Table 5.

Summary of membrane disruption approaches covered in this review. Several are widely used for intracellular delivery while others have barely been attempted.

Modality	Methods	Membrane Disruption Mechanisms	Disruption / Pore Distribution	Disruption / Pore Size	Throughput / Scalability	Suspension / Adherent
DIRECT PENETRATION
Mechanical	Microinjection	Mechanical forces at contact zone. Membranes only tolerate 2–3% lateral strain393. Can be strain rate dependent390,1486	At contact zone	Depends on size of injection tip, usually 0.3 – 1 μm	Low, could be improved via automation	Mostly adherent, suspension cells require second holding pipette
	Penetrating Projectiles (Biolistics)			Depends on size of projectile, usually micron-size	Potentially high	Primarily adherent, some reports on suspension cells
	Nanowires & Nanostraws			Depends on size of nanoneedle tip: reported range 50 – 1000 nm	Potentially high	Mostly adherent, suspension cells must be forced onto the array
PERMEABILIZATION
Mechanical (Solid Contact)	Cell Scraping	Mechanical forces transmitted by contact/cell deformation. Membranes only tolerate 2–3% lateral strain393. Can be strain rate dependent390,1486	Variable: Presumably at contact zone otherwise at weak points/defects due to global membrane strain	Variable: probably depending on force, strain rate, size of contact zone, direction of strain	High	Adherent
	Bead Loading				High	Adherent
	Scratch Loading				Low/Medium	Adherent
	Microfluidic Cell Squeezing				High	Suspension
	Nanowires for Transient Permeabilization				Potentially high	Adherent
	Sudden Cell Shape Changes and Protease Treatments	Possibly tearing forces at adhesion sites	Unknown	Unknown	Potentially high	Adherent
Mechanical (Fluid Shear)	Syringe Loading / Microfluidic Channel	Fluid shear	Unknown	Unknown	Potentially high	Suspension only
	Sonoporation / Shockwaves	Stale Cavitation (Microstreaming), Inertial Cavitation (Jetting), other Acoustic Effects	Presumably a single hole per cavitation bubble	From nanometers to several micron depending on cavitation intensity and stand-off distance	High	Both
	Laser-controlled Cavitation				High	Both
Mechanical (Pressure)	Hypo-osmotic shock	Mechanical forces transmitted by osmotic/hydrostatic pressure. Membranes only tolerate 2–3% lateral strain393. Can be strain rate dependent390,1486	Presumably at weak points or nucleating at membrane defects	Variable: depending on membrane reservoirs, attachment / reinforcement of membrane, and magnitude / rate of pressure	High	Both
	Hydrostatic pressure				High	Both
	Osmotic rupture of endosomes			Limited by endosome	High	Both
Electroporation	Conventional Electroporation	Probability of defect formation for given pulse-strength duration at a given temperature	At cell poles. More permeabilization expected on hyperpolarized side	Nucleate as small defects then grow as a function of voltage and duration	High	Primarily Suspension, but Adherent also possible
	Micro-electroporation		Depends on geometry		Potentially high	Primarily Suspension
	Nano-electroporation		Usually single hole at aperture		Currently Medium / Low	Both, depending on system
Thermal	Freeze-thaw	Expansive mechanical strain due to ice crystal formation	Location of ice crystals	Presumably variable	High	Both
	Rapid temperature transitions	Defect formation due to phase transitions	Probably near lipid domain boundaries and protein clusters	Presumably small defects	High	Both
	Supraphysiological heating	Dissociation of bilayer structure leading to defect formation	Site of maximal heat	Presumably small defects	High	Both
	Laser absorption at membrane or particle/structure	Absorption causes high local temperature to trigger membrane disruption	Laser focal point or location of absorbent structure	Presumably variable depending on local temperature effects	High	Both
Optoporation	Lasers variables: - Continuous wave or pulsed - Wavelength - Frequency - Power / Intensity	Can be a mix of: - Chemical (low energy plasma) - Mechanical (cavitation, shock waves, thermoelastic stress) - Thermal (Heat in focal region)	Maximal in focal region. Usually one hole	Depending on parameters and mechanisms. Nanometers to several micron	Low to high - limited by laser focusing approach	Primarily Adherent, but suspension also possible
Biochemical	Organic solvents and penetration enhancers	Perturb bilayer structure by burying their hydrophobic residues into the membrane	Indiscriminate in bulk, otherwise depends on local concentration	Presumably small defects then disintegration of the whole bilayer at high concentration	High	Both
	Detergents / surfactants – generic	Insert into bilayer and distort the structure, leading to defects, pore formation, and micellization	Indiscriminate in bulk, otherwise depends on local concentration	Presumably small defects then disintegration of the whole bilayer at high concentration	High	Both
	Detergents – saponin family	Extracts of cholesterol out of the bilayer core to form a surface complex, induces curvature and defect/pore formation	Cholesterol rich sites. Indiscriminate in bulk, otherwise depends on local concentration	From nanometers to micron	High	Both
	Pore-forming toxins – CDC family	Insertion and oligermization into pore structure in cholesterol-rich membranes	Cholesterol rich sites. Indiscriminate in bulk, otherwise depends on local concentration	20 – 50 nm	High	Both
	Membrane-active peptides	Adopt active conformation upon membrane binding. Concentration dependent aggregation / insertion	Depend on membrane composition. Indiscriminate in bulk, otherwise depends on local concentration	Presumably small defects, but large holes have been suggested	High	Both
	Chemical destabilization	Lipid peroxidation leads to structural interference / distortion of membranes to form pores and defects	Depends on source of oxidation. If local, can be confined.	Presumably small defects, but large holes are conceivable	High	Both
Gated Channels and Valves	Endogenous or engineered membrane transporters and channels	Appropriate stimuli (e.g. mechanical, chemical, optical) can gate opening and closing activity	Depends on location of the membrane transporters / channels	Limited by size of the channel. Usually only amenable for transport of small molecules < 1 kDa	High	Both
	Synthetic nanodevices	Insertion of constructs into host membrane. Gating may be engineered	Presumably depends on location of insertion and lateral diffusion throughout membrane	Limited by size of the engineered central channel	Potentially High	Both