Section IV: Reflection on guest lecture

Reflection on guest lecture:

(1) The potential of Nanoparticles (NP) in drug delivery

(2) Evaluation of drug delivery before clinical trials.

(1) The potential of NP in drug delivery: -

The strength of Nanoparticles – Dr. Furong focuses her research on gold nanoparticles for theranostics. It is a combination of therapeutic and diagnostic strategy that aims to monitor the response of treatment in real-time instead of before and after; to optimise drug efficacy and safety (Wang et al., 2012). To specifically investigate what cells are killed by administrated drugs, an active Raman reporter (diethylthiatricarbocyaniniodid, DTTC) was encapsulated to an antibody-drug conjugated polymer, that are stable upon drug delivery for Raman imaging, single-cell detection, cancer diagnosis and surface-enhanced Raman spectroscopy (SERS), which identifies where the drug resides at (Conde et al., 2014). Difficulties in gene delivery include gene packaging, serum stability, target specificity, cellular uptake, endosomal release, transportation through the cytoplasm and gene release (Pack et al., 2005).

Gold nanoparticle has been intensively researched due to its capability to conjugate with molecules through alkyl thiol adsorption. The presence of thiol groups allows the formation of disulphide linkage with drugs or DNA. Tumour cells have relatively higher concentration of glutathione (GSH), a reducing agent that cleaves disulphide bonds into two thiol groups, therefore, drugs or nucleic acids are specifically released into the cytoplasm of tumour cells (Wang et al., 2012; Chuard et al., 2015). A research done by Conde et al. (2014) suggested that by conjugating Cetuximab onto a PEGylated gold nanoparticle could efficiently inhibit tumour growth. Cetuximab works as an antagonist that blocks the binding of EGF, halting proliferation, angiogenesis and survival of tumour. Angiogenesis is a process where ‘new’ blood vessels are developed. As the tumour tissue grows larger, it either disperse and circulate in the body as metastasis, or obtain sufficient nutrients by forming new blood vessels to gain access to exchange nutrients and waste.

Besides that, the PEGylation could drastically increase the half-time of the 90nm NP. First, it is hydrophilic and attracts water molecules to surround the NP, that masks the ‘foreign molecule’ from phagocytosis of macrophage. Also, small molecules are usually taken up by macrophage as a debris, the PEGylation could increase the size of particle to avoid rapid engulfment in the bloodstream (Hans & Lowman, 2002).

Besides that, once the nanoparticle enters the bloodstream, it tends to form aggregates with serum proteins that would decrease cellular uptake. Molecules with a diameter between 70 – 90 nm exhibit strong transfection ability (Kozielski, 2013; Anderson et al, 2005). However, Hurst et al. (2006) found that AuNP with the size of more than 250nm could deliver higher DNA loading capacity, two which is two-folds the capacity of small nanoparticles (13 – 30 nm).

The potential of NP are not fully discovered yet, that few decades ago people thought that it was absurd to utilise polymers for controlled release of drugs, but now, not only it can delivery variety of drugs into the body, it can also produce quantitative and qualitative signals to operator, which is amazing.

(2) Evaluation of drug delivery before clinical trials.

Why is it important? – When a drug is designed, evaluation must be carried out to ensure that the drug has the desired structure, pharmacophore, pharmacokinetics (absorption, distribution, metabolism, elimination) and pharmacodynamics; developing the drug through essential modification so that it carries out desired therapeutic effects. Therefore, usually it takes 10 years to bring the drug to the market. The evaluation can be categorised into chemical and biological evaluation. The order of drug development is characterisation of synthesised active compound, cell culture activity, animal testing and clinical trials.

Dr. Christine O’Conner gave a talk about the characterisation of synthesised compound using various instruments. For example, UV-Vis, High Performance Liquid Chromatography (HPLC), Gel Permeation Chromatography (GPC), Nuclear Magnetic Resonance (NMR), zeta sizer, etc.

The importance of compound’s structure – The structure does not only affect drug-target interaction, but also the pharmacokinetics. Depending the choice of administration, the molecules should have functional groups that assist the absorption through barriers such as skin, mucosal membrane, villi, cellular and nuclear membrane, etc. Most drugs that are in the form of protein faces hurdles of aggregation in the blood stream or engulfed by macrophages, hence, unable to distribute uniformly. Langer (2003) mentioned that increasing the size of the molecules, or encapsulate drugs with water molecules by PEGylation, could prevent phagocytosis of macrophages.

The characterisation should be done before and after a reaction – Some compounds require multiple step of reactions, before moving to the next step, it should be characterised to identify whether the functional groups react each other; the growth of polymer reaches the target MW, the size and potential of the compound. Software such as ChemDraw could assist on estimating NMR peaks, if the NMR does not show similar peaks with the estimated, possibly the compound does not have the desired structure. This is very important because a molecule with a different structure will not interact with target site but possibly causing adverse effects by interacting with another site. When something wrong is detected, immediate countermeasures can be carried out. For example, continue the polymerisation, ensure implication of Good Manufacturing Practice (GMP), etc.

Dr. Furong Tian gave a talk about biological evaluation of the accumulation of gold nanoparticles (NP) that has different characteristics. It was thought that the smaller the size of nanoparticle, the more efficient the cellular uptake. However, many researchers have found that NP size could specifically target different cells or tissue in vivo. For instance, bigger gold NP accumulates in the liver, smaller gold NP accumulates in the brain (0.05% can pass the blood brain barrier) and gold NP that are conjugated with ligands can target tissues selectively. When testing a subject, it is essential to keep all parameters controlled to ensure that the manipulated variable is the only factor that affects the result, compared with a control. For example, same amount of food, water, air, pressure and space are given to isolated mouse; while the manipulated variable differs based on research topic.

How to pinpoint the site of drug accumulation (distribution) – The most common method is to isolate and compare tissues of mouse. There are many ways in pinpointing the drug accumulation, which includes conjugating drugs with isotopes that emits specific radiation and conjugating with proteins and carry out western blotting. These methods allow quantification and qualification analysis to compare efficiencies between commercial and test products.

Did the accumulated drug carry out its therapeutic effect? – PET-scan requires pre-injection of radioactive substance, which is craved by cells that generates high energy. Solid tumour is a clump of cells that metabolise and proliferate very rapidly that has higher energy level compared to normal cells, resulting rapid take up of radioactive substance. Next, CT-scan also provides insight of the size and shape of solid tumour tissue (ASCO, 2016). However, PET scan and CT scan are expensive instruments that are meant to be used on human. Usually, bioluminescence imaging is used on animal testing.

Bioluminescence imaging is a method to evaluate how efficient is a drug to kill tumour cells. The amount of tumour cells are quantified with by the intensity of fluorescence. A piece of plasmid that express luciferase (enzyme) is transfected into tumour cells, where the tumour cells will be injected into small animals to create ‘tumour models’. To quantify the amount of tumour cells, Luciferin (substrate) is injected into animal. The luciferin is distributed and interacted with luciferase specifically to emit fluorescence. Hence it is performed before and after treatment on the same animal; when tumour cells decrease, the number of cells that express luciferase decrease, the lower the interaction with luciferin, hence the weaker the fluorescence intensity (Carceles-Cordon et al, 2016).

Metabolism & excretion of drugs – Generally, drugs are metabolised in the liver by Cytochrome P450 through oxidation to make the drug more water soluble. Blood (withdrawn from artery and vein), urine, faeces and exhaled air are collected to check the route and rate of excretion.

Conclusion – The human body is very complex, even if a drug works well on tissue culture and animal models, it may not work the same for human. Researchers should be patient changing parameters and try until the drug works. Ever since the death of Jesse Singer in a clinical trial for viral drug delivery, raises the concern that it is important to carefully evaluate products, in vitro and in vivo testing. Unless promising results are shown, it should not be administered to human as it could possibly cause adverse effects. It is also important to note that possible drug-drug interaction should be evaluated as it might cause unexpected effects even though the drug itself has acceptable risk.

Bibliography

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