Drug delivery and gene therapy Flashcards
Why do we need drug delivery systems?
keep drug plasma levels between the minimal effective level and the maximal required level; the therapeutic window. for selectivity and for targeting.
There are 2 principles of controlled drug delivery systems. Which and give examples of each.
diffusion-controlled, example tablet semipermeable membrane in gastro-intestinal system, inhalation device (?) degradation-controlled, example: gels that release drug when GEL degrades.
Give examples of drug delivery systems.
inhalation devices, patches, tablets and injectables
How small is a nanoparticle? Give relative examples.
DNA (2.5 nm diam) - bacterium (2.5 microm, length) - large raindrop (2.5 mm) carbon nanotube ( 1 nm, diam) - hair (0.1 mirco m) - house (10 m)
What are quantum dots?
No idea
Give 5 examples of organic NPs and 4 examples of inorganic NPs.
polymeric NPs, micelle, liposome, nanogel, dendrimer. magnetic NP, mesoporous silica, gold NP, quantum dot
What is the “smartest” NP nature has made? Describe how it looks like and how it works.
virus (approx 100 nm) - nucleoprotein (with RNA) - capsid - lipid envelope - proteins on outer core (eg neuraminidase , hemagglutinin) -infection: endocytosis - uncoating - to nucleus - replication - transcription: outside the nucleus translation to viral proteins - maturation in Golgi - expression on cell membrane inside the nulceus: RNA is packaged with ribonucleoprotein core - move outside nucleus - budding - virus exocytosis (name correct?) with cell membrane with viral proteins on it.
Give 3 examples of nanoparticles designed for drug delivery.
1) amphiphilic copolymer with hydrophobic drug: polymeric micelle 2) polymer with pendant drug: self assembly to NP 3) lipids with drug : liposomes.
What is a polymeric micelle?
micelle formed from amphiphilic copolymers.
Give examples of drug conjugates with polymers.
drug itself is polymer (?) protein - polymer polymer-DNA complex (with hydrophilic block and cationic block) polmer - drug conjugate (with linker in between) polymeric micelle (with hydrophilic block and hydrophobic block)
Give an example of the use of a polymer drug conjugate in the treatment of a disease. What is the purpose of conjugation with a polymer?
PEGylation of interferon prolonged the half life in plasma and could be used for the treatment of hepatitis C. P.S: info from Wiki: Interferons (IFNs) are a group of signaling proteins made and released by host cells in response to the presence of pathogens, such as viruses, bacteria, parasites, or tumor cells. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses.
What is the advantage of molecular imaging in the treatment of tumours?
localisation and targeting the tumors. Localised therapy.
What is the chemical structure of PEG?
polyethyleneglycol H(OCH2CH2)nOH
Describe some general characteristics of the physiology of cancer.
1) high intake of nutrients (over-expression of receptors) 2) highly vascularised (leaky vessels) 3) acidic and reducing environment (tumor cells are acidic because of the high energy demand and consequently the anaerobe use of glucose) 4) gene mutations
What are the challenges in NP delivery?
1) barriers (removal by kidneys, liver, spleen; blood and cellular clearance) 2) opsonisation 3) endosomal trap 4) P-glycoprotein
Why is the “ideal” size of a NP for the purpose of drug delivery between 100 and 200 nm?
Natural clearance: kidneys (glomerular filtration): smaller than 5 nm liver (entrapment by Kupfer cells): smaller then 100 nm liver and spleen (reticulo-endothelial system): larger than 200 nm
What type of NPs are subject to opsonisation?
neutral or hydrophylic particles
Describe how an endosomal trap works.
endocytic vesicle - acidification inside the vesicle - destabilised membrane. Consequence: 1) membrane rupture and burst, or 2) pore formation, or 3) membrane fusion.
What is P-glycoprotein?
ATP-powered drug efflux pump. removes hydrophobic molecules.
Describe an example with a degradable polymer for the active targeting of cancer.
acid-degradable monomer plus functionalised monomer - polymer - particle formation with a protein antigen (?). In acidic environment (tumor) polymer degrades and antigen protein can destroy tumor cells. If I understood correctly.
Describe how DNA damage in the cell may occur?
UV radiation thymine dimer formation endogenous sources of reactive oxygen species resulting in either tumor promotion and/or in depletion of cellular antioxidants - more mutations due to oxidative stress which cannot be counterbalanced by DNA repair.
What is a tumor suppressor gene? Give 2 examples.
gene which codes for proteins which prevent cell division or which lead to cell death (inhibition of mitosis) Rb gene (retinoblastoma) and p53
What is a proto-oncogene?
gene that codes for proteins which stimulate cell division. Examples: PD growth factor, some ras genes, something with myc and c-jun and c-fos
How may DNA damage lead to tumor formation?
in a tumor suppressor gene: mutation results in dysfuntioning of protein - the inhibition of cell division is blocked = cell growth in a proto-oncogene: mutation results in the gene always “on” = continuous cell division. Examples Ras family protein mutations present in certain cancer types.
Describe how siRNA can counteract the effects of over-expressed proteins?
siRNA - 5’phosphorylation - complex formation with RISC - siRNA unwinding - mRNA targeting - mRNA cleavage.
Design a siRNA drug. Which steps do you need to consider?
1) select correct siRNA (in silico, in vitro) 2) stabilise RNA (chemistry: backbone modification of the “overhang” and 2’-base sugar modification) 3) select delivery method (naked, PEG, liposome, cyclodextrine, dendrimer, peptide conjugated, antibody-siRNA chimera, aptamer -siRNA chimera, 3-way junction pRNA nanoparticle; iron oxide NP and DNA nanoparticle)
Give 2 examples for the targeted delivery of siRNA.
1) direct coupling to siRNA : GalNAc ligands coupled to siRNA. These ligands bind to the asialoglycoprotein receptor on hepatocytes (liver cells). 2) cyclodestrin polymer together with PEG and with PEG coupled to transferrin protein. This protein targets transferrin receptors on tumor cells. Clinical trial results published in Nature 2010. siRNA targets ribonucleotide reductase M2. Paper in Nature: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855406/pdf/nihms183887.pdf
If we want to introduce genes into human cells, the first strategy is to focus on diseases caused by single-gene defects. Mention several such diseases.
cystic fibrosis, hemophilia, muscular dystrophy, sickle cell anemia optice nerve disease, wound repair and regeneration and cardiovascular disease (?!)
What are the challenges of gene therapy in humans?
delivery large DNA molecule, gene intergration to the right site, stability in blood, efficacy.
Describe the principle of gene therapy using viruses.
virus DNA : A - B - C gene A encodes a protein allowing gene integration in human genome. gene B and C: cause a disease in humans replace gene B and C with a beneficial gene: human cell makes now protein(s) encoded by these genes.
Describe ex-vivo gene therapy and in which disease it was successfully applied.
1) therapeutic gene and insert in viral DNA 2) take defect cells from patient 3) culture the cells and infect with the virus 4) re-introduce cell back to patient 5) the genetically altered cells produce the proteins for which the therapeutic genes encode
Succesfully applied in SCID
In which disease was gene therapy shown to work? What was the disadvantage?
severe combined immunodefficiency (SCID), immunity disease in children. - some patients got leukemia instead because the virus inserted the new genes close to an oncogene.
What is hyaluronan?
Sigma: Hyaluronic acid (HA) is the simplest glycosaminoglycan (a class of negatively charged polysaccharides) and a major constituent of the extracellular matrix (ECM)1. Hyaluronan is a scaffold secreted by cells that surrounds them in vivo2. HA is a linear, non-sulfated polysaccharide that provides compression strength, lubrication and hydration within the ECM2. It also regulates cell adhesion and motility3,4 and mediates cell proliferation and differentiation5 making it not only a structural component of tissues, but also an active, signaling molecule.
What is the principle mechanism of the suicide gene approach?
addition of a gene into a cell that results in cell death. example is genes that stimulate apoptosis. Oomen gave example of adding a gene that codes for cytosine deaminase. If you then give the patient 5-fluoro cytosine, the enzyme will convert it to a compound which is cell toxic.
Which aspects do you need to consider when developing a NP for cancer treatment?
- Size and stability of the ligand - NP complex. How long is it in the circulation?
- How effective is the NP to release the drug at the desired site (cancer cells)?
- Does the ligand induce an immunity response?
- How efficiently can you load the NP with anticancer drug?
- Does binding of the ligand to the NP affect binding of the ligand to the target receptor?
- What is the biocompability and toxicity of the NP?
- Is there spatial and temporal variation in receptor overexpression?
Mention 5 modes of cellular internalisation of NPs.
- phagocytocis (large ..)
- clathrin-mediated endocytosis (apporx 120 nm)
- caveolar-mediated endocytosis (approx 60 nm)
- pinocytosis (macropinocytsos - larger than 1000 nm)
- clathrin-independent, caveolin-independent endocystosi (approx 90 nm)
