exam 2- nanopharmaceuticals 2

Question 1

labeling NPs for PK studies, other than radioactivity

Answer 1

instead of measuring radioactive activity, can measure fluorescence

• label NP with fluorescent dyes
• fluorescence is also a sensitive technique - however, background from biological samples is significant and may overlap with tags added to NPs
• excitation = samples absorb energy and release it in the form of light

Question 2

when labeling NPs with fluorescent dyes, what range is optimal?

Answer 2

use dyes in the near-infrared imaging window (in order to track materials in our body, use fluorescence specifically in this window- b/c our bodies already have fluorescence so it can be hard to track)

• NIR wavelengths between 700-900 nm are advantageous for in vivo b/c of the low absorption of biological molecules in this region (reduces the use of radioactivity)
• many mammals are also highly fluorescent - so label the NP with dye in the near-IR window –> can track in body and take in vivo images to see where the material is

Question 3

how to take images to track fluorescent NPs in animals

Answer 3

• NIR molecular probes for in vivo imaging
  can take pics of live animals - MSOT imaging workflow- put in chamber, use anesthesia and shave the animal (b/c hair can interfere with fluorescence)

Question 4

AmBisome vs. DOXrubicin

Answer 4

Doxil - (liposomal doxorubicin) NP medicine used to treat cancer- fits all criteria for prolonged circulation time (~100 nm in size, between neg and pos 25 charge) –> b/c to treat cancer, want to stay in circulation for a while

AmBisome- (liposome loaded with amphotericin B) opposite of doxil in size and surface appearance, used to treat fungal infections, approach is to target macrophages b/c they migrate to site of infection
- non-PEGylated and negatively charged NP used to intentionally target MPS

Question 5

describe NPs function with therapeutic drugs/agents

Answer 5

the NP itself can be a therapeutic drug/agent (ex: AuNPs for arthritis therapies)

in other cases, NP can enhance another therapeutic drug to work better in our bodies, does this by 5 ways:

1- increasing circulation time (ex: doxil tends to clear out body quickly, but NP helps it stay)
2- prevent drug degradation (mRNA degraded very quickly in body by nucleases, but protected from nucleases when encapsulated in NPs)
3- increasing stability/compatibility (some medicines are highly hydrophobic, NPs can increase stability)
4- promoting cellular internalization (certain drugs that in order to be effective, need to enter cell and access cytosol and organelles, the rate of this happening can be very low, NPs help this transport)
5- targeted delivery (chemotherapy agent- only wants to hit tumor and not healthy regions, encapsulate chemo agent in NP to target better)

Question 6

describe the issue of drugs not getting into the cytosol

Answer 6

in order to achieve therapeutic effects, the majority of drugs need to enter the cytosol (it does not matter how efficient a drug is, if it cannot penetrate the cell membrane, it is useless)

• drugs that have high MW or high charge have trouble getting into cell (only gases and small nonpolar molecules can diffuse through cell membrane

Question 7

NP-mediated intracellular delivery, via lipid-based NPs

Answer 7

liposomes are made of phospholipids (similar composition to cell membrane) - favors fusion

• encapsulate drug in liposome and expose to cell –> fusion process, drug released in cell (fusion happens b/c liposome composition v similar to membrane composition)
• this is passive transport, no ATP needed
• computational models can help with the design of liposomes- can predict correct biophysical properties to enhance fusion, which depends on cell type

Question 8

NP-mediated intracellular delivery, via endocytosis

Answer 8

endocytosis = type of active transport that moves particles, such as large molecules, obsolete cells, nutrients, and even fluids into a cell (internalization of material)

• clathrin-dependent: cell membrane receptor not specific, can bind to any material- binds NP, pulls it, vesicle forms with help of specific protein, clathrin
• caveolin-dependent: same process but protein that helps is caveolin
• phagocytosis- binds NP, movement on cell membrane that engulfs that material (performed by phagocytes)
• macro-pinocytosis: movement of cell membrane to internalize materials
• clathrin- & caveolin-independent: the proteins are not clathrin or caveolin, but similar process

Question 9

once vesicle is internalized inside the cell, describes what happens with endosomes

Answer 9

the materials inside vesicle become part of the early endosome –> then late endosome –> material can then be passed to lysosome (degraded) or escape endosome (which is what we want, release NP and drug leaves NP and is accessible to cytosol)

Question 10

mechanisms of endosomal release

Answer 10

1- membrane fusion- NP/drug fuses again with the endosome to leave into the cytosol

2- proton sponge escape (most accepted theory)- endosomes have an acidic environment, cationic NPs become protonated and this process leads to an influx of Cl- counter ions to restore charge neutrality- this influx triggers membrane destabilization (alteration of pH leads to degradation of the endosome, NP and drug released)

• overall the process is not well understood, problems: NP, cargo, and cell dependent!