prime the immune system against future exposure to sars-cov-2. pfizer-biontech covid-19 vaccine (branded as comirnaty) designed to provide protection against covid-19 caused by infection with the sars-cov-2 virus (us emergency use authorization 2020). the mechanism is the same as moderna (above) except that composition of the lipid nanoparticles are different, with pfizer using the ionizable lipid known as alc-0315 while moderna uses an ionizable lipid called sm-102. antisense oligonucleotides (asos): mipomersen (branded as kynamro), for the treatment of homozygous familial hypercholesterolaemia, administered by subcutaneous injection of chemically modified asos that accumulate into the liver with a half-life of ~30 days. the asos gain entry into liver cells to prevent the expression of pathogenic apob mrna (us approval granted 2013). spinraza (branded as nusinersen) for the treatment of spinal muscular atrophy (sma), administered by intrathecal injection of chemically modified asos that enter motor neuron cells, bind smn2 mrna, and alter the alternative splicing of the smn2 gene to restore expression of the protein. in vitro fertilisation (ivf) for human pregnancies: in cases such as low sperm count or motility, or where eggs cannot easily be penetrated by sperm, single sperm may be microinjected directly into the egg using a procedure termed intracytoplasmic sperm injection (icsi). after injection the fertilised egg is incubated in a special growth medium for about 48 hours until the egg consists of six to eight cells. it is then transferred to the patient's uterus through a thin, plastic catheter, which goes through the vagina and cervix. the first human pregnancy generated by icsi was carried out in 1991 by gianpiero palermo and his team. ivf through icsi has facilitated millions of pregnancies worldwide since the 1990s. safety. research is still in the early stages of understanding the immediate and long-term medical effects of intracellular delivery of materials to human cells. for example, investigations have shown that children born through icsi suffer more health problems than those naturally conceived. however, it is not known whether this is due to poorer health of the parents reproductive systems or aspects of the ivf and icsi procedure. hsc-based gene therapies prepared with gamma retroviral and lentiviral vectors have in some cases shown an increased risk of leukemia down the track due to genotoxicity, as occurred with strimvelis. furthermore, the lipids used for intracellular delivery of therapeutic sirna and mrna may cause inflammatory reactions. in the case of patisiran, pretreatment with multiple anti-inflammatory drugs is used to minimize reactions to the nanoparticle. there is currently little data available on the medical impact of intracellular delivery of novel chemical components of mrna vaccines, such as sm-102 and alc-0315, on both the short and long-term health of the recipient population. thus, safety and unintended side-effects will continue to be a topic of importance for medical treatments that utilize intracellular delivery. categories of methods methods of intracellular delivery mapped to mechanism. methods are linked to their mechanism of action, being 1) permeabilization, 2) direct penetration, 3) endocytosis, or 4) fusion . some method categories can operate through multiple mechanisms (e.g. certain pore-forming toxins induce endocytosis and subsequent endosome disruption while others transiently permeabilize the plasma membrane). most of the physical methods are membrane disrupting while most of the biochemical methods are carrier-mediated. current methods of intracellular delivery can be placed into two broad categories: membrane disruption-mediated carrier-mediated membrane disruption membrane disruption-mediated techniques involve creating temporary holes in the cell membrane and delivering the cargo molecules via either a) permeabilization and diffusive influx of materials from the extracellular solution b) direct penetration with a vehicle or carrier that both punctures the plasma membrane and introduces the cargo of interest. the plasma membrane of the cell can be disrupted through mechanical, electrical, chemical, optical or thermal means. intracellular delivery methods that employ permeabilization include: electroporation thermal treatments optoporation (usually with a laser) cavitation effects (usually from ultrasound or laser/particle interactions) fluid shear (usually via microfluidics or cavitation) detergents/surfactants pore-forming toxins osmotic/hydrostatic forces direct mechanical methods like microfluidic cell squeezing, scrape loading, bead loading. intracellular delivery methods that utilize direct penetration include: classic microinjection newer forms of microinjection (e.g. nanopipette, automation etc.) ballistic particles / gene gun nanoneedles and their variations such as nanostraws, nanospears and nanowires. membrane disruption-mediated delivery methods can deliver almost any material that can be dispersed in solution, making them more universal than carrier-mediated methods. the major challenge for membrane disruption-mediated methods is to create holes of the optimal shape, size, location, and duration for the required delivery application. excessive membrane damage should be avoided as it can kill cells or impair their function. historical milestones in the development of intracellular delivery methods and their applications.