Intracellular delivery

Intracellular delivery is the process of introducing external materials into living cells. Materials that are delivered into cells include nucleic acids, proteins, peptides, impermeable small molecules, synthetic nanomaterials, organelles, and micron-scale tracers, devices and objects. Such molecules and materials can be used to investigate cellular behavior, engineer cell operations or correct a pathological function.
Medical applications of intracellular delivery range from in vitro fertilisation and mRNA vaccines to gene therapy and preparation of CAR-T cells. Industrial applications include protein production, biomanufacture, and genetic engineering of plants and animals. Intracellular delivery is a fundamental technique in the study of biology and genetics, such as the use of DNA plasmid transfection to investigate protein function in living cells. A wide range of approaches exist for performing intracellular delivery including biological, chemical and physical techniques that work through either membrane disruption or packaging the delivery material in carriers.
Intracellular delivery is at the intersection of cell biology and technology, and is related to many fields across science and medicine including genetics, biotechnology, bioengineering and drug delivery.

Applications

Analogous to the way computers operate through electronic signals, cells process and transmit information through molecules. Depending on the molecules and materials that are loaded into cells, different outcomes or applications can be achieved. Below are some of the main classifications of cargo materials used to investigate and engineer cells through intracellular delivery.

Cargo types

Nucleic acids

refers to the intracellular delivery of nucleic acids: DNA, RNA and their analogues. Nucleic acids materials that are commonly transfected into cells are plasmid DNA, mRNA, siRNA, and oligonucleotides. The transfection applications span across 3 main areas:

Basic biological research,
biomanufacture, and
gene and cell-based therapies

In basic research, transfection is a cornerstone technique in fields ranging from cell biology and genetics to immunology and drug discovery. In biomanufacture, transfection is used for production of proteins, antibodies, viral vectors, and virus-like particles for vaccines. In cell-based therapies transfection is used for applications such as ex-vivo gene therapy, hematopoietic stem cell engineering, production of induced pluripotent stem cells, and ex-vivo preparation of cells for immunotherapy Over the last 50 years nucleic acid transfection has been the most common subcategory of intracellular delivery.
Plasmid DNA began to be transfected into animal cells for the purpose of gene expression in the late 1970s via microinjection
and calcium phosphate methods. Since then, it has been used to investigate gene and protein function in manifold studies. DNA plasmids are physically large and cumbersome molecules with a 5-10 kilo-basepair plasmid being >100 nm diameter in solution when free and uncondensed. Nevertheless, due to the well-established and relatively low-cost techniques for editing and preparing them, they have been very commonly used in biological research.
In the 1970s it was shown that microinjection of mRNA resulted in protein expression. In certain situations, mRNA transfection is considered advantageous for inducing protein expression compared with DNA plasmids due the following reasons

reduced risk of genomic integration,
does not require nuclear delivery with cytosolic delivery being sufficient,
protein expression is dose-dependent and rapid,
less toxic and immunogenic than DNA vectors when appropriately chemically modified.

Thus, mRNA is considered a better option than DNA for most therapeutic applications although it is more expensive and intrinsically unstable.
Oligonucleotides are single or double-stranded sequences of DNA or RNA of less than 30 nucleotides in length. Small interfering RNA are short 21-22 base pair duplexes of RNA that can be transfected into cells to silence gene expression
. Since their Nobel prize winning discovery in 1998, siRNA have been transfected into cells in thousands of biological studies in order to perturb gene function. Other oligonucleotides of interest for intracellular delivery include antisense oligonucleotides, micro RNAs, and aptamers. Such oligonucleotides can be used to alter cell behaviour through several different mechanisms.
Lipid nanoparticles and electroporation are currently widespread strategies for nucleic acid transfection. However, effective transfection remains a hurdle in many primary cells, stem cells, patient-derived cells and neurons. The ability to conduct biological research and carry out potential medical applications in such cells is often limited by transfection efficiency and tolerance to treatment. Furthermore, there is currently a poor understanding of the long-term effects of performing transfection on cells within the human body.

Proteins and peptides

Delivery of proteins into living cells, such as genome-editing nucleases, active inhibitory antibodies, or stimulatory transcription factors, represents a powerful toolset for manipulating and analyzing cell function. Furthermore, effective intracellular delivery could expand the repertoire of usable protein drugs as most current protein-based therapeutics hit extracellular targets and this is a frontier of current research efforts.
Delivery of purified proteins into cells began as early as the 1960s. Examples include amoeba microinjected with ferritin and mouse eggs microinjected with bovine albumin. Because proteins have diverse size, shape and charge, they cannot easily be delivered into cells with one-size-fits-all solutions that cationic lipids use for nucleic acid transfection. In contrast, a diverse range of methods have been used to deliver proteins into cells including: microinjection, osmotic lysis of pinosomes, hypotonic shock, scrape loading, bead loading, syringe loading, detergent exposure, electroporation, pore-forming toxins, cell penetrating peptides, nanocarriers, cell squeezing, nanoneedles, acoustic perturbations, and vapor nanobubbles. For the purposes of genome editing, Cas9 protein combined with sgRNA has been delivered by methods ranging from electroporation, microinjection, lipid nanoparticle formulations, osmotically induced endocytosis followed by endosome disruption, microfluidic deformation, and cell penetrating peptides among others.

Small molecules

Small molecules requiring intracellular delivery include:

impermeable small molecule drugs,
small molecule probes, and
cryoprotectants.

An example of the former is bleomycin, an anticancer drug with poor permeability due to its positive charge and hydrophobicity. By performing intracellular delivery with electroporation, bleomycin potency can be increased more than a hundred-fold. As for small molecule probes, when delivered to the cell interior, these molecules are capable of reporting cellular properties such as membrane potential, pH, and concentrations of ions. One example is PFBI, a fluorescent dye that can be employed for measurement of intracellular potassium concentration. Finally, some candidate cryoprotectant molecules such as impermeable sugars are highly hydrophilic and do not ready diffuse across cell membranes. For example, trehalose is a natural disaccharide synthesized by a range of organisms to help them withstand desiccation or freezing. Trehalose loaded into animal cells at concentrations up to 200 mM has been shown to provide excellent cryoprotection during freezing and thawing.

Microscale cargo

Cargo materials in the microscale have been successfully delivered into cells for a variety of applications. For a century microinjection has been the dominant method for introducing microscale cargo into cells. A classic example was the transplant of a somatic cell nucleus into a frog egg to demonstrate that nuclei from fully differentiated somatic cells could grow into a new animal when inserted into an egg. Microinjection was first used to inject sperm into eggs as a proof of concept for IVF in animals. Artificial chromosomes have been engineered and transferred into cells by microinjection for proof-of-concept gene therapy. Transplant of mitochondria has also been demonstrated in several cell types via microinjection. More recently laser-triggered cavitation bubbles have been used to open transient holes in the cell membrane for the purpose of delivering bacteria and mitochondria. Using microinjection or ballistic propulsion, micron-scale particles, spheres, and beads have been loaded into cells for cellular microrheology studies that assess internal mechanical behavior of cells. For example, using microinjected PEGylated tracer beads of up to 5.6 micron, it was shown that motor-driven cytoplasmic mixing substantially enhanced intracellular movement of both small and large cellular components.

Others

Other materials of interest for intracellular delivery include carbon nanotubes, quantum dots, magnetic nanoparticles, and nanodevices that serve as sensors or probes

Material	Size	Approx. mass	Dimensions in solution	Charge at neutral pH
Small molecules	N/A	< 900 Da	<1 nm	variable; often neutral to promote permeability
Peptides	<40 amino acids	~110 Da per amino acid	0.2 - 3 nm	variable according to amino acid composition
Proteins	20 - 1000s of amino acids	~110 Da per amino acid	~2 - 25 nm	variable according to amino acid composition
Cas9 ribonucleoprotein	~1400 amino acids + 100 bases RNA	~188 kDa	~12 - 15 nm	~ −80
Antisense Oligonucleotide	13 - 25 bases	4 - 8 kDa	length: 4 - 8 nm if linear	-1 per base
siRNA / miRNA	21 - 23 base pairs	13 - 15 kDa	2 nm wide x 7.5 nm long	-1 per base
mRNA	0.5 - 10 kilo-bases RNA	~320 Da per base	tens to hundreds of nm	-1 per base
plasmid DNA	0.5 - 10 kilo-basepairs DNA	~650 Da per base	hundreds of nm; depends on supercoiling	-1 per base