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Protein transduction technology Jehangir S Wadia and Steven F Dowdy* Intracellular delivery of macromolecules remains problematic because of the bioavailability restriction imposed by the cell membrane. Recent studies on protein transduction domains have circumvented this barrier, however, and have resulted in the delivery of peptides, full-length proteins, iron beads, liposomes, and radioactive isotopes into cells in culture and animal models in vivo. Addresses Howard Hughes Medical Institute, Department of Cellular and Molecular Medicine, UCSD School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0686, USA *e-mail: [email protected] Current Opinion in Biotechnology 2002, 13:52–56 0958-1669/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations HS heparin sulfate PTD protein transduction domain

Introduction Currently, the ability to ectopically express novel proteins that can either alter the cellular phenotype or provide therapeutic benefit is largely limited to recombinant genetic approaches. The introduction of the transgene and its sustained and regulated expression (by either viral or non-viral means) is often difficult to achieve, however, and can result in undesirable consequences such as immunogenicity, toxicity and an inability to target many cell types. These issues have limited the efficacy of transgenes in vivo. Moreover, the ability to circumvent genetic approaches by the delivery of full-length proteins directly into cells is problematic owing to the bioavailability restriction imposed by the cell membrane. In general, the plasma membrane of eukaryotic cells is impermeable to the vast majority of peptides and proteins. However, this dogma has recently been shown to be untrue with the identification of several protein transduction domains (PTDs) that are capable of transducing cargo across the plasma membrane, allowing the proteins to accumulate within the cell. The three most widely studied PTDs are from the Drosophila homeotic transcription protein antennapedia (Antp) [1–3], the herpes simplex virus structural protein VP22 [4], and the human immunodeficiency virus 1 (HIV-1) transcriptional activator Tat protein [5,6]. Transduction across the membrane by these PTDs occurs through a currently unidentified mechanism that is independent of receptors, transporters and endocytosis. Moreover, transduction occurs via a rapid process that at both 37°C and 4°C targets essentially 100% of cells in a concentration-dependent fashion. Significantly, when synthesized as recombinant fusion proteins or covalently

cross-linked to full-length proteins, these PTDs are capable of delivering biologically active proteins, such as β-galactosidase, intracellularly [7•]. These PTD fusion proteins are found both within the cytoplasm and the nucleus. The identification of short basic peptide sequences from these proteins (Antp, RQIKIWFQNRRMKWKK; Tat(47–57), YGRKKRRQRRR; sequences given in single-letter amino acid code) that confer cellular uptake has led to the recent identification and synthesis of numerous new PTDs [8•,9,10]. Although different PTDs show similar characteristics for cellular uptake, it is clear that they vary in efficacy in transporting their cargo into the cell. Although there is limited homology between these PTDs, the rate of cellular uptake has been found to strongly correlate to the number of basic residues present, specifically the number of arginine residues. These results indicate the presence of a common mechanism that probably depends on an interaction between the basic charges on the PTD and negative charges on the cell surface [8•,9]. To date, fusions created with the Tat(47–57) PTD show markedly better cellular uptake than similar fusions using the 16 amino acid sequence from Antp; however, recently devised peptoid transducers, such as the retro-inverso form of Tat(57–48) or homopolymers of arginine, appear to further increase cellular uptake several fold [8•,9]. Moreover, although the Antp PTD can transduce cells when associated with chemically synthesized peptides [11,12], the efficiency dramatically decreases with the incorporation of larger proteins. VP22 transduction is somewhat different from that of Tat or Antp. In this system, the DNA encoding the entire VP22 protein is genetically fused to the gene of interest and transfected into cells. The fusion transgene is then transcribed and the translated protein transduces from the primary transfected cells into the surrounding cells to varying levels [4,13]. Exogenously added VP22 fusion proteins have been reported to be internalized, but little data about the efficiency of this protein delivery mode is available. The direct delivery and efficient cellular uptake of transducing proteins offers several advantages over traditional DNA-based methods for manipulating cellular phenotypes. Consequently, a vast increase in the use of PTD fusion to address biological questions and for the introduction of pharmacologically relevant proteins in vitro and in vivo has now begun. The broad utility of protein transduction technology is illustrated schematically in Figure 1. Here, we review some of the most recent advances in this rapidly expanding area of research.

Mechanisms of PTD-mediated transduction Although the mechanism of PTD-mediated transduction across the cell membrane is largely unknown, recent

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Figure 1 Utility of protein transduction technology. Recently, a wide variety of cargo has been covalently linked to arginine-dependent protein transduction domains (PTDs). Although the exact mechanism of transduction across the cellular membrane is currently unknown, the first step (1) appears to involve a charge–charge interaction of the basic PTD with acidic motifs on the membrane. The second step (2) remains unknown, although it is independent of transporters, receptors and endocytosis. Once inside the cell (3), a plethora of complex events and activities can be performed on the cargo (listed in the figure). Currently, few, if any, molecules have been identified that cannot be transduced into cells when linked to a PTD. Thus, it will be interesting to see if this holds true when the list exceeds 500 molecules, which given the rapidity and creativity of the field may occur within the next year.

Small/ Fullintermediate length molecules Peptides Antisense proteins

40 nm Fe beads

200 nm Liposomes

Cargo Fe

PTD

++ ++

1 Extracellular – – – – – – – –– –– 2 –– 3

Intracellular

Cargo delivery Target binding Protein refolding Post-translational modification (e.g. phosphorylation, myristylation) Macromolecular assembly (e.g. dimer, trimer, tetramer) Intracellular transport: nuclear import/export, cytosolic membrane Separation of cargo from PTD: disulfide reduction, cleavage Molecular Imaging Non-PTD-linked cargo release: drug/DNA delivery Current Opinion in Biotechnology

progress has been made in identifying the key physiochemical requirements of PTDs. Ho et al. [10] modified the arginine content and distribution of Tat(47–87) and found increased transduction efficiency. This was further demonstrated by Futaki et al. [9] when they screened a series of arginine-rich peptides derived from 14 RNA- and DNAbinding proteins, including Tat(48–60). To their surprise, they found that all of these peptides were able to translocate through the cell membrane and accumulate within the cell. As both of these studies showed peptide internalized at 4°C, it is unlikely that uptake required any cellular-mediated process or required physical arrangement. In general, transduction efficiency appears to correspond to the number and location of arginine residues in the PTD sequence. Interestingly, the substitution of a non-charged glutamine residue in Tat(49–57) with alanine showed little effect on cellular uptake, whereas the substitution of any of the basic residues produced a significant (70–90%) decrease in uptake [8•]. Consequently, the transducing capabilities of different length homopolymers of L- and D-arginine were determined by fluorescently labeling the peptides and measuring uptake in Jurkat cells by flow cytometry [8•]. A 9-mer of L-arginine was 20-fold more efficient at internalization than Tat(49–57), whereas the corresponding D-isomer showed an even greater increase in uptake (100-fold). The

degree of internalization corresponded with the number of arginine residues, with six to nine residues showing the highest transduction efficiency. However, Ho et al. [10] noted little difference in transduction efficiency between polyarginine and Tat peptides. Thus, the commonality between these PTDs is the presence of multiple arginine residues. Rothbard’s group [8•,14] demonstrated that the guanidine headgroup of the arginine sidechain is a critical structural feature for transduction. Indeed, homopolymers of citrulline, an arginine isostere in which the nitrogen of the guanidine group is replaced with oxygen, showed no transduction activity. Increasing the distance between the guanine group and the peptide backbone by adding alkyl spacers had no significant effect on cellular uptake. As the guanidine sidechain of arginine has a pKa of approximately 12, each arginine residue is highly charged at physiological pH and, unlike histidine or lysine, the NH moiety of guanidine can donate a hydrogen atom allowing the formation of stable bidendate hydrogen bonds with anions such as phosphate or sulfate [14]. Thus, the internalization of arginine-rich PTDs occurs through a specific interaction between the arginine headgroup and charged members of the cell membrane. Recently, at least one structural cellular membrane component required for PTD internalization has been identified.

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Early work by Mann and Frankel [15] showed that increasing concentrations of heparin could compete with Tat PTD internalization. Not surprisingly given their highly basic charge, Tat peptides bind to heparin [16–18]. This work led to the discovery that cell-surface heparin sulfate (HS) proteoglycans appear to be key mediators of peptide internalization in vivo [19•]. Consistent with this observation, Tyagi et al. [19•] found that cells genetically impaired in the biosynthesis of fully sulfonated HS proteoglycans were selectively impaired for transduction by a Tat fusion protein (Tat–green fluorescent protein). The Tat–HS binding affinity was found to be proportional to the size of the heparin oligosaccharides and the arginine composition of the transduction domain. Binding probably involves both specific ionic and structural interactions, as several highly negatively charged molecules, such as dextran sulfate, were poor Tat antagonists and binding of other macromolecules to HS on the cell surface does not mediate their internalization. In addition, Tat uptake could be competitively inhibited by the selective degradation of the HS sidechains using glycosaminoglycan lyases. The ubiquitous presence of HS might explain the observation that Tat peptides and conjugated proteins are able to enter a wide variety of cells. This interaction at the cell surface is likely to mediate the uptake of other arginine-rich PTDs. Antp, for instance, has also been found to interact strongly with cellular membranes and to translocate across pure lipid bilayers [20]. However, it is still largely unknown how PTD-linked macromolecules are internalized following their interaction at the cell surface. The hydrophobic interior of the lipid membrane poses a significant barrier to the uptake of hydrophilic proteins and any uptake mechanism must overcome this obstacle. The formation of membrane channels similar to those used for the cellular uptake of certain bacterial toxins, such as diphtheria toxin, is unlikely to account for the internalization of large PTD-conjugated macromolecules such as 40 nm iron beads. In addition, Tat-mediated transduction does not appear to involve any disruption of the plasma membrane, as it could not promote the uptake of unrelated non-conjugated peptides present in the incubation media [21]. This observation also demonstrates a key asset of PTD-mediated transduction: it has the ability to deliver therapeutic cargo, while avoiding bringing non-linked molecules into or out of the cell.

Intracellular delivery of PTD-conjugated macromolecules Proteins have been evolutionarily selected to perform specific functions. Thus, the ability to deliver a wide variety of full-length, functional proteins has tremendous potential as a biological tool for studying cellular processes as well as for developing novel and potentially very specific therapeutic agents. To date, a growing list of transducible proteins covering a wide range of sizes and functional classes have been successfully used to study intracellular mechanisms and delivered in vivo. These

include p16 [22], p27 [23], E2F [24], β-galactosidase [7•], Cdk2 [25,26], Cu,Zn superoxide dismutase [27], enhanced green fluorescent protein [28], Cre recombinase [29], p53 [30], caspase-3 [31], PEA-15 [32] CRAC (cholesterol recognition/interaction amino acid consensus) [33], CDC42, Rac and Rho [34–36] and Iκ-B [37]. Schwarze et al. [7•] demonstrated the ability to successfully deliver full-length Tat fusion proteins in excess of 100 000 Da molecular weight to most, if not all, tissues of mouse models. Subsequently, several reports have described the delivery of peptides and proteins in vivo. May et al. [38] demonstrated the ability to inhibit NF-κB activity in vivo by treating mice with Antp–Iκ-B kinase peptides. Moreover, the advantages and versatility of protein transduction over viral transgene delivery were recently illustrated in a pair of papers by van der Noen’s group [39,40]. Previous studies by this group using retroviral vectors expressing β-galactosidase delivered into the rat salivary gland in vivo [39,40] were compared with results from the injection of Tat–β-galactosidase protein [10]. Injection of the Tat-conjugated protein resulted in transduction into 100% of the cells in a concentrationdependent manner, whereas viral delivery could only achieve 30–50% efficiency. The ability of PTDs to deliver cargo into cells is not limited only to proteins. Tat peptides have also been shown to mediate the efficient intracellular accumulation of nonorganic molecules. Indeed, PTDs may be useful for enhancing the delivery of poorly absorbed drugs across tissue barriers. For example, owing to their poor penetration through the skin, many drugs administered intravenously, such as cyclosporine A, are ineffective as topical agents. However, cyclosporine A conjugated to an arginine peptide through a pH-sensitive linker was found to efficiently transduce across human and mouse skin and inhibit cutaneous inflammation [41]. Thus, similar strategies could be employed to enhance the delivery of compounds/drugs with poor bioavailability (in excess of 700 Da) either across the cutaneous barrier, via administration across the alveolar membranes, or by increasing their oral availability. The most impressive aspect of PTD-mediated delivery and its therapeutic potential is its size independence. PTDs have been shown to deliver proteins in excess of 100 000 Da into cells in culture and most, if not all, cells in mammalian model systems. Furthermore, Weisseleder’s group [42–44] recently delivered into cells 40 nm superparamagnetic iron nanoparticles that were conjugated to Tat PTD peptides. These particles were internalized into both hematopoietic and neural progenitor cells at levels up to 10–30 pg per cell. Moreover, the transduction of such a large particle did not affect cell viability, differentiation or proliferation of CD34+ human stem cells. Indeed, the transduced cells could be injected into mice, tracked with magnetic resonance in vivo, and harvested from tissue 24 h post-injection.

Protein transduction technology Wadia and Dowdy

The therapeutic delivery of polar compounds is often inefficient owing to the difficulty of crossing the lipid membrane of cells. As a consequence, delivery of these compounds within liposomal carriers has been the focus of increasing attention, but has been hampered by inefficient cellular uptake and consequent degradation through the endocytic pathway. Recently, Torchilin et al. [45•] delivered 200 nm liposomes coated with Tat PTD. Indeed, confocal analysis showed that the liposomes remained intracellularly intact 1 h after transduction. The attachment of PTDs to liposome carriers and their subsequent cellular uptake and cytoplasmic release may have significant implications for the future of both drug and DNA delivery in vitro and in vivo.

Conclusions The recent explosion of studies both investigating the mechanism of protein transduction and utilizing the methodology to deliver a wide array of compounds, peptides and proteins in vitro and in vivo has begun to propel this once obscure field into mainstream scientific investigations. Understanding the mechanism of transduction and employing that information to further improve the efficacy of PTDs will help to advance both experimental and therapeutic potential. Indeed, if protein transduction can be adapted in humans with the efficacy observed for in vitro and in vivo animal models then, at our fingertips, we now possess the potential to devise entirely new therapeutic compounds, peptides and proteins to combat infectious diseases, complement deficiencies in specific genes and hopefully to specifically kill tumor cells.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest •• of outstanding interest 1.

2.

3.

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Elliott G, O’Hare P: Intercellular trafficking and protein delivery by a herpes virus structural protein. Cell 1997, 88:223-233.

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7. •

Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF: In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 1999, 285:1569-1572. This article demonstrated for the first time the ability to deliver via protein transduction (2000 Da) peptides and large (120 000 Da) proteins into most, if not all, cells and tissues of a mouse. Significantly, to become enzymatically active, the transduced Tat–β-galactosidase required intracellular refolding and homotetramerization.

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Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB: The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc Natl Acad Sci USA 2000, 97:13003-13008. This paper identified a series of structural requirements and optimal sequence lengths for arginine-dependent protein transduction domains. Moreover, this paper synthesized synthetic peptidyl-mimetics that demonstrated the absolute requirement for the guanidine group on the end of the arginine group. 9.

Futaki S, Suzuki T, Ohashi W, Yagami T, Tanaka S, Ueda K, Sugiura Y: Arginine-rich peptides. An abundant source of membranepermeable peptides having potential as carriers for intracellular protein delivery. J Biol Chem 2001, 276:5836-5840.

10. Ho A, Schwarze SR, Mermelstein SJ, Waksman G, Dowdy SF: Synthetic protein transduction domains: enhanced transduction potential in vitro and in vivo. Cancer Res 2001, 61:474-477. 11. Kato D, Miyazawa K, Ruas M, Starborg M, Wada I, Oka T, Sakai T, Peters G, Hara E: Features of replicative senescence induced by direct addition of antennapedia-p16INK4A fusion protein to human diploid fibroblasts. FEBS Lett 1998, 427:203-208. 12. Chen YN, Sharma SK, Ramsey TM, Jiang L, Martin MS, Baker K, Adams PD, Bair KW, Kaelin WG Jr: Selective killing of transformed cells by cyclin/cyclin-dependent kinase 2 antagonists. Proc Natl Acad Sci USA 1999, 96:4325-4329. 13. Elliott G, O’Hare P: Intercellular trafficking of VP22–GFP fusion proteins. Gene Ther 1999, 6:149-151. 14. Mitchell DJ, Kim DT, Steinman L, Fathman CG, Rothbard JB: Polyarginine enters cells more efficiently than other polycationic homopolymers. J Pept Res 2000, 56:318-325. 15. Mann D, Frankel A: Endocytosis and targeting of exogenous HIV-1 Tat protein. EMBO J 1991, 10:1733-1739. 16. Rusnati M, Coltrini D, Oreste P, Zoppetti G, Albini A, Noonan D, di Fagagna F, Giacca M, Presta M: Interaction of HIV-1 Tat protein with heparin. Role of the backbone structure, sulfation, and size. J Biol Chem 1997, 272:11313-11320. 17.

Rusnati M, Tulipano G, Urbinati C, Tanghetti E, Giuliani R, Giacca M, Ciomei M, Corallini A, Presta M: The basic domain in HIV-1 Tat protein as a target for polysulfonated heparin-mimicking extracellular Tat antagonists. J Biol Chem 1998, 273:16027-16037.

18. Hakansson S, Jacobs A, Caffrey M: Heparin binding by the HIV-1 tat protein transduction domain. Protein Sci 2001, 10:2138-2139. 19. Tyagi M, Rusnati M, Presta M, Giacca M: Internalization of HIV-1 • requires cell surface heparan sulfate proteoglycans. J Biol Chem 2001, 276:3254-3261. This paper proposes that extracellular membrane-bound heparin sulfate proteoglycans are involved in the transduction of Tat–GFP into cells. However, it remains unclear if this is only true for Tat–GFP or for other/all Tat-mediated transduction molecules. 20. Thoren PE, Persson D, Karlsson M, Norden B: The antennapedia peptide penetratin translocates across lipid bilayers – the first direct observation. FEBS Lett 2000, 482:265-268. 21. Vives E, Brodin P, Lebleu B: A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem 1997, 272:16010-16017. 22. Ezhevsky SA, Nagahara H, Vocero-Akbani AM, Gius DR, Wei MC, Dowdy SF: Hypo-phosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb. Proc Natl Acad Sci USA 1997, 94:10699-10704. 23. Nagahara H, Vocero-Akbani AM, Snyder EL, Ho A, Latham DG, Lissy NA, Becker-Hapak M, Ezhevsky SA, Dowdy SF: Transduction of full-length TAT fusion proteins into mammalian cells: TAT–p27Kip1 induces cell migration. Nat Med 1998, 4:1449-1452. 24. Lissy NA, Davis PK, Irwin M, Kaelin WG, Dowdy SF: A common E2F-1 and p73 pathway mediates cell death induced by TCR activation. Nature 2000, 407:642-645. 25. Ezhevsky SA, Ho A, Becker-Hapak M, Davis PK, Dowdy SF: Differential regulation of retinoblastoma tumor suppressor protein by G(1) cyclin-dependent kinase complexes in vivo. Mol Cell Biol 2001, 21:4773-4784. 26. Klekotka PA, Santoro SA, Ho A, Dowdy SF, Zutter MM: Mammary epithelial cell-cycle progression via the α2β1 integrin: unique and

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synergistic roles of the α2 cytoplasmic domain. Am J Pathol 2001, 159:983-992. 27.

Kwon HY, Eum WS, Jang HW, Kang JH, Ryu J, Ryong Lee B, Jin LH, Park J, Choi SY: Transduction of Cu,Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into mammalian cells. FEBS Lett 2000, 485:163-167.

28. Caron NJ, Torrente Y, Camirand G, Bujold M, Chapdelaine P, Leriche K, Bresolin N, Tremblay JP: Intracellular delivery of a Tat–eGFP fusion protein into muscle cells. Mol Therapy 2001, 3:310-318. 29. Jo D, Nashabi A, Doxsee C, Lin Q, Unutmaz D, Chen J, Ruley HE: Epigenetic regulation of gene structure and function with a cellpermeable Cre recombinase. Nat Biotechnol 2001, 19:929-933. 30. Wills KN, Atencio IA, Avanzini JB, Neuteboom S, Phelan A, Philopena J, Sutjipto S, Vaillancourt MT, Wen SF, Ralston RO et al.: Intratumoral spread and increased efficacy of a p53-VP22 fusion protein expressed by a recombinant adenovirus. J Virol 2001, 75:8733-8741. 31. Vocero-Akbani AM, Heyden NV, Lissy NA, Ratner L, Dowdy SF: Killing HIV-infected cells by transduction with an HIV proteaseactivated caspase-3 protein. Nat Med 1999, 5:29-33. 32. Embury J, Klein D, Pileggi A, Ribeiro M, Jayaraman S, Molano RD, Fraker C, Kenyon N, Ricordi C, Inverardi L et al.: Proteins linked to a protein transduction domain efficiently transduce pancreatic islets. Diabetes 2001, 50:1706-1713. 33. Li H, Yao Z, Degenhardt B, Teper G, Papadopoulos V: Cholesterol binding at the cholesterol recognition/interaction amino acid consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV TAT–CRAC peptide. Proc Natl Acad Sci USA 2001, 98:1267-1272. 34. Chellaiah MA, Soga N, Swanson S, McAllister S, Alvarez U, Wang D, Dowdy SF, Hruska KA: Rho-A is critical for osteoclast podosome organization, motility, and bone resorption. J Biol Chem 2000, 275:11993-12002. 35. Hall DJ, Cui J, Bates ME, Stout BA, Koenderman L, Coffer PJ, Bertics PJ: Transduction of a dominant-negative H-Ras into human eosinophils attenuates extracellular signal-regulated kinase activation and interleukin-5-mediated cell viability. Blood 2001, 98:2014-2021. 36. Soga N, Namba N, McAllister S, Cornelius L, Teitelbaum SL, Dowdy SF, Kawamura J, Hruska KA: Rho family GTPases regulate

VEGF-stimulated endothelial cell motility. Exp Cell Res 2001, 269:73-87. 37.

Abu-Am Y, Dowdy SF, Ross FP, Clohisy JC, Teitelbaum SL: TAT κBα α arrest fusion proteins containing tyrosine 42-deleted Iκ osteoclastogenesis. J Biol Chem 2001, 276:30499-30503.

38. May MJ, D’Acquisto F, Madge LA, Glockner J, Pober JS, Ghosh S: κB activation by a peptide that blocks Selective inhibition of NF-κ κB kinase complex. Science the interaction of NEMO with the Iκ 2000, 289:1550-1554. 39. Barka T, van der Noen H: Retrovirus-mediated gene transfer into rat salivary glands in vivo. Hum Gene Ther 1996, 7:613-618. 40. Barka T, van der Noen H: Retrovirus-mediated gene transfer into rat salivary gland cells in vitro and in vivo. J Histochem Cytochem 1997, 45:1533-1545. 41. Rothbard JB, Garlington S, Lin Q, Kirschberg T, Kreider E, McGrane PL, Wender PA, Khavari PA: Conjugation of arginine oligomers to cyclosporin A facilitates topical delivery and inhibition of inflammation. Nat Med 2000, 6:1253-1257. 42. Josephson L, Tung CH, Moore A, Weissleder R: High-efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugates. Bioconjug Chem 1999, 10:186-191. 43. Lewin M, Carlesso N, Tung CH, Tang XW, Cory D, Scadden DT, Weissleder R: Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat Biotechnol 2000, 18:410-414. 44. Dodd CH, Hsu HC, Chu WJ, Yang P, Zhang HG, Mountz JD Jr., Zinn K, Forder J, Josephson L, Weissleder R et al.: Normal T-cell response and in vivo magnetic resonance imaging of T cells loaded with HIV transactivator-peptide-derived superparamagnetic nanoparticles. J Immunol Methods 2001, 256:89-105. 45. Torchilin VP, Rammohan R, Weissig V, Levchenko TS: TAT peptide on • the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors. Proc Natl Acad Sci USA 2001, 98:8786-8791. This paper demonstrates the ability to transduce liposomes in excess of 200 nm diameter directly across the cell membrane and into cells by anchoring Tat PTDs to the liposome surface. Significantly, this results in by-passing the endocytic pathway and thereby avoids lysosomal degradation/exposure of the liposome contents. In addition, this is the current size record holder for transduction.