal aqueous environment in the core of the liposome. Each of the non-polar tail groups are protected in between the head groups.

Question 2

How does the structure of phospholipids differ to the structure of micelles?

Answer 2

Micelles have one non-polar tail group per one polar head whilst phospholipids have two non-polar tail groups per one polar head.

Question 3

What are phospholipids and micelles both examples of?

Answer 3

Amphiphile- a compound that has both hydrophilic and hydrophobic properties

Question 4

Thinking about the differences in structure between micelles and phospholipids, what shapes are they capable of forming?

Answer 4

Micelles only have one tail group per polar head and therefore by itself more likely to assemble into a cone formation whereas phospholipids have two tail groups per polar head so by itself adopts a cylinder shape.

Question 5

What does aggregate formation depend on?

Answer 5

Mainly dependent on the structure of the amphiphile.
It is possible to predict which type of aggregate is formed depending on the packing parameter.

Packing parameter is equal to:
Volume / Area of the head group x length of the alkyl chain

Shape of the aggregates is not exclusively determined by structure but also temperature, ionic strength and pH of solution.

Question 6

Why do phospholipids form aggregates in aqueous (hydrophobic) environments?

Answer 6

When phospholipids are dispersed in an aqueous solution, their non-polar tails are unable to interact with the water molecules, which reduces the volume available for water molecule to occupy. Their movement becomes restricted and their formation becomes more ordered.
When the non-polar tails parts of the phospholipids aggregate, the surface area of the molecular parts facing the water is reduced and the number of ordered water molecules is decreased as the volume they can occupy is less restricted, causing an increase in the entropy of the system.

Question 7

What packing parameters favour liposome formations?

Answer 7

Liposomes are spherical vesicles and therefore a packing parameter of between 0.5 and 1 forms flexible phospholipid bilayers and hence vesicles.

Question 8

A drug has a log P of 1.6 which has been identified for liposomal formulation, describe the manufacturing process? How would this differ if the drug had a log P of 6?

Answer 8

A drug with a log P of 1.6 is a hydrophilic molecule. Whereas a drug with a log P of above 5 (6) is a lipophilic molecule.
The manufacturing process of preparing liposomal formulations are the same, the log P only affects when you add the drug to the formulation.
Firstly raw lipids are dissolved in organic solvents such as ethanol. The solvent is then evaporated during heating in a water bath and is dried by purging with drug nitrogen. A thin liposomal film forms around the inner wall of the flask and water/buffer are added and heated to above the melting temperature (only if the liposome has a high melting temperature). Water/buffer is then added to hydrate the liposome in a closed rotatory flask and undergoes vigorous shaking and potential sonification in ultrasonic bath, which enables the film to peel of the flask and form liposomes specifically multi-lamella vesicles. Conversion to SUV or LUV is completed by sonification, homogenisation or extrusion.
Lipophilic drug molecules (Log P 6)that need to be encapsulated into liposomes can be added at the beginning of the preparation process with the raw lipids. Hydrophilic drug molecules (Log P of 1.6) are added in with the water and buffer/solution.

Question 9

How are liposomes classified?

Answer 9

Liposomes are classified by size (their diameter) as well as their number of lamella.

There are both small uni-lamella vesicles (25-100 nanometres) and large uni-lamella vesicles (up to 1 micron in size).

Multi lamelli vesicles contain concentric or muti-encompessed lipid bilayers.

Question 10

State an example of a bilayer modification that could be made to a liposome.

Answer 10

Addition of cholesterol

Question 11

How would the example of a phospholipid bilayer modification alter the properties of the liposome?

Answer 11

Cholesterol when added to the liposomal formulation, occupies the internal tail groups of the phospholipid bilayer due to its extended planar hydrophobic group. This increases the rigidity of the interface, reducing the permeability of the liposomal surface, results in a higher retention of the drug for longer periods of time.

Question 12

State an example of a surface modification that could be made to a liposome.

Answer 12

PEGylation of the liposome (addition of polyethylene glycol to the polar head group)

Question 13

How would the example of a phospholipid surface modification alter the properties of the liposome?

Answer 13

The addition of polyethylene glycol molecules to the polar head groups of the phospholipid bilayer, that protrudes into the aqueous environment, this disguises the liposome.
This can have many effects on the properties of the liposome:
Prevents aggregation of the liposome due to steric hinderance of the protruding PEG molecules.
Hydrophilicity of the surface increases reducing the affinity to the phagocytes, so it is not uptake in the reticulo-endothelial system
Reduce antigenicity and immunogenicity
Influences the pharmacokinetic properties of the drug: Reduces rate of liver clearance and prolongs the half life (and volume of distribution), reducing the patient’s dosing frequency.

Question 14

Where do different drugs with different log P values reside within the liposome?

Answer 14

Drugs with a log P less than 1.7 are hydrophilic molecules and therefore will reside within the aqueous compartment in the core of the liposome.
Drugs with a log P greater than 5 are hydrophobic/lipophilic molecules and therefore will reside within the phospholipid bilayer.
Drugs that have a log P between these two values, known as an intermediate log P, will partition between the aqueous compartment and the phospholipid bilayer.

Question 15

Why is a drug with a known intermediate log P value known to be problematic when attempting to incorporate into the liposomal formulation?

Answer 15

This is because the drug will partition into both the aqueous compartment in the core of the liposome in addition to the phospholipid bilayer. In vivo when the liposome enters the aqueous environment (plasma) the drug contained within the phospholipid bilayer partition out into the plasma. This can lead to dose dumping, an increase in potential adverse effects, of potentially very cytotoxic drugs.

Question 16

How can the manufacturing process be altered to avoid potential dose dumping of drug in lipid formulations that an intermediate log P values?

Answer 16

Utilisation of a remote drug loading (active loading) leads to the higher retention of the drug until it is delivered to its target site and it is released. Firstly a preformed liposome is formulated, with the aqueous compartment also including a so called ‘trapping agent’. A trapping agent could be a gelling agent, pH or ions.
The transmembrane gradient of the pH or ionic concentration is the driving force which facilitates the diffusion of the drug across the phospholipid bilayer to the inner aqueous core, causing the encapsulation of the drug, increasing its retention and stability until it is delivered to its target site.

Question 17

Is the remote loading process only used for drugs with an intermediate log P?

Answer 17

No not necessarily just for drugs that partition across both the phospholipid bilayer and aqueous compartment, it is an effective method in order to improve stability and retention of drugs within their liposomes.
This is an effective method to prevent leakage of lipophilic/hydrophobic molecules with a log P greater than 6 from the phospholipid bilayer.
Idarubicin, an anthracycline used in cancer chemotherapy is an example of this, in which the hydrophobic drug (resides within the phospholipid bilayer) undergoes interactions within the bilayer with cholesterol and charged lipids. Remote drug loading introduced EDTA disodium or diammonium salt as an agent to form low solubility complexes between the drug and EDTA molecules which increased its retention.

Question 18

What are drugs with an intermediate log P also called?

Answer 18

Amphiphatic drugs

Question 19

Where are drugs held once a trapping agent has been introduced?

Answer 19

In the aqueous compartment regardless of their log P

Question 20

What does the passive loading of drug molecules involve?

Answer 20

Passive loading is the process that is used in the liposomal preparation of both drugs with a log P of below 1.7, or a log P above 5, where the drug is added at the appropriate stage of the liposomal formation (either at the beginning with the raw lipids or when the aqueous compartment is added).

Question 21

Describe some of the reasons behind the need for drug encapsulation within a liposome.

Answer 21

• When a drug is encapsulated within a liposome, the pharmacokinetics of the drug (which may be unfavourable) is no longer determining the fate of the drug but instead the pharmacokinetics of the liposome. This can overcome pharmacokinetic problems associated with the formulation of the drug itself without having to alter its molecular structure.
• As the vehicle is now considered as the external drug this can alter the absorption, distribution, metabolism, excretion of the drug as well as reduction in potential side effects, leading to reduced clearance from the body and enhanced therapeutic effects but a reduction of adverse side effects.
• The liposome can be modified to ensure sustained or modified release and hence acts as a drug reservoir, giving a patient better therapeutic control and a reduction of dosing frequency.
• Liposomes also protects newer drugs such as peptides and nucleotides from enzymes that cause their degradation such as DNAases, RNAases and peptidases.

Question 22

Describe how liposomal encapsulation can overcome problems associated with poor solubility.

Answer 22

Drugs with a poor solubility will precipitate out in an aqueous environment such as in the plasma. However liposomal formulation provides both a hydrophilic and a hydrophobic environment for the drug to partition into, preventing precipitation and the adverse effects associated with that. Liposomal formulation ensures that the drug retained and only released when it reaches its biological target, minimising problems associated with solubility.

Question 23

Describe how liposomal encapsulation can overcome problems associated with tissue damage on extravasation.

Answer 23

When a drug has the potential immunogenic properties or associated toxicity associated with it by encapsulating the drug into a liposome, there is reduced bio-distribution of the drug and depositing into unwanted tissue which could provoke an immune response. Moreover the drug is only released and exposed at its biological target from the liposome so is unable to cause tissue damage from extravasation.

Question 24

Describe how liposomal encapsulation can overcome problems associated with rapid metabolism of drug in vivo.

Answer 24

The quicker drugs are metabolised, the shorter their duration of action and hence reduced therapeutic effect. Liposomes are formulated for drugs to be retained in either their hydrophobic or hydrophilic environment to ensure a sustained release profile. If there is still an instability issue with drug and it is rapidly metabolised, introduction of remote loading techniques that includes a trapping agent ensures that the drug is retained within the liposome for longer as it is encompassed within the aqueous environment of the liposomal core.

Question 25

Describe how liposomal encapsulation can overcome problems associated unfavourable pharmacokinetics.

Answer 25

A drug that has unfavourable pharmacokinetic properties when enclosed within a liposome is no longer the issue, as it is the pharmacokinetic properties of the liposome which manipulates its volume of distribution, half life etc. Drugs with poor pharmacokinetic properties may be rapidly cleared from the body, enclosing the drug within a liposome reduces this clearance, as the determining pharmacokinetics become that of the vehicle.

Question 26

Describe how liposomal encapsulation can overcome problems associated with poor biodistribution .

Answer 26

In drugs with a poor biodistribution, this results in a widespread distribution throughout the body, increasing the potential for adverse side effects and limiting its therapeutic effect. In liposomal formulations there is much smaller and more targeted distribution of of the liposomes, reducing the potential for adverse effects.

Question 27

Describe how liposomal encapsulation can overcome problems associated with lack of selectivity.

Answer 27

In drugs where there is a lack of selectivity to the target tissue this can result in lower uptakes of drug within the therapeutic target tissue, resulting in a sub-optimal response. Liposomal formulations can be uptaken via the EPR which increases its selectivity.

Question 28

List some of the conditions in which liposomal formulations have been developed for.

Answer 28

Small molecule cancer drugs
Ultrasound contrast agents
Vaccines
Anaesthetics
Fungal treatments
Macular degeneration

Question 29

Describe the main drug release mechanism of cell like liposomes following IV administration?

Answer 29

Once liposomes have been intravenously injected into the bloodstream, opsonins (antibodies) recognise the cell like structure and mark them to be targeted for phagocytosis in which the liposomes are attacked and removed. This results in the accumulation of drug containing liposomes that have been opsonised within the mononuclear phagocytotic system. This essentially becomes a drug release depot, where the liposomes begin to be broken down via phagocytosis, causing the slow drug release mimicking a slow release transfusion.

Question 30

How can a liposome be modified to ensure MPS uptake?

Answer 30

Enhanced opsonisation occurs when the liposome appears more cell like therefore increased liposome size, and its lipid composition and surface charge matches that of a cell.

Question 31

What modifications may you make to a liposome when you want to avoid accumulation into the MPS system?

Answer 31

To avoid uptake of liposomes into the MPS, opsonisation needs to be avoided. Surface modifications, normally PEGylation is used to retain the liposome in systemic circulation. The densely packed hydrophilic chains of PEG reduces interparticle attractive forces but most importantly causes a steric repulsion towards circulating plasma proteins and antibodies, suppressing opsonisation and phagocytosis causing the liposomes to be ‘inert’ they become stealth liposomes. By reducing the uptake into the MPS system, PEGylated liposomes remain in circulation for longer.

Question 32

What is achieved if liposomes remaining in circulation for longer?

Answer 32

The longer liposomes remain in circulation by avoiding opsonisation and uptake into the MPS, the higher the chance the liposomes will accumulate at pathogenic sites by the EPR effect.

Question 33

What are the most important factors to consider when attempting to prolong retention in circulation?

Answer 33

Stability
Reducing clearance rate
Targeted tissue distribution

Question 34

What is Myocet (Doxorubicin) indicated for?

Answer 34

First line treatment for metastatic breast cancer

Also undergoing investigation for treatment of:
Breast cancer
Lymphoma
Ovarian cancer

Question 35

Describe the features of the liposomal formulation of Myocet.

Answer 35

The liposome is composed of egg phosphatidyl choline and cholesterol
It is a large uni-lamella vesicle of 180nm
non-PEGylated

Question 36

Describe how the features of Myocet relate to its passive targeting approach.

Answer 36

Myocet a liposomal formulation of Doxorubicin, has enhanced cell like characteristics of its liposome. This includes its large uni-lamella structure of 180nm in addition to its liposomal composition of egg phosphatidyl choline/cholesterol within the phospholipid bilayer which enhances its cell like properties and therefore increases opsonin recognition and uptake into the mononuclear phagocytic system when injected intravenously into the bloodstream. The increased accumulation of opsonised drug-containing liposomes within the MPS results (drug depots) in a slow drug-release as the liposomes are broken down, that of mimicking a slow drug transfusion as drug is released into the bloodstream.

Question 37

What is Caelyx (Doxorubicin) indicated for?

Answer 37

Breast cancer
Ovarian cancer (secondary to platinum based therapies)
HIV-associated Kaposi’s sarcoma (secondary to chemotherapy)
Multiple myeloma (secondary)

Question 38

Describe the features of the liposomal formulation of Caelyx?

Answer 38

It is a small uni-lamella structure of less than 100 nanometres
PEGylated liposomal surface attached to the polar heads within the phospholipid bilayer (PEG2000 and distearoyl phosphatidyl ethanolamine DSPE)

Question 39

Describe how the features of Caelyx relate to its active targeting approach.

Answer 39

Caelyx, a liposomal formulation of Doxorubicin is formulated in attempt to avoid opsonin recognition, uptake into the MPS and resulting in longer retention with the bloodstream.
The smaller uni-lamella vesicle of less than 100 nanometres in addition to having liposomal surface modifications (PEG 2000 and DSPE) presents the liposome as less cell like in addition to the hydrophilic chains causing steric repulsion towards plasma proteins and antibodies (such as opsonins) which prevents opsonins being able to target Caelyx liposomes for phagocytosis, and accumulate in the MPS as opsonised liposomes in depots and slower transfusion into the bloodstream. Instead due to inhibited uptake into the MPS, the liposome has longer retention within the bloodstream which increase its chance of accumulation at the tumour by the EPR effect.

Question 40

How can use of liposomal formulations of Doxorubicin also be beneficial in terms of potential for adverse effect?

Answer 40

One of the main side effects of Doxorubicin and other anthracyclines, which resulted in its lifetime exposure limit is cardiotoxicity due to the ability of the cytotoxic drugs to causing apoptosis of the cardiomyocytes. Liposomal formulations of the drug have been shown to reduce this cardiotoxicity due to lower drug distribution and deposition within the cardiomyocyte. In both Myocet and Caelyx coating the surface of the drug in a liposome avoids direct contact of the cytotoxic drug within the vasculature. In additon Caelyx in particular, has a more narrowed biodistribution in the body despite retaining in the body for longer due to the EPR effect, exploting the difference in vasculature between the healthy and tumour cells.

Question 41

What is the most common barrier in providing effective treatments for solid tumours?

Answer 41

Being able to achieve an effective local drug concentration (narrowed biodistribution) and limiting adverse effects.

Question 42

What is Abraxane (Paclitaxel) indicated for?

Answer 42

Advanced nonsmall cell lung cancer
(surgery or radiation is not an option)
Metastatic breast cancer (secondary)
Metastatic pancreatic cancer (primary)

Question 43

Describe the features of the nanoparticle Abraxane?

Answer 43

Abraxane consists of the active pharmaceutical ingredient Paclitaxel which is bound to human serum albumin nanoparticles.
The hydrophobic drug becomes essentially encapsulated within the human serum albumin, the hydrophilic head groups face outwards into the aqueous plasma environment.
Mean particle size is approximately 130 nanometres.

Question 44

Why can formulations such as Abraxane can be preferential to liposomal formulation?

Answer 44

Abraxane is formulated of Paclitaxel bound to human serum albumin. This is advantageous due to albumin being a component of the blood as a transport plasma protein and therefore is not foreign material so provokes no antigenic/immunogenic response unlike PEG chains in liposomal formulations.
In liposomes additionally to evade MPS uptake, PEGylation is required, however as a component of the blood albumin is recognised as self and therefore not marked by opsonins for phagocytosis and can remain in the bloodstream for long periods of time.

Question 45

How is use of human serum albumin exploited in terms of its drug delivery to cells?

Answer 45

Albumin is also known as a carrier protein and is usually responsible for the transport of steroids, fatty acids and thyroid hormones. Albumin undergoes gp60 mediated transcytosis across the vascular endothelium upon binding to a gp60 receptor on the cell surface. Activated gp60 by either albumin binding or gp60 crosslinking phosphorylates gp60 and caveolin 1 causing invagination and pinching off of the endothelial cell membrane enabling the transport of albumin across the cell via vesicles known as caveolae. gp60 transcytosis method of transporting albumin into tissues is exploited in the design of Abraxane to ensure extravasation into tissues from the bloodstream of Paclitaxel via Albumin binding to gp60.

Question 46

What are examples of vaccines that have nanoparticle formulations?

Answer 46

COVID-19 vaccines have all exploited nanoparticle technologies

Question 47

What are some of the barriers to drug delivery for mRNA based vaccinesand how can they be overcome?

Answer 47

mRNA is positively charged and is hydrophilic and therefore is unable to diffuse across the cell membrane to ensure effective drug delivery to the nucleus.
In addition mRNA is prone to degradation by nucleases or is removed quickly from circulation by phagocytosis.
Liposome are able to overcome all of these issues due to the phospholipid layer allowing the easy permeation of the liposome across the cell membrane. The encapsulation of the drug within the liposome also protects it from nucleases and can undergo surface modifications such as PEG to prevent opsonin detection and hence phagocytosis.

Question 48

What are some of the ongoing problems associated with vaccine drug delivery?

Answer 48

Improved stability and hence storage of vaccines should be improved in the future, to maximise efficiency and availability/roll out worldwide.

Question 49

When are solid lipid nanoparticles (SLN) and nano-structured lipid carriers (NLC) used?

Answer 49

To improve bioavailability of drugs with a:
Poor solubility
Improve stability
Absorption
Overall therapeutic effect of drugs via the oral route

Question 50

What are the benefits of using SLNs and NLCs?

Answer 50

Ease of large scale production
Increased biocompatibility
Biodegradability of the materials
Low toxicity potential
Possibility of controlled and modified release
Enhanced drug solubility
Possibility of hydrophilic and lipophilic incorporation

Question 51

How do SLNs and NLCs differ in composition?

Answer 51

In solid lipid nanoparticles, drugs are incorporated into fatty acids or mono-, di-, or triglycerides (solid lipid matrices) alongside surfactants on the outer coating to reduce the surface tension.
Nanoparticle lipid carriers are formulated by replacing a fraction of solid lipids with liquid lipids to form drug incorporated matrix. Surfactants still coat the surface of the lipid carrier.
SLNs have a perfect crystalline structure whereas NLCs have an unstructured-matrix due to the different moieties of the constituents of NLCs.

Question 52

What advantages does NLCs have over SLNs?

Answer 52

The perfect crystalline structure in SLNs mean they have a low drug loading capacity whereas in NLCs replacement of a fraction of solid lipids with liquid lipids reduces the melting point and allows more space for drug dissolution and payload increasing the drug loading capacity.
Furthermore in SLNs there is a risk of drug expulsion when there are polymorphic changes in the solid lipid structures during storage periods and during manufacturing resulting in the previously dissolved drug being expulsed. In NLC, lipid crystallisation is minimised due to the presence of liquid lipids.
NLCs also increase drug solubility in the lipid matrix and have good controlled release profiles.

Question 53

How much does the drug solubility improve by using SLNs?

Answer 53

2 to 25 fold

Question 54

What is the main purpose of self nano-emulsifying drug delivery system (SNEDDS)?

Answer 54

Enhance the solubility and bioavailability of poorly water-soluble drugs via oral administration.
They can also significantly improve the permeability or transport of poorly permeable drugs across the GI epithelium and into target tissues in addition to preventing degradation of drugs in physiological environments (harsh GI conditions).

Question 55

What does SNEDDS consist of?

Answer 55

SNEDDS generally consists of an oil, a surfactant, and a cosurfactant, spontaneously forming an oil-in-water nanoemulsion upon mild agitation. With oral drug delivery this nano-emulsion usually occurs on exposure to GI fluids.

Question 56

How does SNEDDS work in vivo?

Question 57

Compare the size of the Pfizer/BioNTech lipid nanoparticles in the vaccine to the AstraZeneca vaccine.

Answer 57

The nanoparticles in the Pfizer/BioNTech vaccine is 70-100 nanometres compared to 80-120 nanometres in the AstraZeneca vaccine.

Question 58

Describe the lipid composition of the nanoparticles in the Pfizer/BioNTech vaccine.

Answer 58

The lipid nanoparticles used in the Pfizer/BioNTech formulation share many similarities with that of the SLN/NLC used in oral administration of poorly soluble drugs. The mRNA is enclosed within a lipid core and is surrounded by chargeable (cationic) ionisable lipids. The spherical outer core surface of the nanoparticle consists of of phospholipid bilayer containing cholesterol and PEGylated polar heads.

Question 59

Explain how the nanoparticle formulation of the Pfizer/BioNTech overcomes some of the barriers associated with drug delivery of genetic material.

Answer 59

The first problem associated with mRNA drug delivery is that its positively charged and is hydrophilic. This inhibits its diffusion across the cell membrane to reach the nucleus. Encapsulating the drug within a liposome with an overall neutral phospholipid bilayer containing cholesterol ensures its uptake and diffusion across the membrane. Encapsulation within the liposome prevents mRNA being exposed to nucleases which induce its degradation.
The size of the mRNA nanoparticles for vaccine is between 70-100 nanometres so it is known as a small nanoparticle and its size alone deviates from a cell like structure which reduces its recognition by opsonins which target it for phagocytosis via MPS uptake. The surface modification of PEGylation also prevents opsonin detection and hence phagocytosis.

Question 60

Describe the composition of the nanoparticles used in the AstraZeneca virus.

Answer 60

The nanoparticles used in the AstraZeneca virus mimic that of adenoviruses (such as the chimp adenovirus) but has been genetically modified such as to present COVID spike proteins on its surface.

Question 61

What is an advantage of the adenoviral nanoparticles in comparison to the lipid nanoparticles?

Answer 61

Can be stored for longer, and are seemingly more stable than the lipid nanoparticles.

Question 62

Why nanoparticle formulations need to be retested to meet standard criteria before entering the market?

Answer 62

Nanoparticles due to their size and composition can have different A.D.M.E.T and pharmacokinetic properties.
They can have additional toxicity due to:
Their composition
Surface coating (PEG can induce immunogenic responses)
Concentration
Exposure time

Different biodegradability
Altered metabolism and excretion

Question 63

Define clearance.

Answer 63

The volume of plasma that can be completely cleared of a substance (drug in this case) per unit of time

Question 64

Explain how changing the size of the liposome can increase/decrease potential for adverse drug reactions.

Answer 64

The size of the liposome has a direct influence of its method of elimination from the body. Liposomes that are within the 6-8 nanometre range can effectively be renally cleared by the glomerulus. Renal clearance is very rapid, meaning that liposomes that are cleared by this method have a short duration of action and hence therapeutic effect. However this short duration of action, reduces the potential for adverse events (drug reactions/side effects).
Liposomes that have a diameter greater than 6-8 nanometers are unable to undergo rapid clearance by the kidneys, and are either up taken and accumulate into the mononuclear phagocytic system (which favours large, cell like liposomes) which initiates a slow drug transfusion, retaining the drug for months to years or if not are hepatically cleared from the body, which also takes longer. The longer the liposome-drug complex is retained within the body, there is an increased exposure to it, which in some cases can enhance the therapeutic effect but due to its increased exposure also increases potential for adverse effects.

Question 65

Explain how surface modifications of the liposome can increase/decrease potential for adverse drug reactions.

Answer 65

The most common liposomal surface modification is PEGylation. PEGylation, due to the densely packed hydrophilic chains causes steric repulsion with plasma proteins and antibodies, such as opsonins which target and mark liposomes for phagocytosis and uptake and accumulation in the mononuclear phagocytic system, which induces a slow drug transfusion. With PEGylation inhibiting the uptake into the MPS, this means that liposomes are either renally cleared (if they are 6-8 nanometers) or if larger are hepatically cleared. Whilst haptic clearance is not as rapid as renal clearance, it is clears the body of the drug faster than uptake into the MPS, meaning that there is a reduction in the time of exposure to the drug. Whilst in some cases this means that there is no long term therapeutic effect this also reduces its potential for adverse effects.

Question 66

Explain how the different liposomal formulations of Doxorubicin have altered routes of elimination.

Answer 66

There are two liposomal formulations of Doxorubicin that are in clinical use - Myocet (non-PEGylated) and Caelyx (PEGylated).
Myocet possesses many cell like properties such as egg phosphatidyl choline and cholesterol composition, and a large diameter of 180nm. It avoids renal clearance due to a large size of 180nm (only 6-8 nanometres can pass through the glomerulus) but most importantly its enhanced cell like properties increase recognition by opsonins and are marked for uptake and accumulation into the MPS, where it undergoes a slow drug transfusion, retaining the drug for months to years.
Caelyx on the other hand is a PEGylated liposome (PEG2000 and DSPE) and has a diameter of less than 100 nanometers. Whilst being too large to undergo rapid renal clearance, PEGylation cause steric repulsion to plasma proteins and antibodies in addition to being smaller in size, reduces its recognition by opsonins and uptake into the MPS. This means that the drug is hepatically cleared from the body, which does take longer than renal clearance especially due to the targeting the EPR and impaired lymphatic system, but is eliminated more quickly than when uptaken into the MPS.

Question 67

Would you argue that Caelyx has reduced cardiotoxicity in comparison to Myocet?

Answer 67

I would agree with the statement that Caelyx has reduced cardiotoxicity in comparison to Myocet for two reasons. Caelyx has a smaller liposomal diameter (less than 100nm) in comparison to Myocet (180nm) resulting in more targeted accumulation in the tumour site by the EPR effect, prolongation in circulation, increasing the chance to accumulate at the tumour site, rather than a slow drug transfusion of Myocet uptake into the MPS. Whilst both drugs via passive and active targeting increase the retention of the drug to enhance the therapeutic effect, long-term, increased retention (months to years) of Myocet within the MPS can be problematic as long term exposure to Doxorubicin, increases the risk of cardiotoxicity.

Question 68

How could the associated cardiotoxicity with Myocet be reduced?

Answer 68

Reducing the size of the liposome nanoparticles, which leads to more accurate accumulation within the tumour.

Question 69

How small do molecules have to be, in order to be cleared for the glomerulus?

Answer 69

6-8 nanoparticles

Question 70

Describe what is meant by passive targeting.

Answer 70

Passive targeting, is when a liposome is transported, distributed, absorbed, elimination etc under the body’s natural physiological processes. The drug is not modified to ensure specific tumour targeting.
An example of this includes, drug targeting via the EPR effect, where as the drug is retained longer in circulation and is not uptaken into the MPS, there is a higher chance of the drug accumulating at the tumour site.

Question 71

Describe what is meant by active drug targeting.

Answer 71

This is when there is modification of the exterior of the liposome surface which induces the presence of targeting properties. For example this could be presence of an antibody on the surface of the liposome which has an affinity to an epitope present on the surface of a organ which a tumour, which upon binding initiates drug release.

Question 72

Discuss the benefit of using nanomedicines in comparison to small molecules.

Answer 72

1) Reduced clearance

Smaller molecules are more prone to undergo rapid clearance from the body via the glomerulus (6-8nm) whereas nanomedicines such as those in liposomal formulations are too large to undergo this type of clearance and so are retained longer in the body, increasing their therapeutic effect via passive targeting (EPR effect associated with longer retention in vivo)

2) More targeted deposition

Many small molecules are designed balancing their hydrophobic and hydrophilic properties, especially if administered orally and so many struggle to permeate cell membranes and reach their biological target. Nanomedicines however which are administered intravenously, the drugs can be encapsulated within a liposome and therefore the pharmacokinetics of the drug are substituted for the pharmacokinetic properties of the liposome which due to their phospholipid bilayer, easily permeate cell membranes and also due to their increase retention in the bloodstream have more target deposition (EPR or modifications to the surface of the liposome causing active targeting).

3) Reduced side effects

Slightly large nanomedicines are unable to permeate through the tight junctions that small molecules are able to, which reduces its deposition in unwanted tissues which lead to side effects. An example of this is reduced myocardium deposition of PEGylated Caelyx in comparison to non-liposomal Doxorubicin. Furthermore in this example, prolonged half-life of Caelyx reduces the peak concentration of Doxorubicin accumulating in vivo which is associated with the dose-dependent cardiotoxicity.
Liposomes can be modified to undergo an active targeting approach, increasing affinity to certain tissue types.

Question 73

In acknowledgement of the potential benefits of using nanomedicines, what is the clinical significance of their use in chemotherapies?

Answer 73

Nanomedicines in chemotherapies, result in a more targeted approach leading to an enhanced therapeutic effect and improved clinical outcomes (tumour shrinkage etc). This is because achieving a local drug concentration at the site of the solid tumour is one of the main barriers in drug delivery of solid tumours.
In addition, the more targeted tumour treatment leads to a reduction of side effects associated with the chemotherapies. Small molecules are unable to target the tumour well, no preference for the tumour over healthy tissues and the drug is rapidly eliminated, so any drug that is able to accumulate does so in smaller concentrations.

Question 74

Compare the pharmacokinetic parameters between nanomedicines and small molecules.

Answer 74

Nanomedicines there is a higher blood concentration of drug for longer periods of time (enhanced passive targeting).
It accumulates more slowly but reaches a higher total drug concentration in the tumour for a longer period of time.

Question 75

Explain how enhanced permeation improves drug delivery of nanoparticles in tumours.

Answer 75

Nanomedicines in comparison to small molecules have an increased retention in circulation due to avoid rapid renal clearance. This can lead to higher accumulation at tumour sites due to the EPR effect.
Within normal vasculature nanomedicines are not able to easily permeate the endothelial cells, however the tumour microenvironment, as the tumour grows angiogenesis is initiated due to the increasing supply demands of the tumour. This results in distorted endothelial cell growth and in effect ‘leaky vasculature’. This enables nanomedicines to easily permeate and accumulate within tumours.

Question 76

Explain how enhanced retention improves drug delivery of nanoparticles in tumours.

Answer 76

The lymphatic system is usually responsible for the drainage of waste materials from a tissue, this can include drug molecules. The lymphatic system drains to the liver and then drugs are eliminated hepatically through bile/faeces. In tumours there is often impaired lymphatic drainage, which results in the impaired removal of waste materials from the tumour cells. However this can be beneficial in terms of the accumulation of drug concentrations at the tumour site, as they are not removed so readily, leading to the retention of the drug and hence enhanced therapeutic effects.

Question 77

Briefly describe the tumour microenvironment (in the context of nanomedicine optimisation).

Answer 77

Cancer is defined as the uncontrolled abnormal growth of mutated cells, that have the ability to metastasise to other areas of the body.
This rapid, uncontrolled growth causes an increase metabolic demand, meaning that once the tumour reaches a couple of millimetres in diameter it is no longer supported by the host vasculature. This results in the secretion of pro-angiogenic factors such as VEGF, resulting in the rapid but distorted growth of new blood vessels (enhanced permeation), that have no structure resulting in areas within the tumour of normoxia, hypoxia and a necrotic core.
The rapid growth also causes areas of high pressure within the tumour resulting in defective or absent lymphatic systems (resulting in enhanced retention).

Question 78

What is the main function of the lymphatic system?

Answer 78

The lymphatic system absorbs extravasated molecules that have been secreted from the tissues in the interstitial spaces. After these molecules have been resorbed into lymphatic capillaries they join to form lymphatic trunks and then into the bloodstream via the left lymphatic duct.

Question 79

What are some of the waste molecules that can be resorbed into the lymphatic capillaries?

Answer 79

Protein rich fluids, lipids, macromolecules and immunocompetent cells

Question 80

Aside from the resorption of waste products, what else does the lymphatic system do?

Answer 80

Maintains plasma volume
Prevents increased tissue pressure
Permit passage of leukocytes, and hence functioning of the immune system

Question 81

Briefly describe the evidence available for the EPR effect.

Answer 81

Often using a dye complex such as the Evans blue-albumin or a lipid contrast agent attached to the nanomedicine, enabled a clear observation of the accumulation of of the nanomedicine because of the EPR effect.

Question 82

Describe some of the issues associated with the EPR effect.

Answer 82

In pre-clinical trials, the EPR effect appeared to be very promising however when liposomal formulations reached clinical trials it appeared to only increase specificity by 20-30% in compared to critical normal organs, and only 0.7% accumulates at the tumour (however this could be effective).

Question 83

What is the EPR effect likely to depend on?

Answer 83

It is believed that the EPR effect is highly heterogenous - and can have different outcomes within different tumour types, different patients, different tumours within the same patient.
Their effect can also change over the course of the tumour development.