How to spray dry nucleic acids

This post is a homage to all my colleagues in molecular biology, biotechnology and biochemistry. After discussing proteins and peptides, another nod in the direction of biologists, today I turn to a long overdue topic, nucleic acids. I explore one of the hottest applications of nucleic acids, gene therapy and discuss several approaches to delivery of nucleic acids, including viral and non-viral delivery systems.

As a chemist, I’ve always envied the terminology connected to my colleagues in molecular biology. I mean, “It’s in your DNA”, “it’s baked in your DNA”, “the deep end of the gene pool”, “the building blocks of life”, the list goes on and on. Okay, fair enough, DNA might be the “molecule of life”, but then maybe it’s time to have a blog post on this topic. We’ve already honored their offspring proteins and peptides by having posts on concentration of proteins, and formulation of proteins. It is really time to talk about how to spray dry nucleic acids!

But why would you want to spray dry nucleic acids in the first place?

Since the discovery of nucleic acids, new DNA-based therapeutics have been developed. These approaches make use of transgene-containing plasmids, oligonucleotides or small interfering RNA (siRNA). Gene therapy is most frequently achieved by:

  • Transfecting cells with an encapsulated plasmid DNA (pDNA), where pDNA must enter the nucleus to replace a defective gene in the genome
  • Using RNA to silence harmful genes: siRNA travels to the cytoplasm and induces degradation of its complementary mRNA to prevent the synthesis of a targeted protein

The siRNA approach offers several advantages over use of plasmid DNA or oligonucleotide molecules, including higher degree of specificity to mRNA, nonimmongenic properties, high resistance to ribonucleases. Since siRNAs do not integrate into the genome, using these types of molecules for gene therapy offers greater safety than using pDNA.

How do we administer nucleic acids?

You need a pretty good delivery system to promote cellular internalization and to protect nucleic acids from degradation. Your ideal system should:

  • have high transfection efficiency
  • have high degree of cell specificity
  • is neither cytotoxic nor immunogenic
  • ideally biodegradable
  • be able to maintain high drug concentrations over time by preventing the recognition by macrophages or expression through the kidney.
  • be easy to formulate
  • be easy to modify for customized nucleic acid release, delivery and expression

Most available delivery systems can be classified into two types, depending on their origin: biological viral delivery systems or chemical non-viral delivery systems.

The viral delivery system is based on the ability of viruses to enter cells and transfer nucleic acids into these cells efficiently. Gene therapy frequently relies on viral machinery for internalization of genetic material by using attenuated viruses as delivery systems. Here is how it works. The nucleic acids of interest are assembled in the viral genome and the innate mechanism of infection of the virus is used to enter the cell and to release genetic material there. Viral delivery systems often contain plasmid DNA since they make it possible for DNA molecules to enter the nucleus, where the DNA is integrated into the host gene pool and eventually expressed.

The biggest advantage of viral delivery systems is that they have extremely high transfection efficiency in human tissue. These types of systems are also easier to design and manufacture. However, there are also serious concerns to using viral-based approaches. For example, the toxicity of viruses could triger immune responses. Pre-existing antibodies against the virus would provoke an immune response and neutralize the delivery system and the molecule it carries, decreasing the efficiency of the therapy. The integration of the nucleic acids in the host genome by the virus cannot be controlled and occurs randomly. Depending on the insertion site, this could have serious consequences, such as mutagenesis that may inhibit expression of normal cellular genes or tumorigenesis.

Non-viral delivery systems can be used to circumvent some of the disadvantages associated with viral delivery. Because non-viral systems do not trigger an immune response and are relatively easy to formulate and assemble, they are emerging as a favorable alternative to viral delivery vectors. Lipid-based and polymer-based systems are frequently used as non-viral delivery systems. The physiochemical properties of lipids and polymers can be modified to improve biocompatibility, increase internalization and tailor the exact requirements for nucleic acid delivery.

When designing non-viral delivery systems, most researchers use nucleic acids in the form of nanoparticles that are packaged by methods such as spray drying, freeze drying, liposome entrapment, emulsion/ double emulsion or a combination of the previous techniques.

Spray drying, with its one-step process, scalability and control of particle properties is an excellent option to process solutions, suspensions or emulsions into dry powder formulations with engineered functional properties, such as defined particle size and density.

Spray drying is commonly used in gene therapy research to produce a delivery system for both pDNA and siRNA therapy, with most of the research focusing on inhalable dry powder for lung delivery. Whenever you are trying to design your formulation, you must consider factors, such as the carrier material and spray drying parameters.

But I don’t want to give away all my goodies at once. I do have several tips on how you can optimize the spray drying process for nucleic acids, but they are saved for my most loyal fans. Keep dropping by to read the blog and you will find them…sooner rather than later.

Till next time,

The Signature of Bart Denoulet at Bart's Blog