Nanotechnology

Question 1

What are the other names for 3D printing?

Answer 1

Additive manufacturing
Additive layer manufacturing
Freeform fabrication

Question 2

What is the general process of 3D printing?

Answer 2

1. Generate a 3D model, for example using CAD software, MRI, or X-ray computed tomography.
2. Use a printer software to slice the model into layers.
3. Plan a print path for each layer, each with a start point, end point, and link point.
4. 3D print the structure layer-by-layer.

Question 3

What is the difference between additive and subtractive manufacturing?

Answer 3

Additive: you start with a material and it undergoes the additive manufacturing process to produce a 3D object and a small amount of waste.

Subtractive: start with an object and chisel away at it to produce the preferred structure, leaving more waste.

Question 4

What are the advantages and disadvantages of additive manufacturing compared to subtractive manufacturing?

Answer 4

Pros:
* Can make complex structures with no additional costs related to complexity.
* Can fit individual needs.
* Minimal waste

Cons:
* Slower process
* Objects have lower quality e.g., rougher surface finishing, worse mechanical properties.
* Quality insurance can be challenging due to customization

Question 5

How are powder-based 3D printed tablets produced?

Answer 5

1. Powder bed containing API (drug) and excipients.
2. Spreading bar spreads a thin layer of powder on the sample platform (glidants allow smooth spreading).
3. Printhead jets tiny liquid droplets of binder solution.
4. Ideally, there will be good wetting and good penetration (necessary to bind not only particles on the same layer but to bind the layers together) of the binder solution through the powder bed is required to bind the powder particles together.

Question 6

What excipients does the powder bed for powder-based 3D printing usually contain?

Answer 6

• Disintegrants e.g., sodium carboxymethyl starch, poly vinylpyrrolidone, alginic acid, croscarmellose sodium
• Glidants e.g., magnesium stearate, cornstarch, slilica derivatives, talc
• Binders e.g., PVP, hydroxypropyl cellulose, microcrystalline cellulose, polyethylene glycol, gelatin, starch,
• Surfactants e.g., Tween 20, sodium dodecyl sulphate

Question 7

What does the binder solution for powder-based 3D printing consist of?

Answer 7

Polyvinylpyrrolidone (PVP),
A solvent - water or water/ethanol mixture
A surfactant - Tween 20 and sodium dodecyl sulphate

Question 8

What are spinal fusion cages and how do they work?

Answer 8

3D printed metal cages used in the treatment of lower back pain caused by issues with the intervertebral disc. The degenerate disc is removed and replaced by the cage which is placed between the spinal bones. The bones essentially grow into the porous structure of the cage and fuse together.

Question 9

How are spinal fusion cages produced by 3D printing?

Answer 9

using a powder-based 3D printing technique known as selective laser sintering.
1. A thin and even layer of metal powder is placed on the fabrication piston using a roller or blade.
2. A high energy laser is shone upon selected locations causing partial melting of the powders and consequently the fusion of them to form a consolidated layer.
The materials used in SLS include metals (Titanium, Ti), polymers (Nylon) and ceramics (alumina).

Question 10

What is an advantage of 3D printed powder-based tablets?

Answer 10

Their porous internal structure means they disperse almost instantly, and significantly faster than a conventional fast-melt tablet.

Question 11

What is Polypill?

Answer 11

A 3D printed tablet containing Ramipril, Pravastatin, Atenolol, Aspirin, and Hydrochlorothiazide which allows a complex medication regimen to be combined in a single personalised tablet, which may improve patient adherence.

Question 12

How does Polypill control delivery of different drugs?

Answer 12

The pills consists of different compartments:
* Immediate release compartment – containing a formulation of aspirin (acetylsalicylic acid, ASA) and Hydrochlorothiazide (HCT)
* Individual extended-release compartments:
o Atenolol
o Ramipril
o Pravastatin
The immediate release and extended-release compartments are separated by a controlled porosity cellulose acetate membrane to ensure faster release of some compartments and slower release of others

Question 13

What changes in viscosity occur during extrusion-based 3D printing?

Answer 13

The ink (formulation) needs to flow like liquid (low viscosity) to be able to move easily under pressure in the cartridge and the needle. However, to form a freestanding structure which won’t collapse under its own weight and have sufficient mechanical properties, the ink needs to rapidly increase in viscisity after exiting the nozzle to form a filament.

Question 14

How does inkjet printing work?

Answer 14

1. A piezoelectric actuator responds to a pulsed voltage with a mechanical response which causes it to shrink or expand.
2. These periodic changes of volume creates a pressure wave that is propagated throughout the capillary.
3. When the positive pressure wave approaches the nozzle, the fluid ink is pushed outwards.
4. As a droplet touches the substrate it forms a sessile drop, and multiple drops connect to form a line (spacing dependant).
5. The ink is solidified usually by UV exposure, as most inks used in inkjet printing polymerise/crosslink under UV.
6. Another layer can then be printed on top of the solidified ink.

Question 15

What is the composition of extrusion-based 3D printing formulation such as Polypill?

Answer 15

• Cellulose acetate (hydrophobic membrane)
• D-mannitol (filler)
• Polyethylene glycol (PEG6000)(plasticizer)
• Acetone and dimethyl sulfoxide (DMSO) (solvents) – acetone has a low boiling point so the after exiting the nozzle, it will evaporate quickly, causing the ink viscosity to increase. DMSO is an organic solvent and has a much higher boiling point.

Question 16

What is the composition of inkjet printing formulation?

Answer 16

• The drug (cardvedilol, beta-blocker)
• N-vinyl pyrollidine
• Polyethylene glycol diacrylate
• Irgacure 2959

Question 17

How does UV solidify the ink in inkjet printing?

Answer 17

The ink/formulation contains Irgacure 2959, a photo initiator which is activated by UV to form radicals. The unpaired electrons on the radicals attack carbon double bonds in the polyethylene glycol diacrylate and N-vinyl pyrrolidone causing them to bond together and form a cross-linked latticework structure. This crosslinked structure causes the mixture to go from a liquid to a solid.

Question 18

What is photon-based 3D printing used to produce?

Answer 18

Drug-loaded hollow microspheres which acts as a drug delivery device.

Question 19

How does photon-based 3D printing work?

Answer 19

1. Two-photon microfabrication – a light based technology which uses a laser beam and a substrate to produce a hollow 3D-printed resin sphere.
2. Magnetic decoration – Ni/Ti spluttering is used to coat the sphere in a magnetic coating.
3. Drug loading – a centrifugal force is used to load the drug solution into the centre of the sphere.
4. Automated sealing – NIR-responsive sealing layer is applied to the gap where the drug is inserted, to seal it into the microsphere.

Question 20

How do photon-based 3D printed microspheres work?

Answer 20

The spherical device containing the drug is taken orally and passes through the stomach to the intestine. Since the sphere has a magnetic coating, its movement can be controlled with an external magnetic field, allowing it to be tracked to particular areas, such as a tumour. Drug release can be done on-demand once it reaches the target site using NIR (near-infrared spectroscopy) which removes the NIR-response seal.

Question 21

What is stereolithography also known as?

Answer 21

Micro-projection lithography
Digital light processing

Question 22

What is the process of stereolithography

Answer 22

1. A UV light is projected from a lens and reflected by a digital micromirror device (DMD) at pre-decided positions (by tilting the mirror) in the resin tank (containing liquid-state polymer/monomer) according to the 3D structure to be printed.
2. The monomers at the target point polymerise and solidify under the UV light.
3. The stage is moved to allow another layer of monomers to fill the space.
4. The process is repeated layer-by-layer to form a 3D structure.

Question 23

What are some methods of improving the solubility of a poorly soluble drug?

Answer 23

• Chemical modification – formulate drug as salt form.
• Use of prodrugs with higher solubility
• Increase surface area of drug.
• Formulate an amorphous solid dispersion

Question 24

Why is Itraconazole formulated as an amorphous solid dispersion?

Answer 24

Itraconazole, an antifungal which is class II of BCS due to its poor solubility (1-4ng/mL). It is formulated/dispersed in a polymer carrier composed of hydroxypropyl methylcellulose at a concentration of up to 40%. The use of an amorphous dispersion means that there is no high lattice energy from a crystalline structure which needs to be overcome for dissolution.

Question 25

Whag is the process of hot melt extrusion?

Answer 25

1. API/drug is mixed with polymer.
2. Mixture is melted and inserted into extruder.
3. Extruder mixes the API and polymer and releases them where they can either:
   a. Cool to form a solid and be cut into pellets.
   b. Exit via a nozzle which forms a viscous filament which can be layered to form a tablet.

Question 26

What are the general steps of tissue engineering?

Answer 26

1. Biopsy – extract cells either from patient (autologous) or donor (allogeneic).
2. Cell isolation – isolate cells of interest.
3. Cell cultivation – grow the cells in the lab.
4. Cell proliferation – expand the number of cells so there is a sufficient number for dosage. These cells can then be put back into the patient (cell therapy) or can progress to next step.
5. Tissue development – cells are combined with a scaffold which provides a surface for them to survive on. This cell-covered scaffold can then be cultured with growth factors and mechanical stimulus in vitro to produce a tissue.
6. Implantation – tissue implanted into patient.

Question 27

How can 3D printing be used in tissue engineering?

Answer 27

3D printing can produce 3D shapes to recapitulate the anatomical structures in the body. For example, the liver is composed of many small hexagonal-shaped units called lobules. Within the lobules are smaller units called acinus in which different vessels are situated. In order to engineer a tissue or organ which will have the same physiological functions, it will need this same structure, and this can be achieved with 3D printing. 3D printing can also be involved in production of the vasculature for solid organs.

Question 28

How can the viscosity of an ink be measured?

Answer 28

With a rheometer. The material is placed between 2 plates. The top plate then rotates back and forth and the device measures 3 values: the force required to rotate the plate, how much rotation occurs, and the speed at which it rotates. These values can then be used to calculate the shear rate (speed of rotation – low=slow, high=fast rotation) and viscosity of the material.

Question 29

What are the 2 main types of viscosity?

Answer 29

Zero shear viscosity - stationary will still flow even at low shear rates e.g., shower gel.

Yield stress - when shear rate reduced, the viscosity continues to increase e.g., hand cream.

Question 30

What are the 3 relationships between viscosity and shear rate?

Answer 30

• Newtonian – viscosity unaffected by shear rate e.g., water.
• Shear thinning – Viscosity decreases as shear rate increases. This is common for hydrogels as it means in the cartridge under pressure (low shear rate) the ink can flow, but when it leaves the nozzle and experiences no pressure (high shear rate) the viscosity increases and allows filaments to be made.
• Shear thickening – rare. Viscosity increases as shear rate increases.

Question 31

What are hydrogels?

Answer 31

Non-fluidic colloidal or polymer networks that are physically or chemically crosslinked (physical or chemical links between chains) and contain a large amount of water.

Question 32

How can polymer solutions be turned into hydrogels?

Answer 32

Individual polymer chains dispersed in water can be crosslinked to forma hydrogel.

Question 33

Name 2 hydrogels used in extrusion-based bioprinting.

Answer 33

Alginate
PolyNIPAM
Extracellular matrix

Question 34

What is alginate and how does it form a hydrogel?

Answer 34

Alginate is a polysaccharide isolated from seaweed. It forms a solution in water which forms a hydrogel upon introduction of cations as it crosslinks the chains and increase the viscosity of the solution

Question 35

What is PolyNIPAM and how does it forma a hydrogel?

Answer 35

PolyNIPAM is thermos-responsive material.
At room temperature, the material can be dissolved in water to form a solution as the polyNIPAM chains form H-bonds with the water molecules. However, if the temperature is increased to body temperature, the polyNIPAM forms a white solid.
This is because, as the temperature increases, the hydrogen bonds between the chains and the water molecules break, and the polyNIPAM groups form hydrogen bond between each other to form globules. These globules associate with each other to form a hydrogel.

Question 36

How does the extracellular matrix form a hydrogel?

Answer 36

1. ECM isolated by removing embedded cells.
2. Decellularised ECM solution is freeze dried into a powder.
3. ECM powder dissolved in buffer.
4. Solution forms a gel in response to a particular temperature.

Question 37

What is a bio-ink?

Answer 37

Where cells are mixed with an ECM solution to form a hydrogel bio-ink for 3D printing.

Question 38

What are the 2 types of needle shape and how do they affect cell viability?

Answer 38

Conical – higher peak shear stress but cells experience this magnitude of shear stress for a shorter period, and ultimately cell viability is better.

Cylindrical – lower peak shear stress but cells experience this magnitude of shear stress for a longer period, and ultimately cell viability is worse.

Question 39

What can affect cell viability during bioprinting?

Answer 39

Cell viability decreases with decreasing needle diameter and increasing pressure used for extruding the ink. Can lead to shear-stress-induced death/

Question 40

What is the principle of hot melt extrusion 3D printing of polymers?

Answer 40

Thermoplastic polymers are heated, melted, and extruded through a nozzle and, upon cooling, forms a solid filament. The thermoplastic polymers are non-crosslinked polymers which become soft and malleable then heated and harden again once cooled.

Question 41

Name 2 thermoplastics for hot melt extrusion 3D printing.

Answer 41

• Polycaprolactone – can be used to improve properties of bioprinting hydrogels. For example, it can improve shape fidelity and viscosity of ECM hydrogel.
• Poly(lactic-co-glycolic) - often used to control drug release.

Question 42

What properties are better thermoplastic polymers than hydrogels?

Answer 42

They’re mechanically more robust than hydrogels, therefore improving shape fidelity and mechanics.

Question 43

What is the difference between stereolithography and two photon stereolithography?

Answer 43

In two photon stereolithography, the excitation volume is much smaller, resulting in a higher resolution (as small as 1 micron). Only photo initiators in the excitation volume will be activated to facilitate crosslinking (solidification) of the resin. In TPS, two photons with longer wavelength (e.g., 760nm) is needed (absorbed) simultaneously to activate the photo initiator compared to one photon (380nm) needed in normal stereolithography. This means a much higher resolution (submicron, <1 μm) can be achieved by TPS. However, TPS uses a slower speed compared to one photon stereolithography due to less volume being crosslinked at a given time.

Question 44

What is an issue with using IV to crosslink photo-crossslinkable hydrogel inks to solidify them?

Answer 44

UV irradiation-induced cell death by apoptosis. As UV increases, the number of live cells decreases and the number of dead cells increases.
Therefore, an optimal UV exposure needs to be struck for balancing cell damage and mechanical properties of the 3D printed constructs.

Question 45

What is biocompatability?

Answer 45

The ability of a biomaterial to perform its desired function with respect to medical therapy, without eliciting any undesirable local or systemic effects in the recipient or beneficiary of that therapy, but generating the most appropriate beneficial cellular or tissue response in that specific situation and optimising the clinically relevant performance of that therapy.

Question 46

What is ISO10993?

Answer 46

An international standard for evaluating biocompatibility of biomaterials and medical devices, including in vitro cytotoxicity, genotoxicity, carcinogenicity, systemic toxicity, and local effects after implantation. ISO10993 tests the adverse effects, but not the active functionalities that the biomaterials are designed to perform.

Question 47

What are microneedles?

Answer 47

An array of micro-size needles that are much smaller than hypodermic needles. They enhance drug absorption by penetrating the stratum corneum (often the barrier to drug absorption through the skin) via intradermal delivery.

Question 48

What are some advantages of microneedles?

Answer 48

• Painless administration - Silicon microneedles cause less pain compared to a 26-guage hypodermic needle.
• Easy to use by patients themselves.
• Safe needle disposal
• Eliminate spread of pathogens by re-use of needles.
• Higher bioavailability and faster absorption than oral formulations.

Question 49

How do microneedles allow for painless administration?

Answer 49

MNs penetrate the stratum corneum and into the viable epidermis, only just entering the dermal layer, avoiding contact with nerve fibres and blood vessels that reside primarily in the dermal layer.

Question 50

What route do microneedles use?

Answer 50

Intradermal administration

Question 51

What pharmacokinetic profile does microneedle intradermal delivery mimic?

Answer 51

Subcutacaneous.

Question 52

How do solid microneedles work?

Answer 52

Microneedles are inserted and removed to form microscale pores in the skin surface. A topical drug formulation can then be applied to the skin and diffuse through the pores into the body.
This increases absorption of topical drugs by allowing penetration through the stratum corneum.

Question 53

What materials are solid microneedles made from?

Answer 53

Silicon
Metal
Polymers

Question 54

How are silicon solid microneedles produced?

Answer 54

1. Silicon wafer
2. Apply oxide and nitride layers on top of wafer.
3. Photoresist polymer spun on top.
4. Photo mask applied to cover certain areas and expose others.
5. Exposed areas of photoresist layer exposed to light and removed.
6. Developer removes newly exposed oxide and nitride layers, to leave pillars of layers.
7. Reactive ion etching removes remaining oxide and nitride.
8. Photoresist remaining layers removed.
9. Etching with KOH to form needles.

Question 55

How are polymer solid microneedles produced?

Answer 55

Photolithography:
1. Silicon wafer
2. Apply oxide and nitride layers on top of wafer.
3. Photoresist polymer spun on top.
4. Photo mask applied to cover certain areas and expose others.
5. Exposed areas of photoresist layer exposed to light and removed.
6. Developer removes newly exposed oxide and nitride layers, to leave pillars of layers.
7. Reactive ion etching removes remaining oxide and nitride.
8. Photoresist remaining layers removed.
10. Etching with KOH to form mould.

Micro moulding:
1. Omnicoat applied
2. UV curable monomer added to mould.
3. Photo mask applied to cover certain areas and expose others.
4. Exposed areas of photoresist layer exposed to light and removed.
5. Microneedles left.

Question 56

What are some considerations for drug-coated microneedles?

Answer 56

• Microneedles need to possess sufficient mechanical strength. Different polymers have different success rates, PolyAMPS was by the far the least successful as it possesses a sulphide group which is hydrophilic (absorbs water quickly) making it less robust.
• Controlled wetting and spreading of drug solution on microneedles – otherwise can lead to non-uniform or inaccurate drug doses.
• Adhesion between dried drug coating microneedle should be enough during insertion into skin – otherwise can lead to non-uniform or inaccurate drug doses.
• Coating excipients and solvent should be safe.
• Coating process is compatible with the drug – ensure drug properties won’t be altered.

Question 57

What are some limitations of drug-coated microneedles?

Answer 57

• Small dose (small surface area) so requires more potent drugs.
• Loaded cargo may lessen the strength of the MNs, resulting in low penetration ability.

Question 58

What are drug-coated microneedles?

Answer 58

MNs ciated in drug by dipping or spraying, allowing direct insertion of the drug into the skin.

Question 59

What are dug-encapsulated microneedles?

Answer 59

Microneedles filled with drug solution which can be inserted into the skin and release the drug.

Question 60

How can the release rate of drug-encapsulated microneedles be controlled?

Answer 60

• Rapid dissolving - Water-soluble sugars and polymers (dextran, polyvinylpyrrolidone), photopolymerisable liquid monomers (methacrylic acid).
• Slow dissolving – biodegradable polymers (PLGA)

Question 61

What are some considerations for drug-encapsulated microneedles?

Answer 61

• Microneedles need to possess sufficient mechanical strength. Incorporation of drugs within the MNS may compromise the mechanical strength of the MNs.
• Thermo-sensitive compounds (e.g., proteins) should be encapsulated at moderate conditions
• The speed of dissolution of microneedles decides the time that the microneedles need to be inserted into skin.

Question 62

How do hydrogel-forming microneedles work?

Answer 62

The drug is loaded in the backing membrane, allowing sustained drug release (although some hydrogel-forming MNs have drug loaded in the needles or both). Upon insertion into the skin, interstitial fluid is rapidly absorbed by the hydrophilic polymers (e.g., Poloxamer) which causes the polymer to swell and form a hydrogel. This generates continuous hydrogel conduits for drug permeation into the skin. The rate of drug delivery can be controlled by altering the crosslink density of the hydrogel-forming materials, permitting controlled drug delivery kinetics.

Question 63

What are the Pros and cons of hollow microneedles?

Answer 63

Pros:
Enable great control over the amount and timing of drug delivery.
Separation of the microneedles from drug during manufacturing
Can use already existing drug liquid formulations.

Cons:
Require a drug reservoir, pump, and microfluidic networks, making the whole system more complex and expsensive.
Must ensure leak-free infusion into the skin.

Question 64

How are hollow microneedles produced?

Answer 64

Reactive ion etching: The silicon wafer is covered with a photoresistor and the areas around it are etched away to leave a needle plate.

Electroplating using a master mould, which uses elecrtochemical deposition of metals:
1. Tapered holes are laser drilled through a polymer substrate to form a mould.
2. A conductive seed layer (Ti-Cu-Ti) is deposited on the top and side walls of the mould.
3. Metal (nickel) is electroplated onto the seed layer.
4. The polymer mould is removed to release the metal hollow microneedles.

2 photon stereolithography: has a high resolution to allow fabrication of microneedles with high fidelity and more complexity. Can be slow making mass production difficult.