Enhanced cellular uptake with a cobaltacarborane-porphyrin-HIV-1 Tat 48-60 conjugate

A series of four porphyrin-cobaltacarborane conjugates have been synthesized, containing three or four cobaltabisdicarbollide anions linked by O(CH(2)CH(2)O)(2) groups to the porphyrin macrocycle and one of them containing a HIV-1 Tat 48-60 peptide sequence linked via a low molecular weight poly(ethylene glycol) (PEG) spacer. The cellular uptake, cytotoxicity, and preferential sites of intracellular localization of the conjugates were evaluated in human HEp2 cells. All conjugates are nontoxic in the dark at the concentrations studied. Upon exposure to low light dose (1 J cm(-)(2)) only the porphyrin-cobaltacarborane-HIV-1 Tat 48-60 conjugate showed 30% inhibition of cell proliferation at a concentration of 10 microM. The cellular uptake was dependent on the number of carborane cages and was significantly enhanced by the presence of the cell penetrating peptide sequence HIV-1 Tat 48-60. All conjugates preferentially localized in the cell lysosomes.     

Permeation peptide conjugates for in vivo molecular imaging applications

Rapid and efficient delivery of imaging probes to the cell interior using permeation peptides has enabled novel applications in molecular imaging. Membrane permeant peptides based on the HIV-1 Tat basic domain sequence, GRKKRRQRRR, labeled with fluorophores and fluorescent proteins for optical imaging or with appropriate peptide-based motifs or macrocycles to chelate metals, such as technetium for nuclear scintigraphy and gadolinium for magnetic resonance imaging, have been synthesized. In addition, iron oxide complexes have been functionalized with the Tat basic domain peptides for magnetic resonance imaging applications. Herein we review current applications of permeation peptides in molecular imaging and factors influencing permeation peptide internalization. These diagnostic agents show concentrative cell accumulation and rapid kinetics and display cytosolic and focal nuclear accumulation in human cells. Combining methods, dual-labeled permeation peptides incorporating fluorescein maleimide and chelated technetium have allowed for both qualitative and quantitative analysis of cellular uptake. Imaging studies in mice following intravenous administration of prototypic diagnostic permeation peptides show rapid whole-body distribution allowing for various molecular imaging applications. Strategies to develop permeation peptides into molecular imaging probes have included incorporation of targeting motifs such as molecular beacons or protease cleavable domains that enable selective retention, activatable fluorescence, or targeted transduction. These novel permeation peptide conjugates maintain rapid translocation across cell membranes into intracellular compartments and have the potential for targeted in vivo applications in molecular imaging and combination therapy.     

Imaging of human glioma cells by means of a Syndecan-4 directed DOTA-conjugate

The extracellular glycoprotein Tenascin-C (TN-C) is highly upregulated in gliomas. Therefore, many chemotherapies with radiolabeled antibodies against TN-C have been performed. However, TN-Cs binding partner Syndecan-4 did not play any role as a therapeutic or imaging target in gliomas. We constructed an imaging compound containing the magnetic resonance imaging (MRI) contrast agent gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), the fluorescence dye sulforhodamine and a synthetic Syndecan-4-specific 21 amino acid peptide derived from TN-C. Magnetic resonance relaxometry, confocal laser scanning microscopy, and flow cytometry showed that the Syndecan-4-DOTA-Rhodamine conjugate was taken up into the cytoplasm of human U373 glioma cells without any cytotoxic effects. Competition experiments indicate that this uptake was receptor-mediated. This conjugate might be used for future MRI studies of brain tumors after systemic or intraoperative local application.     

Synthesis, cellular uptake and HIV-1 Tat-dependent trans-activation inhibition activity of oligonucleotide analogues disulphide-conjugated to cell-penetrating peptides

Oligonucleotides composed of 2′-O-methyl and locked nucleic acid residues complementary to HIV-1 trans-activation responsive element TAR block Tat-dependent trans-activation in a HeLa cell assay when delivered by cationic lipids. We describe an improved procedure for synthesis and purification under highly denaturing conditions of 5′-disulphide-linked conjugates of 3′-fluorescein labelled oligonucleotides with a range of cell-penetrating peptides and investigate their abilities to enter HeLa cells and block trans-activation. Free uptake of 12mer OMe/LNA oligonucleotide conjugates to Tat (48–58), Penetratin and R₉F₂ was observed in cytosolic compartments of HeLa cells. Uptake of the Tat conjugate was enhanced by N-terminal addition of four Lys or Arg residues or a second Tat peptide. None of the conjugates entered the nucleus or inhibited trans-activation when freely delivered, but inhibition was obtained in the presence of cationic lipids. Nuclear exclusion was seen for free delivery of Tat (48–58), Penetratin and R₉ conjugates of 16mer phosphorothioate OMe oligonucleotide. Uptake into human fibroblast cytosolic compartments was seen for Tat, Penetratin, R₉F₂ and Transportan conjugates. Large enhancements of HeLa cell uptake into cytosolic compartments were seen when free Tat peptide was added to Tat conjugate of 12mer OMe/LNA oligonucleotide or Penetratin peptide to Penetratin conjugate of the same oligonucleotide.     

Synthesis and properties of cell-targeted Zn(II)-phthalocyanine-peptide conjugates

Two Zn-Pc-peptide conjugates bearing either a short linker or a long PEG-linker between the macrocycle and a bifunctional peptide containing the nucleoplasmin and HIV-1 Tat 48-60 sequences have been synthesized in order to increase the Pc cell-targeting ability and to evaluate the effect of the linker. The presence of the peptide chain increased the water solubility of the Pc macrocycle and, consequently, its fluorescence in aqueous solutions. The highest fluorescence quantum yields were observed at low pH (5.0) for both conjugates and were always higher for the conjugate bearing the short linker. Both conjugates were found to have low dark cytotoxicity toward human HEp2 cells (IC50 > 77 microM) but were highly phototoxic (IC50 < 2 microM at 1 J cm-2). The conjugate bearing the long PEG-linker accumulated the most within cells (26 times more than the unconjugated Zn-Pc), followed by the short linker conjugate (17 times more than the unconjugated Zn-Pc). Both conjugates were found to localized preferentially within the cell lysosomes.     

Enhanced delivery of cell-penetrating peptide-peptide nucleic acid conjugates by endosomal disruption

Improvement of cellular uptake and cellular localization is still one of the main obstacles to the development of antisense-antigene therapeutics, including peptide nucleic acid (PNA). Cell-penetrating peptides (CPPs) such as Tat peptide and polyarginine have been widely used to improve the cellular uptake of PNA and other antisense agents. Cellular uptake of most CPP conjugates occurs mainly through endocytotic pathways, and most CPP conjugate is retained in the endosomal compartments of the cell. Several methods to induce endosome disruption have been shown to improve the bioavailability of CPP conjugates to the cytosol and/or nucleus by facilitating escape from the endosomal compartments. Here we describe protocols for the delivery of CPP-PNA conjugates to adherent cultured cells using photodynamic treatment (photochemical internalization), Ca2+ treatment or chloroquine treatment to potentiate the antisense effects of CPP-PNA conjugates through increased release of CPP conjugates into the cytoplasm. This protocol, consisting of CPP-mediated delivery assisted by an endosome-disruption agent, allows the delivery of the CPP-PNA conjugates to the nucleus and/or cytosol of cultured cells. The endosome-disruption treatment improves the nuclear antisense effects of CPP-PNA conjugates by up to two orders of magnitude using 24-hour delivery.     

Mitochondrial targeting of farnesyl diphosphate synthase is a widespread phenomenon in eukaryotes

Cellular internalization of cell-penetrating peptide HIV-1 Tat basic domain (RKKRRQRRR) was studied in Triticale cv AC Alta mesophyll protoplasts. Fluorescently labeled monomer (Tat) and dimer (Tat(2)) of Tat basic domain efficiently translocated through the plasma membrane of mesophyll protoplast and showed distinct nuclear accumulation within 10 min of incubation. Substitution of first arginine residue with alanine in Tat basic domain (M-Tat) severely reduced cellular uptake of the peptide (3.8 times less than Tat). Tat(2) showed greater cellular internalization than Tat (1.6 times higher). However, characteristics of cellular uptake remained same for Tat and Tat(2). Cellular internalization of Tat and Tat(2) was concentration dependent and non-saturable whereas no significant change in cellular uptake was observed even at higher concentrations of M-Tat. Low temperature (4 degrees C) remarkably increased cellular internalization of Tat as well as Tat(2) but M-Tat showed no enhanced uptake. Viability test showed that peptide treatment had no cytotoxic effect on protoplasts further indicating involvement of a common mechanism of peptide uptake at all the temperatures. Endocytic inhibitors nocodazole (10 muM), chloroquine (100 muM) and sodium azide (5 mM) did not show any significant inhibitory effect on cellular internalization of either Tat or Tat(2). These results along with stimulated cellular uptake at low temperature indicate that Tat peptide is internalized in the plant protoplasts in a non-endocytic and energy-independent manner. Competition experiments showed that non-labeled peptide did not inhibit or alter nuclear accumulation of fluorescent Tat or Tat(2) suggesting active transport to the nucleus was not involved. Studies in mesophyll protoplasts show that internalization pattern of Tat peptide is apparently similar to that observed in mammalian cell lines.     

Cellular trajectories of peptide-modified gold particle complexes: comparison of nuclear localization signals and peptide transduction domains

Gold nanoparticles modified with nuclear localization peptides were synthesized and evaluated for their subcellular distribution in HeLa human cervical epithelium cells, 3T3/NIH murine fibroblastoma cells, and HepG2 human hepatocarcinoma cells. Video-enhanced color differential interference contrast microscopy and transmission electron microscopy indicated that transport of nanoparticles into the cytoplasm and nucleus depends on peptide sequence and cell line. Recently, the ability of certain peptides, called protein transduction domains (PTDs), to transclocate cell and nuclear membranes in a receptor- and temperature-independent manner has been questioned (see for example, Lundberg, M.; Wikstrom, S.; Johansson, M. (2003) Mol. Ther. 8, 143-150). We have evaluated the cellular trajectory of gold nanoparticles carrying the PTD from HIV Tat protein. Our observations were that (1) the conjugates did not enter the nucleus of 3T3/NIH or HepG2 cells, and (2) cellular uptake of Tat PTD peptide-gold nanoparticle conjugates was temperature dependent, suggesting an endosomal pathway of uptake. Gold nanoparticles modified with the adenovirus nuclear localization signal and the integrin binding domain also entered cells via an energy-dependent mechanism, but in contrast to the Tat PTD, these signals triggered nuclear uptake of nanoparticles in HeLa and HepG2 cell lines.     

Pharmacokinetic analysis of polyamide nucleic-acid-cell penetrating peptide conjugates targeted against HIV-1 transactivation response element

We have demonstrated that polyamide nucleic acids complementary to the transactivation response (TAR) element of HIV-1 LTR inhibit HIV-1 production when transfected in HIV-1 infected cells. We have further shown that anti-TAR PNA (PNA(TAR)) conjugated with cell-penetrating peptide (CPP) is rapidly taken up by cells and exhibits strong antiviral and anti-HIV-1 virucidal activities. Here, we pharmacokinetically analyzed (125)I-labeled PNA(TAR) conjugated with two CPPs: a 16-mer penetratin derived from antennapedia and a 13-mer Tat peptide derived from HIV-1 Tat. We administered the (125)I-labeled PNA(TAR)-CPP conjugates to male Balb/C mice through intraperitoneal or gavage routes. The naked (125)I-labeled PNA(TAR) was used as a control. Following a single administration of the labeled compounds, their distribution and retention in various organs were monitored at various time points. Regardless of the administration route, a significant accumulation of each PNA(TAR)-CPP conjugate was found in different mouse organs and tissues. The clearance profile of the accumulated radioactivity from different organs displayed a biphasic exponential pathway whereby part of the radioactivity cleared rapidly, but a significant portion of it was slowly released over a prolonged period. The kinetics of clearance of individual PNA(TAR)-CPP conjugates slightly varied in different organs, while the overall biphasic clearance pattern remained unaltered regardless of the administration route. Surprisingly, unconjugated naked PNA(TAR) displayed a similar distribution and clearance profile in most organs studied although extent of its uptake was lower than the PNA(TAR)-CPP conjugates.     

A comprehensive model for the cellular uptake of cationic cell-penetrating peptides

The plasma membrane represents an impermeable barrier for most macromolecules. Still some proteins and so-called cell-penetrating peptides enter cells efficiently. It has been shown that endocytosis contributes to the import of these molecules. However, conflicting results have been obtained concerning the nature of the endocytic process. In addition, there have been new findings for an endocytosis-independent cellular entry. In this study, we provide evidence that the Antennapedia-homeodomain-derived antennapedia (Antp) peptide, nona-arginine and the HIV-1 Tat-protein-derived Tat peptide simultaneously use three endocytic pathways: macropinocytosis, clathrin-mediated endocytosis and caveolae/lipid-raft-mediated endocytosis. Antennapedia differs from Tat and R9 by the extent by which the different import mechanisms contribute to uptake. Moreover, at higher concentrations, uptake occurs by a mechanism that originates from spatially restricted sites of the plasma membrane and leads to a rapid cytoplasmic distribution of the peptides. Endocytic vesicles could not be detected, suggesting an endocytosis-independent mode of uptake. Heparinase treatment of cells negatively affects this import, as does the protein kinase C inhibitor rottlerin, expression of dominant-negative dynamin and chlorpromazine. This mechanism of uptake was observed for a panel of different cell lines. For Antp, significantly higher peptide concentrations and inhibition of endocytosis were required to induce its uptake. The relevance of these findings for import of biologically active cargos is shown.     

Translocation and nuclear accumulation of monomer and dimer of HIV-1 Tat basic domain in triticale mesophyll protoplasts

Cellular internalization of cell-penetrating peptide HIV-1 Tat basic domain (RKKRRQRRR) was studied in Triticale cv AC Alta mesophyll protoplasts. Fluorescently labeled monomer (Tat) and dimer (Tat₂) of Tat basic domain efficiently translocated through the plasma membrane of mesophyll protoplast and showed distinct nuclear accumulation within 10 min of incubation. Substitution of first arginine residue with alanine in Tat basic domain (M-Tat) severely reduced cellular uptake of the peptide (3.8 times less than Tat). Tat₂ showed greater cellular internalization than Tat (1.6 times higher). However, characteristics of cellular uptake remained same for Tat and Tat₂. Cellular internalization of Tat and Tat₂ was concentration dependent and non-saturable whereas no significant change in cellular uptake was observed even at higher concentrations of M-Tat. Low temperature (4 °C) remarkably increased cellular internalization of Tat as well as Tat₂ but M-Tat showed no enhanced uptake. Viability test showed that peptide treatment had no cytotoxic effect on protoplasts further indicating involvement of a common mechanism of peptide uptake at all the temperatures. Endocytic inhibitors nocodazole (10 μM), chloroquine (100 μM) and sodium azide (5 mM) did not show any significant inhibitory effect on cellular internalization of either Tat or Tat₂. These results along with stimulated cellular uptake at low temperature indicate that Tat peptide is internalized in the plant protoplasts in a non-endocytic and energy-independent manner. Competition experiments showed that non-labeled peptide did not inhibit or alter nuclear accumulation of fluorescent Tat or Tat₂ suggesting active transport to the nucleus was not involved. Studies in mesophyll protoplasts show that internalization pattern of Tat peptide is apparently similar to that observed in mammalian cell lines.     

Cellular uptake [correction of utake] of the Tat peptide: an endocytosis mechanism following ionic interactions

The cellular delivery of various biological compounds has recently been improved by conjugating them to short peptides known as protein transduction domains or cell penetrating peptides. These peptides include Tat, Antennapedia and arginine-rich peptides. The common feature of these peptides is their highly cationic nature. Up to now, the cellular uptake of about 50 different peptides and proteins coupled to Tat or Antennapedia peptides has been reported. The ability to deliver molecules into cells is not limited to peptide moieties, since oligonucleotides, peptide nucleic acids or other low molecular weight entities have been successfully internalized. Moreover, most of these examples have been accompanied by the expected biological response. More surprisingly, the uptake of large structures such as liposomes, phages, nanoparticles or adenoviruses has also been documented. Indeed the mechanism by which these very different entities could enter cells following a putative common pathway appeared more and more intriguing after each new reported example of cellular uptake mediated by these peptides. After a long period of uncertainty regarding the mechanism of entry, data from several groups now argue for an energy-dependent process of entry. The entry of most of these molecules is likely to be inhibited by low temperature incubation or in the presence of various drugs applied to inhibit the energy-dependent pathway of cell entry. Moreover, the binding of the highly cationic Tat peptide to various anionic membrane components probably initiates the first step of the cell internalization process.     

Cell-dependent differential cellular uptake of PNA,peptides, and PNA-peptide conjugates

Peptide nucleic acid (PNA) oligomers were conjugated to cell-penetrating peptides: pAnt, a 17-residue fragment of the Drosophila protein Antennapedia, and pTat, a 14-amino acid fragment of HIV protein Tat. A 14-mer PNA was attached to the peptide by disulfide linkage or by maleimide coupling. The uptake of (directly or indirectly, via biotin) fluorescein-labeled peptides, PNAs, or PNA-peptide conjugates was studied by fluorescence microscopy, confocal laser scanning microscopy, and fluorometry in five cell types. In SK-BR-3, HeLa, and IMR-90 cells, the PNA-peptide conjugates and a T1, backbone-modified PNA were readily taken up (2 microM). The PNA was almost exclusively confined to vesicular compartments in the cytosol. However, the IMR-90 cells also showed a weak diffuse staining of the cytoplasm. In the U937 cells, we observed a very weak and exclusively vesicular staining with the PNA-peptide conjugates and the T(lys)-modified PNA. No evident uptake of the unmodified PNA was seen. In H9 cells, both peptides and the PNA-peptide conjugates quickly associated with the membrane, followed by a weak intracellular staining. A cytotoxic effect resulting in artificial staining of the cells was observed with fluoresceinated peptides and PNA-peptide conjugates at concentrations above 5-10 microM, depending on cell type and incubation time. We conclude that uptake of PNAs in many cell types can be achieved either by conjugating to certain peptides or simply by charging the PNA backbone using lysine PNA units. The uptake is time, temperature, and concentration dependent and mainly endocytotic. Our results also show that proper controls for cytotoxicity should always be carried out to avoid misinterpretation of visual data.     

Tat-conjugated synthetic macromolecules facilitate cytoplasmic drug delivery to human ovarian carcinoma cells

We have synthesized N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-cell penetrating peptide Tat conjugates and evaluated their subcellular distribution in A2780 human ovarian carcinoma cells by confocal fluorescence microscopy and subcellular fractionation. Our data indicate the transport of these conjugates by a single Tat molecule to both the cytoplasm and nucleus via a nonendocytotic and concentration independent process. The uptake was observed to occur within 3 min, as confirmed by live cell microscopy. In contrast, HPMA copolymers lacking the Tat peptide were internalized solely by endocytosis. For the first time, Tat-mediated cytoplasmic delivery of a polymer bound anticancer drug, doxorubicin, was also demonstrated. These findings establish the feasibility of overcoming major cellular and subcellular obstacles to intracellular macromolecular delivery and hold great promise for the development of polymer-based systems for the cytoplasmic delivery of therapeutic molecules.     

Synthesis of radiometal-labeled and fluorescent cell-permeating peptide-PNA conjugates for targeting the bcl-2 proto-oncogene

The B-cell lymphoma/leukemia-2 (bcl-2) proto-oncogene has been associated with the transformation of benign lesions to malignancy, disease progression, poor prognosis, reduced survival, and development of resistance to radiation and chemotherapy in many types of cancer. The objective of this work was to synthesize an antisense peptide nucleic acid (PNA) complementary to the first six codons of the bcl-2 open reading frame, conjugated to a membrane-permeating peptide for intracellular delivery, and modified with a bifunctional chelating agent for targeting imaging and therapeutic radiometals to tumors overexpressing bcl-2. Four peptide-PNA constructs were synthesized by a combination of manual and automated stepwise elongation techniques, including bcl-2 antisense conjugates and nonsense conjugates with no complementarity to any known mammalian gene or DNA sequence. The PNA sequences were synthesized manually by solid-phase 9-fluorenylmethoxycarbonyl (Fmoc) techniques. Then a fully protected lysine monomer, modified with 1,4,7,10-tetraazacyclododecane-N,N',N',N'"-tetraacetic acid (DOTA) for radiometal chelation, was coupled manually to each PNA sequence. Synthesis of the DOTA-PNA conjugates was followed by automated elongation with a peptide sequence (PTD-4-glycine, PTD-4-G), known to mediate cellular internalization of impermeable effector molecules, or its retro-inverso analogue (ri-PTD-4-G). Preparation of the four conjugates required an innovative synthetic strategy, using mild acid conditions to generate hydrophobic, partially deprotected intermediates. These intermediates were purified by semipreparative reversed-phase HPLC and completely deprotected to yield pure peptide-PNA conjugates in 6% to 9% overall yield. Using modifications of this synthetic strategy, the ri-PTD-4-G conjugate of bcl-2 antisense PNA was prepared using a lysine derivative of tetramethylrhodamine (TMR) for fluorescence microscopy. Plasma stability studies showed that (111)In-DOTA-labeled ri-PTD-4-G-anti-bcl-2 PNA was stable for 168 h at 37 degrees C, unlike the conjugate containing the parent peptide sequence. Scanning confocal fluorescence microscopy of TMR-labeled ri-PTD-4-G-anti-bcl-2 PNA in Raji lymphoma cells demonstrated that the retro-inverso peptide was active in membrane permeation and mediated cellular internalization of the antisense PNA into the cytoplasm, where high concentrations of bcl-2 mRNA are expected to be present.     

HIV-1 Tat protein-mediated transactivation of the HIV-1 long terminal repeat promoter is potentiated by a novel nuclear Tat-interacting protein of 110 kDa, Tip110

Human immunodeficiency virus type 1 (HIV-1) gene expression and replication is highly dependent on and modulated by interactions between viral and host cellular factors. Tat protein, encoded by one of the HIV-1 regulatory genes, tat, is essential for HIV-1 gene expression. A number of host cellular factors have been shown to interact with Tat in this process. During our attempts to determine the molecular mechanisms of Tat interaction with brain cells, we isolated a cDNA clone that encodes a novel Tat-interacting protein of 110 kDa or Tip110 from a human fetal brain cDNA library. GenBank BLAST search revealed that Tip110 was almost identical to a previously cloned KIAA0156 gene with unknown functions. In vivo binding of Tip110 with Tat was confirmed by immunoprecipitation and Western blotting, in combination with mutagenesis. The yeast three-hybrid RNA-protein interaction assay indicated no direct interaction of Tip110 with Tat transactivating response element RNA. Nevertheless, Tip110 strongly synergized with Tat on Tat-mediated chloramphenicol acetyltransferase reporter gene expression and HIV-1 virus production, whereas down-modulation of constitutive Tip110 expression inhibited HIV-1 virus production. Northern blot analysis showed that Tip110 mRNA was expressed in a variety of human tissues and cells. Moreover, digital fluorescence microscopic imaging revealed that Tip110 was expressed exclusively in the nucleus, and within a nuclear speckle structure that has recently been described for human cyclin T and CDK9, two critical components for Tat transactivation function on HIV-1 long terminal repeat promoter. Taken together, these data demonstrate that Tip110 regulates Tat transactivation activity through direct interaction, and suggest that Tip110 is an important cellular factor for HIV-1 gene expression and viral replication.     

Acid wash in determining cellular uptake of Fab/cell-permeating peptide conjugates

Successful intracellular delivery of various bioactive molecules has been reported using cell-permeating peptides (CPPs) as delivery vectors. To determine the effects of CPPs on the cellular uptake of immunoglobulin Fab fragment, conjugates of a radio-iodinated Fab fragment with CPPs (CPP-(125)I-Fab) derived from HIV-1 TAT, HIV-1 REV, and Antennapedia (ANP) were prepared. These vectors are rich in basic amino acids, and their strong adsorption on cell surfaces often results in overestimation of internalized peptides. Cell wash with an acidic buffer (0.2M glycine-0.15M NaCl, pH 3.0) was thus employed in this study to remove cell-surface adsorbed CPP-(125)I-Fab conjugates. This procedure enabled clearer understanding of the methods of internalization of CPP-(125)I-Fab conjugates. The kinetics of internalization of REV-(125)I-Fab conjugate was rapid, and a considerable fraction of REV-(125)I-Fab was taken up by HeLa cells as early as 5 min after administration. It was also shown that cellular uptake of these conjugates was significantly inhibited in the presence of endocytosis/ macropinocytosis inhibitors, in the order REV-(125)I-Fab > or = TAT-(125)I-Fab > or = ANP-(125)I-Fab; this order was the same as for effectiveness of intracellular delivery. Simultaneous cell washing with phosphate-buffered saline (PBS) and this acidic buffer effectively separated the internalized conjugates from the cell-surface-adsorbed ones, and considerable differences were observed in these amounts dependent on the employed CPPs.