intracellular delivery intracellular delivery intracellular delivery is the process of introducing external materials into living cells. materials that are delivered into cells include nucleic acids (dna and rna), 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. intracellular delivery through viral vectors, non-viral carriers, and membrane disruption. membrane disruption-mediated delivery can introduce diverse materials by direct penetration or permeabilisation mechanisms. viral vectors are capable of dna or rna delivery through engineering. non-viral nano-carriers have mostly been used for nucleic acid delivery but can also be designed for delivery of more diverse materials. medical applications of intracellular delivery range from in vitro fertilisation (ivf) 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 applications of intracellular delivery. depending on the input materials delivered into cells, different outcomes or applications can be achieved. tcr = t cell receptor, car = chimeric antigen receptor, hscs = hematopoietic stem cells, cnt = carbon nanotube 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 (see figure "applications of intracellular delivery" for examples). below are some of the main classifications of cargo materials used to investigate and engineer cells through intracellular delivery. nucleic acids transfection 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 (sirnas) 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 (asos), micro rnas (mirnas), 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 (mw = 342 da) 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 properties of common molecules of interest for intracellular delivery materialsize (units)approx. mass (da)dimensions in solution (nm)charge at neutral ph small moleculesn/a< 900 da<1 nmvariable; often neutral to promote permeability peptides<40 amino acids~110 da per amino acid0.2 - 3 nmvariable according to amino acid composition proteins20 - 1000s of amino acids~110 da per amino acid~2 - 25 nmvariable according to amino acid composition cas9 ribonucleoprotein (rnp)~1400 amino acids + 100 bases rna~188 kda (~158 kda protein + ~30 kda rna)~12 - 15 nm~ −80 (+22 protein, −100 sgrna) antisense oligonucleotide (aso)13 - 25 bases (single stranded)4 - 8 kdalength: 4 - 8 nm if linear-1 per base sirna / mirna21 - 23 base pairs13 - 15 kda2 nm wide x 7.5 nm long-1 per base mrna0.5 - 10 kilo-bases rna (single-stranded)~320 da per basetens to hundreds of nm-1 per base plasmid dna0.5 - 10 kilo-basepairs dna (double-stranded)~650 da per basehundreds of nm; depends on supercoiling-1 per base medical applications the following are examples of medical treatments that rely on intracellular delivery in at least one step. hematopoietic stem cell-based gene therapies: strimvelis for the treatment of adenosine deaminase severe combined immunodeficiency (ada-scid), produced through ex vivo gamma retroviral vector gene delivery of a functional adenosine deaminase (ada) gene (european approval granted 2017). strimvelis was the first ex vivo autologous gene therapy to gain approval from the european medicines agency. atidarsagene autotemcel (branded as libmeldy) for the treatment of metachromatic leukodystrophy (mld), produced through ex vivo lentiviral gene delivery of a functional human arylsulfatase a (arsa) gene (european approval granted 2020). betibeglogene autotemcel (branded as zynteglo) for the treatment of transfusion-dependent beta thalassemia (tdt), produced through ex vivo lentiviral gene delivery of a functional human hbb gene (european and us approvals granted 2022). car-t cell immunotherapies: tisagenlecleucel (branded as kymriah) for the treatment of b-cell acute lymphoblastic leukemia (b-cell all), produced through ex vivo lentiviral gene delivery of a car gene targeting cd-19 (us approval granted 2017). axicabtagene ciloleucel (branded as yescarta) for the treatment of large b-cell lymphoma, produced through ex vivo gamma retroviral gene delivery of a car gene targeting cd-19 (us approval granted 2017). brexucabtagene autoleucel (branded as tecartus) for the treatment of adult patients with relapsed/refractory mantle cell lymphoma (r/r mcl), produced through ex vivo gamma retroviral gene delivery of a car gene targeting cd-19 (us approval granted 2021). in vivo viral vector-mediated gene therapy: onasemnogene abeparvovec (branded as zolgesma) for the treatment of spinal muscular atrophy, administered by intravenous infusion of a aav9 viral vector that delivers a functional smn1 transgene to the affected motor neurons (us approval granted 2019). voretigene neparvovec (branded as luxturna) for the treatment of leber congenital amaurosis (an inherited retinal disorder causing progressive blindness), administered by subretinal injection of a aav2 viral vector that delivers a functional copy of the rpe65 gene to eye cells to compensate for the rpe65 mutation (us approval granted 2017). sirna medicines: patisiran (onpattro) for the treatment of polyneuropathy in people with hereditary transthyretin-mediated amyloidosis, administered by intravenous infusion of sirna formulated into lipid nanoparticles that gain entry into liver cells to silence the expression of pathogenic transthyretin mrna (us and european approval granted 2018). givosiran (givlaari) for the treatment of adults with acute hepatic porphyria, administered by administered by intravenous infusion of sirna-galnac conjugates that gain entry into liver hepatocytes to silence the expression of pathogenic aminolevulinate synthase 1 (alas1) mrna (us approval granted 2019). mrna vaccines: moderna covid-19 vaccine (branded as spikevax) designed to provide protection against covid-19 caused by infection with the sars-cov-2 virus (us emergency use authorization 2020). it is administered by intramuscular injection of 0.5 ml doses of mrna-lnp complexes in saline. local injection has been reported to deliver mrna-lnp complexes to resident/infiltrating apcs, related immune cells, and muscle cells, stimulating local expression of covid spike protein to