Nanoparticle formulation
An ethanolic mixture of an ionisable lipid, a helper phospholipid, cholesterol and a PEG-lipid is mixed rapidly against an acidic aqueous phase containing nucleic acid. The protonated ionisable lipid binds the cargo as the ethanol is diluted out, and buffer exchange to pH 7.4 leaves a near-neutral, PEGylated particle with the cargo in its core.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Lipid nanoparticles
An ethanolic mixture of an ionisable lipid, a helper phospholipid, cholesterol and a PEG-lipid is mixed rapidly against an acidic aqueous phase containing nucleic acid. The protonated ionisable lipid binds the cargo as the ethanol is diluted out, and buffer exchange to pH 7.4 leaves a near-neutral, PEGylated particle with the cargo in its core.
How it differs from the standard method
- Mix at pH 4 or below and buffer-exchange afterwards; N/P around 6 is a usual starting point.
- Size is set by the mixing time, so total flow rate and flow-rate ratio are formulation parameters.
- Measure encapsulation with a dye that cannot cross the membrane, before and after lysis.
1Packaging a payload into something the body handles differently
A nanoparticle formulation puts a drug or a nucleic acid inside a carrier tens to hundreds of nanometres across, so that where the payload goes, how long it survives and how fast it is released are set by the carrier rather than by the molecule. That matters for two kinds of cargo in particular: hydrophobic small molecules that cannot be dosed in water, and nucleic acids, which are large, charged and destroyed by nucleases within minutes of injection.
Lipid nanoparticles
An ionisable lipid is charged at pH 4, condenses RNA as ethanol is diluted out, and becomes near-neutral at pH 7.4. The format behind siRNA and mRNA medicines.
PLGA particles
A biodegradable polyester precipitated from an organic solvent around a hydrophobic drug, giving release over days to months.
Liposomes
Phospholipid bilayers enclosing an aqueous core: water-soluble cargo inside, hydrophobic cargo in the membrane. Cholesterol seals the bilayer.
Size, PDI, EE, zeta
Four numbers decide whether a batch is worth taking further, and all four depend on how the batch was purified before measurement.
2Why mixing speed sets the size
Nanoprecipitation and microfluidic mixing both work the same way: a water-miscible solvent carrying lipid or polymer meets water, the solute becomes insoluble, nuclei form, and they grow until the solvent is diluted below the point where anything can still deposit. Particles therefore grow for as long as mixing takes.
3The four numbers, and how they mislead
Dynamic light scattering reports an intensity-weighted size and a polydispersity index, which is essentially the square of the relative width of the distribution: below about 0.1 is one narrow population, 0.2 is usually the limit for a nucleic-acid carrier, and above 0.3 the number is barely meaningful. Because scattering goes as the sixth power of diameter, a few specks of dust or a handful of aggregates dominate the signal, so samples are filtered and cuvettes kept clean, and sizes are worth confirming by an orthogonal method.
4How batches fail
- Too little ionisable lipid, or formulation at neutral pH, so nucleic acid never gets inside.
- A drug load above what the carrier phase can dissolve: the excess ends up in the washings or as crystals.
- No surfactant, so growing particles coalesce and the batch creams or sediments.
- Organic solvent left in, keeping particles soft and letting the payload partition back out.
- Cholesterol-free liposomes, which leak small water-soluble cargo within hours.
- Extrusion below the lipid transition temperature, where the bilayer will not pass or reseal.
Key terms
Practise these as flashcards →Width of the size distribution from DLS cumulant analysis, roughly the square of the relative standard deviation of diameter.
Fraction of the offered payload that ends up inside the particles, measured after the free payload is separated.
Mass of payload as a percentage of the total particle mass; a different quantity from encapsulation efficiency.
Moles of ionisable amine per mole of nucleic-acid phosphate; the main control on nucleic-acid encapsulation.
A lipid with an amine of pKa near 6.5, cationic at pH 4 and near-neutral at pH 7.4.
Electrokinetic potential at the slipping plane, measured from electrophoretic mobility; depends on the medium as much as on the particle.
Aqueous to organic volumetric ratio in a micromixer; higher ratios mix faster and leave less residual solvent.
Drying lipid from organic solvent and hydrating it above the transition temperature to form multilamellar vesicles.
Repeated passage through a polycarbonate membrane to bring vesicles to a defined size.
The layer of plasma proteins that adsorbs on a particle in blood and determines much of its fate.
When to use it
- To deliver a nucleic acid, which cannot cross membranes or survive in plasma on its own.
- To dose a hydrophobic drug that has no acceptable aqueous formulation.
- To change where a drug goes or how long it circulates, for example to reduce a dose-limiting toxicity.
- To release a payload over days to months from a single administration.
Compared with alternatives
The next experiment on any batch made here: what comes out, how fast, and what happens to the carrier.
The instrument and the theory behind the size, PDI and zeta numbers quoted for every batch.
When uniform particles have to be made one at a time as droplet templates rather than by precipitation.
The bench alternative for getting nucleic acid into cultured cells, where circulation and stability do not matter.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
An ethanolic mixture of an ionisable lipid, a helper phospholipid, cholesterol and a PEG-lipid is mixed rapidly against an acidic aqueous phase containing nucleic acid. The protonated ionisable lipid binds the cargo as the ethanol is diluted out, and buffer exchange to pH 7.4 leaves a near-neutral, PEGylated particle with the cargo in its core.
- Mix at pH 4 or below and buffer-exchange afterwards; N/P around 6 is a usual starting point.
- Size is set by the mixing time, so total flow rate and flow-rate ratio are formulation parameters.
- Measure encapsulation with a dye that cannot cross the membrane, before and after lysis.