More than 40 nanomedicines are already in routine clinical use with a growing number following in preclinical and clinical development. The therapeutic objectives are often enhanced disease-specific targeting (with simultaneously reduced access to sites of toxicity) and, especially in the case of macromolecular biotech drugs, improving access to intracellular pharmacological target receptors. Successful navigation of the endocytic pathways is usually a prerequisite to achieve these goals. Thus a comprehensive understanding of endocytosis and intracellular trafficking pathways in both the target and bystander normal cell type(s) is essential to enable optimal nanomedicine design. It is becoming evident that endocytic pathways can become disregulated in disease and this, together with the potential changes induced during exposure to the nanocarrier itself, has the potential to significantly impact nanomedicine performance in terms of safety and efficacy. Here we overview the endomembrane trafficking pathways, discuss the methods used to determine and quantitate the intracellular fate of nanomedicines, and review the current status of lysosomotropic and endosomotropic delivery. Based on the lessons learned during more than 3 decades of clinical development, the need to use endocytosis-relevant clinical biomarkers to better select those patients most likely to benefit from nanomedicine therapy is also discussed.

The signaling lipid, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), likely functions in multiple signaling pathways. Here, we report the characterization of a mouse mutant lacking Vac14, a regulator of PI(3,5)P 2 synthesis. The mutant mice exhibit massive neurodegeneration, particularly in the midbrain and in peripheral sensory neurons. Cell bodies of affected neurons are vacuolated, and apparently empty spaces are present in areas where neurons should be present. Similar vacuoles are found in cultured neurons and fibroblasts. Selective membrane trafficking pathways, especially endosome-to-TGN retrograde trafficking, are defective. This report, along with a recent report on a mouse with a null mutation in Fig4, presents the unexpected finding that the housekeeping lipid, PI(3,5)P 2, is critical for the survival of neural cells.T he low-abundance signaling lipids, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P 2 ) and phosphatidylinositol 5-phosphate (PI(5)P), were discovered relatively recently (1-3). Because of their low abundance and the limited number of tools available for their study, relatively little is known about these lipids.An interesting property of PI(3,5)P 2 occurs in yeast, where a stimulus of hyperosmotic shock induces dramatic and transient changes in the levels of PI(3,5)P 2 . The levels of PI(3,5)P 2 transiently rise Ͼ20-fold (4). Within 1 minute, the levels rise 5-fold; by 5 minutes, they increase Ͼ20-fold; there is a short plateau of 10 min, and then PI(3,5)P 2 levels decrease at a rate similar to their increase. The rapid decrease in PI(3,5)P 2 levels occurs even though the cells remain in hyperosmotic media. Vacuole volume undergoes transient changes that parallel PI(3,5)P 2 levels. That these rapid and transient changes occur even in the presence of a sustained stimulus strongly suggests that PI(3,5)P 2 plays a major role in signaling pathways related to adaptation.Several proteins are required for the synthesis and turnover of PI(3,5)P 2 . PI(3,5)P 2 is synthesized from PI(3)P by the PI(3)P 5-kinase Fab1/PIKfyve/PIP5K3 (5, 6). Fab1 is stimulated by a regulatory complex that contains Vac14 (7, 8) and Fig4 (4, 9). Surprisingly, the Vac14/Fig4 complex plays two opposing roles in the regulation of steady-state levels of PI(3,5)P 2 . Vac14/Fig4 both activate Fab1 and also function in the breakdown of PI(3,5)P 2 through the lipid phosphatase activity of Fig4 (4, 9-11).In mammals, generation of PI(3,5)P 2 is predicted to impact PI(5)P production. In vitro studies have shown that PI(5)P can be generated from PI(3,5)P 2 through the PI(3,5)P 2 3-phosphatase activity of members of the myotubularin family and related proteins including MTM1, MTMR1, MTMR2, MTMR3, MTMR6, and hJUMPY/MTMR14 (12-15). In addition, PIKfyve/Fab1 can generate both PI(3,5)P 2 and PI(5)P in vitro (16). The source of PI(5)P in vivo has not been established. However, the generation of PI(5)P from either pathway requires PIKfyve/ Fab1 activity, either to produce the substrate for myotubularin [PI(3,5)P 2 ] or to produce PI(5...

Both heterotypic and homotypic fusion events are required to deliver endocytosed macromolecules to lysosomes and remodel late endocytic organelles. A trans-SNARE complex consisting of Q-SNAREs syntaxin 7, Vti1b and syntaxin 8 and the R-SNARE VAMP8 has been shown by others to be responsible for homotypic fusion of late endosomes. Using antibody inhibition experiments in rat liver cell-free systems, we confirmed this result, but found that the same Q-SNAREs can combine with an alternative R-SNARE, namely VAMP7, for heterotypic fusion between late endosomes and lysosomes. Co-immunoprecipitation demonstrated separate syntaxin 7 complexes with either VAMP7 or VAMP8 in solubilized rat liver membranes. Additionally, overexpression of the N-terminal domain of VAMP7, in cultured fibroblastic cells, inhibited the mixing of a preloaded lysosomal content marker with a marker delivered to late endosomes. These data show that combinatorial interactions of SNAREs determine whether late endosomes undergo homotypic or heterotypic fusion events.

Fusogenic peptides derived from viral coat proteins cause perturbation of the endosomal membrane and are often used to improve the transfection efficiency of non-viral vectors in vitro. However, fusogenic peptides have limited potential for use in vivo due to their inherent immunogenicity. Totally synthetic polymers that are endosomolytic should circumvent this problem and could be useful as components of non-viral delivery systems as long as they do not immediately localise in the liver after intravenous (i.v.) injection. Linear poly(amidoamine) polymers (PAAs) having amido- and tertiary amino-groups along the main polymer undergo pH-dependent conformational change and thus provide an ideal opportunity for design of polymers that display membrane activity at low pH. Here we describe four PAAs, ISA 1 (Mn = 6900 Da) and ISA 23 (Mn = 10,500 Da) and their analogues ISA 4 and ISA 22 (Mn approximately 8000 Da) containing approximately 1 mol% 2-p-hydroxyphenyl ethylamine to allow radioiodination and thus monitoring of their biodistribution. In vitro cytotoxicity was assessed by MTT assay after incubation of PAAs with B16F10 and Mewo cell lines. The IC50 values observed for all PAAs were > 2 mg/mL in comparison with poly(L-lysine) which displayed an IC50 in the range 0.01-0.1 mg/mL. At pH 7.4 none of the PAAs studied was haemolytic at 1 h at concentrations below 3 mg/mL. PAAs were subsequently incubated with rat red blood cells for 24 h (1 mg/mL) at different pHs. In contrast to poly(L-lysine) which was haemolytic at pH 7.4, 6.5 and 5.5, none of the PAAs was lytic at pH 7.4, but they became membrane active at lower pH (approximately 45% for ISA 4, 50% for ISA 22 and 90% for ISA 23). These observations were substantiated by SEM and confirm the pH-dependence of membrane activity. After i.v. injection to rats 125I-labelled ISA 4 was immediately taken up by the liver (> 80% recovered dose at 1 h) whereas 125I-labelled ISA 22 was not (liver uptake was < 10% recovered dose at 5 h). Furthermore, biodistribution studies in mice bearing subcutaneous B16F10 melanoma showed that 125I-labelled ISA 22 was still accumulating in tumour tissue after 5 h (2.5% dose/g). PAAs have potential as endosomolytic agents and quantitation of the endosome to cytoplasm transfer is warranted after i.v. administration.

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Five poly(amido-amine)s (PAAs) carrying two ter-amino groups and one carboxyl group per repeating unit were prepared by hydrogen-transfer polyaddition of 2-methylpiperazine (ISA 23), 1,2bis(N-methylamino)ethane (DMEDA-BAC), 1,2-bis(N-ethylamino)ethane (DEEDA-BAC, 1,3-bis(N-methylamino)propane (DMEPDA-BAC), or 1,6-bis(N-methylamino)hexane (DMEXA-BAC), in each case to 2,2-bis(acrylamido)acetic acid (BAC). The resultant PAAs had an M n in the range 7 985-24 980 g/mole and an Mw in the range 11 420-42 710 g/mole. Considerable differences were observed in the basicity of the amino groups present (log K°1 ) 7.5-9.5; log K°2 ) 3.2-8.4), whereas the log K°3 value (2-3) of the carboxyl groups was consistent with that of a fairly strong acid. DEEDA-BAC, DMEDA-BAC, and ISA 23 were nontoxic (IC50 > 5 mg/mL). Those PAAs with the highest log K°2 values were more cytotoxic (IC50 ) 3.55 mg/mL for DMEPDA-BAC, and IC50 ) 0.23 m/mL for DMEXA-BAC). All the PAAs displayed pH-dependent haemolysis (most lytic at pH 5.5), consistent with their proposed use as endosomolytic polymers.

In Saccharomyces cerevisiae, the class C vacuole protein sorting (Vps) proteins, together with Vam2p/Vps41p and Vam6p/Vps39p, form a complex that interacts with soluble N-ethylmaleimide-sensitive factor attachment protein receptor and Rab proteins to "tether" vacuolar membranes before fusion. To determine a role for the corresponding mammalian orthologues, we examined the function, localization, and protein interactions of endogenous mVps11, mVps16, mVps18, mVam2p, and mVam6. We found a significant proportion of these proteins localized to early endosome antigen-1 and transferrin receptor-positive early endosomes in Vero, normal rat kidney, and Chinese hamster ovary cells. Immunoprecipitation experiments showed that mVps18 not only interacted with Syntaxin (Syn)7, vesicle-associated membrane protein 8, and Vti1-b but also with Syn13, Syn6, and the Sec1/Munc18 protein mVps45, which catalyze early endosomal fusion events. Moreover, anti-mVps18 antibodies inhibited early endosome fusion in vitro. Mammalian mVps18 also associated with mVam2 and mVam6 as well as with the microtubule-associated Hook1 protein, an orthologue of the Drosophila Hook protein involved in endosome biogenesis. Using in vitro binding and immunofluorescence experiments, we found that mVam2 and mVam6 also associated with microtubules, whereas mVps18, mVps16, and mVps11 associated with actin filaments. These data indicate that the late Vps proteins function during multiple soluble N-ethylmaleimidesensitive factor attachment protein receptor-mediated fusion events throughout the endocytic pathway and that their activity may be coordinated with cytoskeletal function. INTRODUCTIONSoluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins are central to membrane fusion, forming tight 4 helix bundles that pull opposing membranes together (Hay, 2001). Although much is known about how SNARE complexes form, less is known about the upstream processes that regulate SNARE assembly. Some specificity is contributed by the SNARE proteins themselves, which catalyze fusion only in particular combinations due to the conformation of both the SNARE domain and N-terminal regulatory domain (Sollner, 2003). However, this intrinsic specificity is unlikely to suffice in vivo, and many other SNARE-interacting proteins have been proposed to contribute to the specificity of membrane fusion (Wickner and Haas, 2000;Chen and Scheller, 2001; Hey, 2001;Wendler and Tooze, 2001;Sollner, 2003).One important function that controls fusion specificity is the aggregation of compartments before the formation of trans-SNARE pairs or SNARE-pins. So-called "tethering" factors provide this function and are typically comprised of oligomeric complexes that associate directly or indirectly with SNARE proteins, Rab proteins, and Sec1/Munc-like (S/M) proteins (Waters and Pfeffer, 1999;Whyte and Munro, 2002). Only limited sequence or mechanistic conservation has been observed between tethering complexes, implying that each complex is tailored to provide and integrate ...

Poly(amidoamine)s (PAAs) are water-soluble polymers that display pH-dependent membrane activity. PAAs have the potential to act as a synthetic alternative to fusogenic peptides and thus promote endosomal escape. The purpose of this study was to investigate for the first time whether PAA have the ability to complex DNA, protect it from nuclease degradation and to promote transfection in vitro. PAAs ISA 1 (Mn 6900) and ISA 23 (Mn 10,500) and their 2-phenylethylamine containing analogues ISA 4 and ISA 22 (Mn approximately 8000) were studied. All PAAs retarded the electrophoretic mobility of lambda Hind III DNA demonstrating interpolyelectrolyte complex (IPEC) formation and toroids of 80-150 nm in diameter (10:1 polymer excess) were visible using TEM. DNase II inhibition was observed. At a polymer:DNA ratio of 10:1, this was ISA 1(89.6 +/- 6.1%), ISA 4 (92.2 +/- 11.2%), ISA 22 (69.4 +/- 3.7%), and ISA 23 (58.0 +/- 10.0%). PAAs demonstrated the ability to mediate pSV beta-galactosidase transfection of HepG2 cells. At a vector:DNA mass ratio of 5:1, ISA 23 showed equivalent transfection ability compared with polyethylenimine and LipofectIN and was more effective than LipofectACE. These properties suggest that PAAs warrant further development as endosomolytic vectors.