Table 1.

Summary of the mechanisms of uptake and toxicity studies of transportans and their conjugates with bioactive cargos; see the details in the text.

TP Analog Used	System	Result in Brief
Visualization
N-ε13-biotinyl-TP/Fl-streptavidin, 125I-TP	fluorescence microscopy, gamma-counting	visualization in the fixated Bowes cells [20,36]; cellular internalization of TP is not an artefact caused by cell fixation
biotinyl-, Fl-, Abz-TP, -TP10	fluorescence microscopy, Transwell	visualization of crossing a Caco-2 human colon cancer cell layer in vitro by a transcellular pathway [118]
Fl-transportan antisense conjugates	confocal microscopy	mainly endocytic, macropinocytotic pathway for cellular uptake [46]
TP	FACS, spectrofluorimetry	protein uptake by endocytotic mechanism in HeLa cells of CPP-avidin complexes [73]
Fl-TP, -TP10	fluorescence microscopy, spectrofluorometry and FACS	visualization in different mammalian and plant cells [119]
TP	FACS	similar kinetic uptake profiles in HeLa, A549 and CHO cell lines [68]
111In- or 68Ga-TP	micro-PET imaging	uptake by six tumor cell lines and biodistribution in PC-3 tumor-bearing nude mice [120]
TP	fluorescence polarization, quenching and CD spectroscopy	in small phospholipid vesicles the helical penetratin and transportan lie along the vesicle surface, penetratin variants appear to penetrate deeper into the membrane [121]
AU NP-functionalized TP- and TP10-protein	TEM	cell entry mechanisms and intracellular trafficking of constructs were studied [122]
myristoylated TP-Tf	fluorescence images and functional gene silencing by siRNA	targeting of siRNA over BBB [99]
TAMRA-TP10	fluorescence microscopy, cell toxicity	complexes of with cisplatin (cPt) in (HEK293, HEL299, HeLa OS143B cell lines were visualized [123]
Alexa488-TP10	flow cytometry, live-cell imaging and image analysis	showed that the glycine-phenylalanine switch was most dramatic in TP10 [79]
125I-TP10 and -TP10-2	in vivo injection	BBB delivery [124]
TP10	spheroid model of the BBB, ex vivo imaging	showed increased delivery to mouse brains [125]
TP- isoniazid	Langmuir balance technique and AFM imaging	conjugates showed similar internalization rate into EBC-1 human squamous cell carcinoma in imaging of penetrated lipid layers [126]
Structure and interactions of transportan
TP	CD	random coil structure in water, in SDS micelles 60% induced helix [127]
TP	CD	60% alpha-helix in phospholipid vesicles [128]
TP	CD	helical structure in small phospholipid vesicles [121]
TP	NMR	in neutral bicelles alpha-helix in the C-terminus and tendency to form an alpha-helix in the N-terminus [129]
TP	CD	structure in neutral DMPC bicelles and negative DMPG-containing bicelles different from each other [130]
TP	CD	obtain amphiphilic α-helix when bound to membranes of vesicles composed of typical eukaryotic lipids [131]
TP10	solid-state 19F-NMR, CD	a range of conformations in the DMPC/DMPG vesicle membrane-bound state, C-terminal α-helix is embedded in the membrane being tilted [132]
TP10 and 5 analogs	molecular dynamics simulation	forming α-helical conformation, the higher membrane disturbance yields higher cellular uptake in cells [133]
TP	CD	increased membrane affinity with DPPC and DPPC + mycolic acid mixed monolayers [126]
TP10	Gibbs energy studies	peptide-induced efflux, becoming faster with decrease of the Gibbs energies for binding and insertion [134]
TP and analogs	PepLook algorithm	peptide polymorphism showed common conformational polymorphic characteristics [135]
Cys-TP	EPR	in DMPC/cholesterol caused lipid ordering and a large increase in permeation [136]
TP10	molecular dynamics	interactions with POPC bilayer initiated α-helix with hydrophobic side facing the hydrophobic lipid core [137]
TP10	confocal microscopy	interaction of GPMVs revealed association with liquid-disordered membrane areas [138]
PF6	DLS	mean diameter of PF6/siRNA NPs was shown to be below 200 nm [27]
PF14	DLS	PF14/SCO NPs possessed a net negative charge [28]
PFs	CD, DLS, QSAR	PFs/Luc-plasmid NPs for study of structural requirements for cell penetration [95]
His-PFs, PF132	DLS, CD, calcein leakage	complexes formed were small at pH 7 and grew under acidic conditions [96]
Parallel mechanisms of endocytosis and direct translocation in cells
biotinyl-TP	fluorescence microscopy	cellular uptake by unrelated mechanisms [139]
TP, TP10	Transwell model	translocation across a Caco-2 human colon cancer cell layer by transcellular pathway [118]
TP	CRM and AFM	entered SK-Mel-2 cells within 5 min and widespread distribution via a nonendocytic mechanism [140]
TP, TP10	confocal laser scanning microscopy, GMPV	showed the interactions with glycosaminoglycans [141]
TP, TP10	fluorescence microscopy	uptake, when conjugated to cargoes, involved both endocytosis and direct translocation [20,70]
Biotinyl-TP, -TP10/avidin	fluorescence microscopy	cellular internalization in HeLa and Bowes cellc by endocytosis with different pathways [142]
biotinyl-TP, -TP10/avidin	confocal microscopy	entered Cos-7 cells by caveolin-dependent endocytosis [72]
biotinyl-TP, -TP10/avidin	confocal microscopy	entered HeLa cells via caveolin-1-dependent pathway [122]
biotinyl-TP, -TP10/avidin	FACS analysis and spectrofluorimetry	clathrin-dependent and -independent endocytosis uptake in HeLa cells with partial depolarization [73]
TP-PNA	fluorescence microscopy	rapid cellular uptake by non-receptor-dependent endocytosis [143]
TP-PNA	splice correction assay	splice correction in HeLa/Luc cells with mainly endocytotic, particular macropinocytotic mechanism [46]
TP-, TP10-PNA	splice correction assay	showed cellular translocation by endocytosis [47]
TP/NPs, /BSA, /dextran	fluorescence microscopy	complexed with NPs showed in vitro and ex vivo cell entry via a receptor-dependent macropinocytosis process [144]
Mechanisms of PepFects and NickFects
PF3, PF6	interaction studies with lipid membranes	increased amphipathicity and their ability to insert into a lipid monolayer composed of zwitterionic phospholipids [145]
NFs/pDNA	membrane perturbation study	pDNA cargo inhibited membrane perturbation by NFs [114]
PF32/pDNA	Transwell model	uptake by brain endothelial cells via LRP-1 receptor-mediated endocytosis and scavenger receptors [146]
PF14/SCO	fluorescence microscopy	SR-A3 and SR-A5 recruited by PF14/SCO complexes [147]
PF14/Cy5-siRNA	FCS, FCCS	coexistence of monomers, self-aggregates of peptide/ON in complexes in solution and at the plasma membrane of live cells [148]
NF1/, NF51/pDNA	analysis of separated endosomal vesicles by differential centrifugation	NF51 facilitates rapid internalization of complexes into the cells, NF1 is less capable to induce endosomal release [149]
Delivery of the cargo
TP-BSA	toxicity study	showed no toxicity or initiation of an immune response in epithelial cells [78]
TP6, TP7-PNA	toxicity study	significant cellular toxicity above 3−5 μM in TP-PNA conjugates [49]
St-TP10/SCO	splice-correcting assay, toxicity study	delivery of SCOs for functional splice correction with no toxicity [92]
St-TP10/plasmid	toxicity study, gene expression in vivo	entered different cells with high gene expression level with no toxicity and no nonimmunogenecity in vivo [60]
TK- and TH- camptothecin	toxicity study	TH-camptothecin showed cytotoxicity to cancer cells [150]
TP10, PFs/plasmid, /siRNA	toxicity study	peptide/plasmid and /siRNA showed no cytotoxic and immunogenic response, in vitro and in vivo [151]
TP, TP10, TP-biot1, TP-biot13, TP10-biot1	toxicity study	no significant cytotoxic effect at 0.5–5 µM [75]
T9(dR)/siRNA	cells, in vivo toxicity	showed cellular delivery of siRNA and in mice infected with PR8 influenza virus, and antiviral activity [57]
chloroquine-TP10	antimalarial activity	higher antiplasmodial activity in safe delivery of antimalarial aminoquinolines [152]
TP10-ciprofloxacin or -levofloxacin	antifungal in vitro activity	TP10-ciprofloxacin or -levofloxacin showed antifungal in vitro activity against human pathogenic yeasts [153]