The role of spray drying in gene therapy

Just a few weeks ago I introduced the topic of spray drying nucleic acids to the blog. But before you forget all about it, I want to offer you more insights into delivery systems for gene therapy and how you can use spray drying to help you create safer and more efficient vectors. Read on and be inspired.

You know when you are waiting for a sequel of a book or a movie and so much time passes in between that you forget the whole plot to begin with? I’ve done two-part posts before. For example, I’ve explored ELS detectors in chromatography in part 1 and part 2. And I just promised you a follow-up on how to spray dry nucleic acids. And this time, I don’t want to make you wait forever, so here is part 2 immediately on the footsteps of its predecessor.

As a refresher, spray drying is commonly used in gene therapy to produce delivery systems for pDNA and siRNA therapy. Most applications focus on developing inhalable dry powder for lung delivery.

I want to continue our discussion by firstly exploring the carrier materials you could use.

Lipid-based vectors

Lipid-based delivery systems typically use cationic lipids or liposomes to create complexes with negatively charged nucleic acids through spontaneous electrostatic interactions. But to use lipids, you need to optimize the composition of the liposome or lipoplex to reduce any toxic side effects and inflammatory responses caused by lipid-based deliver systems. Generally, neutral and anionic lipid-based systems are safer, but less effective and thus have limited use as a delivery vector for gene therapy.

To reduce the inflammatory response associated with cationic lipid-based systems, the surface charges of cationic lipoplexes or liposomes can be shielded with polyethylene glycol (PEG).

I just want to mention that specialized lipid-based systems, such as lipid-like molecules or pH-sensitive lipids are also showing promising results in nucleic acid encapsulation.

Polymer-based vectors

Polymer-based vectors have a versatile nature, so it is possible for you to modify their physiochemical characteristics to fit your purpose in gene therapy. For the delivery of nucleic acids, polymers such as poly (D,L-lactide-co-glycolide) (PLGA) or polycations such as polyethylenimine (PEI) or chitosan are often used. Polyacations show good potential for DNA entrapment but are less efficient for siRNA entrapment or in protection siRNA molecules from nucleases.

A potential solution for siRNA protection is to encapsulate the siRNA in particles made of chemically modifiable hydrophobic polymer, such as PLGA and its derivative molecules.

Peptide-based vectors

DNA and siRNA molecules have a limited ability to enter cells due to their negative charges. Cell penetrating peptides (CPP) and pH-responsive peptides can be used to overcome this limitation in gene therapy. CPP molecules could contain of small amino acid sequences composed of arginine, lysine and histidine which provide a positive charge to help mediate interactions with the cell membrane. Alternatively, CPP molecules have structures with both lipophilic and hydrophilic sides that can mediate translocation across the membrane. pH responsive peptides are used to overcome endosomal and liposomal degradation of the nucleic acid after internalization by endocytosis. The pH responsive peptides achieve this by destabilizing the membrane activity the endosomes, provoking endosomal escape and avoiding degradation of the genetic material used in gene therapy.

Spray drying considerations for gene therapy delivery systems

To produce good quality powder by spray drying with the above polymers in gene therapy, you could additionally use thermoprotectants stabilizing adjuvants and excipients. Examples of these include sucrose, glycine, agarose, trehalose, PEG, bovine serum albumin (BSA)and several amino acids (arginine, lysine, and histidine).
Here are several spray drying parameters you need to optimize to produce gene therapy delivery systems:

  • Inlet temperature – is the temperature of the heated drying gas. The more energy is put into the system, the faster the solvent is evaporated, which in turn increases the drying efficiency
  • Outlet temperature – represents the temperature of the dry powder before it enters the collection vessel. This is an important parameter for heat-sensitive samples. The outlet temperature is the result of a combination of many parameters such as inlet temperature, aspirator flow rate, peristaltic pump setting (feed rate) and concentration of the material being sprayed
  • The peristatlic pump – feeds the sample solution to the nozzle. The speed affects the temperature difference between the inlet temperature and outlet temperature since the pump rate directly corresponds to the inlet mass. A higher throughput requires more energy to evaporate the droplet from the particles, so the outlet temperature will decrease.
  • The atomizing air flow – or spray flow rate – is the amount of compressed air needed to disperse the sample. A gas other than compressed air can be used if you need to work in an inert environment.
  • The aspirator capacity – regulates the amount of air available (drying air flow) for the drying process. Because the amount of energy available for vaporization changes with the amount of drying air, the aspirator speed has a substantial effect on the drying performance of the instrument

And here is a table my colleagues at BUCHI have gathered from literature with what spray drying parameters have been used to spray dry and encapsulate nucleic acids for gene therapy:

Nucleic acidsSpray DryerMain ParametersCarrier MaterialMicroparticle size (uM)
DNABUCHI B-191Tin(°C): 120 - 160
Atomizing air flow (L/h): 500 - 700
Feed rate (mL/min): 3-9
Aspirator capacity: not specified
Tout(°C): not specified
3 - 11.8
DNABUCHI B-191Tin(°C): 150
Atomizing air flow (L/h): 600
Feed rate (mL/min): 7.5
Aspirator capacity - 35m3/h
Tout(°C): 80-85
DOTAP liposome
Protamine sulphate
1 - 10
DNABUCHI B-191Tin(°C): 78-79
Atomizing air flow (L/h): 600
Feed rate: 10%
Aspirator capacity - 75%
Tout(°C): not specified
3 - 4
DNABUCHI B-191Tin(°C): 50
Atomizing air flow (L/h): 750
Feed rate (mL/min): 2
Aspirator capacity - 60
Tout(°C): 28
LAH or LADap peptides
DNABUCHI B-191Tin(°C): 50
Atomizing air flow (L/h): 800
Feed rate (mL/min): 1
Aspirator capacity: 70%
Tout(°C): 39 - 40
2.2 - 8.3
Tin(°C): 50
Atomizing air flow (L/h): 740
Feed rate (mL/min): 2
Aspirator capacity: 100%
Tout(°C): 34±2
LAH or LADap peptides
siRNABUCHI B-290Tin(°C): 45
Atomizing air flow (L/h): 473
Feed rate (mL/min): 0.3
Aspirator capacity: not specified
Tout(°C): 30
PLGA / DOTAP-modified PLGA
3.7 - 4.99
siRNAUltrasonic nozzle; Sono-TecTin(°C): 120±2
Atomizing air flow (L/h): not specified
Feed rate (mL/min): 1
Aspirator capacity: 100%
Tout(°C): 65±2
6.2 - 9.8
siRNABUCHI B-90Tin(°C): 30-60
Spray power: not specified
Feed rate (mL/min): not specified
Aspirator capacity: 118 - 121 L/min
Tout(°C): not specified
0.58 - 0.77

I hope this post has been a useful wrap-up of our discussion on how to spray dry nucleic acids for gene therapy. If you need more information on specific spray drying methods, check out an application note on how to spray dry lipids, polycations and pDNA , or another application note on spray drying of plasmid DNA alone.

Should there be a part 3 to this post or did you get all the information you need from the two-part series? Let me know if you have any open questions in the comments below!

Till next time,

The Signature of Bart Denoulet at Bart's Blog