AMPHIPHILIC CYCLIC CELL-PENETRATING PEPTIDES AS DRUG DELIVERY VEHICLES AND ANTIMICROBIAL PEPTIDES

Cell membrane is a barrier to be overcome for efficient delivery of therapeutics into a target site in cytoplasm or nucleus. The hydrophobic phospholipids are major components of the cell membrane that obstruct the transportation of therapeutics. Thus, various delivery systems, such as liposomes, nanoparticles and viral vectors, have been developed to transfer small molecules, peptides, proteins, and oligonucleotides across the membrane. Negatively charged phosphopeptides, oligonucleotides, and siRNAs have emerged as potential therapeutic agents. Phosphopeptides mimic phosphoproteins, which give on/off signal to many enzymes through interactions with protein kinases. For example, phosphopeptide pTyr-Glu-Glu-Ile (pYEEI) is an optimal peptide ligand for binding to the Src tyrosine kinase SH2 domain. Oligonucleotides have been introduced as antisense drugs to inhibit the translation of mRNA that transfers the coding information from genes. Small interfering RNA (siRNA)-based therapy has been also spotlighted since the discovery of RNA interference (RNAi) phenomenon. However, the cellular delivery of phosphopeptides, oligonucleotides, and siRNAs is a major obstacle despite many advantages of these compounds. Phosphopeptides contain negatively charged phosphate group, and/or negatively charged amino acids, such as glutamic acid or aspartic acid, in their sequences. Oligonucleotides and siRNAs are polymers composed of nucleosides, which are connected through negatively charged phosphodiester groups. These negatively charged molecules are hard to enter cancer cells by diffusion because cancer cell membranes are composed of negatively charged lipids. In addition, when a naked siRNA is administered in vivo, it does not show efficient cellular uptake in most mammalian cells and is quickly disappeared in the blood. Thus, developing carriers to improve the cellular uptake delivery of negatively charged cell-impermeable compounds has become a subject of major interest. Novel strategies are urgently needed to circumvent the problems associated with the delivery of these compounds. Cell-penetrating peptides (CPPs) have become one of the emerging vehicles for delivery of cargo drugs. CPPs are short hydrophilic or amphiphilic peptides that have plenty of positively charged amino acids, such as lysine or arginine, which can penetrate cell membranes. CPP-drug conjugates have been reported to help the cellular uptake of some drugs. Alternatively, they have been used as non-covalent drug delivery systems. CPPs have been investigated for improving the intracellular delivery of negatively-charged molecules. By physical interaction between positive charges in CPPs and negative charges in phosphopeptides, oligonucleotides, and siRNAs, the cell penetration could be improved. Among many CPPs, arginine-rich peptides have been the subject of major focus because it has been known that the guanidine group of arginine side chain shows better interaction with the negatively charged phospholipid in the cell membrane. Tryptophan is also a key amino acid found in CPPs that enhances the interaction of peptides with lipids in the cell membrane. Parang’s laboratory has previously shown that monocyclic CPPs containing alternative arginine and tryptophan have potential applications for drug delivery. Cyclic peptides have several benefits compared to linear peptides, such as stability against proteolytic enzymes and rigidness of structure. The rigidity of the structure can enhance the binding affinity of ligands toward receptors by reducing the freedom of possible structural conformations. Cyclic peptides are also present in nature and have been developed as therapeutics. Cyclosporin, gramicin S, polymoxin B, and daptomycin are well-known examples of cyclic peptides. Parang’s laboratory designed amphiphilic cyclic CPPs containing alternative tryptophan and arginine residues as the hydrophobic and positively charged residues, respectively. The peptides were efficient in improving the cellular delivery of anticancer and antiviral drugs. In this dissertation, we designed novel classes of amphiphilic cyclic peptides for improving the intracellular delivery of cell-impermeable phosphopeptides, and their antimicrobial activities were investigated. The hypothesis of this dissertation is that amphiphilic cyclic peptides, having positively charged arginines on one side of structures and hydrophobic tryptophan (or fatty acid) on the other side, can enhance intracellular drug delivery and/or act as antimicrobial agents having synergy with other antibiotics. In Manuscript I (Submitted to Angewandte Chemie International Edition), we designed amphiphilic bicyclic peptides as cellular delivery agents. The objective of this manuscript was to design a novel class of bicyclic CPPs containing two monocyclic peptides of tryptophan and arginine amino acids. Two bicyclic peptides [W5G]-(triazole)-[KR5] and [W5E]-(-Ala)-[KR5] were synthesized by conjugation of two monocyclic peptides using click chemistry and amide synthesis, respectively. A corresponding linear peptide, W5G-(triazole)-KR5, and a monocyclic peptide with a linear component, [W5G]-(triazole)-KR5, were synthesized as controls. Among all peptides, [W5E]-(-Ala)-[KR5] improved the cellular delivery of fluorescein-labeled phosphopeptide, F-GpYEEI by 19.3-fold. Confocal microscopy showed that the corresponding fluorescein-labeled bicyclic peptide F-[KW4E]-(-Ala)-[KR5] was localized in the cytosol and nucleus in human ovarian adenocarcinoma (SK-OV-3) cells. Studying the cellular uptake of F-[KW4E]-(-Ala)-[KR5] in the presence of endoycytosis inhibitors indicated that the clathrinand caveolin-dependent endocytosis were the main pathways for cellular uptake. [W5E]-(-Ala)-[KR5] enhanced the intracellular uptake of fluorescein-labeled phosphopeptide, F-GpYEEI by 4.5and 3.0-fold compared to those of well-known cell-penetrating peptides (CPPs), TAT and CR7, respectively. The bicyclic peptide was able to improve antiproliferative activity of doxorubicin by 20%. Thus, this manuscript suggests that amphiphilic bicyclic peptides containing tryptophan and arginine can be utilized as a new class of cell-penetrating peptides and potential cellular delivery tools. In Manuscript II (Submitted to Molecular Pharmaceutics), we investigated the role of fatty acylation and cyclization for intracellular transport of phophopepides in short-length polyarginine peptides Most of the reported arginine-rich CPPs to enhance intracellular drug delivery are linear peptides, and have more than seven arginines to retain cell penetrating properties. Herein, we synthesized penta and hexaarginine peptides (R5 and R6), and explored the effect of acylation and cyclization on the cell penetrating properties of the peptides. The fluorescence-labeled acylated cyclic peptide dodecanoyl-[R5] and linear peptide dodecanoyl-(R5) showed approximately 13.7and 10.2-fold higher cellular uptake than that of control 5(6)-carboxyfluorescein, respectively. The mechanism of the peptide internalization into cells was found to be energy-dependent endocytosis. The molecular transporter property of fatty acylated cyclic peptides was compared with those of fatty acylated linear peptide and nonacylated cyclic peptide using flow cytometry. The combination of acylation and cyclization (dodecanoyl-[R5]) enhanced intracellular delivery of a fluorescencelabeled phosphopeptide (F′-GpYEEI) in human SK-OV-3 cancer cell line. Dodecanoyl-[R5] and dodecanoyl-[R6] enhanced the intracellular uptake of a fluorescence-labeled cell impermeable negatively charged phosphopeptide (F′GpYEEI) in human ovarian cancer cells (SK-OV-3) by 3.4-fold and 5.5-fold, respectively. The cellular uptake of F′-GpYEEI in the presence of hexadecanoyl-[R5] was 9.3and 6.0-fold higher than that of in the presence of octanoyl-[R5] and dodecanoyl-[R5], respectively. A comparative FACS results showed that dodecanoyl[R5] enhanced the cellular uptake of the phosphopeptide by 1.4-2.5 fold higher than the corresponding linear peptide dodecanoyl-(R5) and those of representative CPPs, such as hepta-arginine (CR7) and TAT peptide. In this manuscript, we found that a combination of acylation by long chain fatty acids and cyclization on short argininecontaining peptides can improve their cell-penetrating property, possibly through efficient interaction of rigid positively charged R and hydrophobic dodecanoyl moiety with the corresponding residues in the cell membrane phospholipids. In Manuscript III (to be submitted to Molecular Pharmaceutics), the antimicrobial activities of cyclic CPPs were investigated against multidrug resistant pathogens. Antimicrobial peptides and CPPs share similar structural features. Based on the intracellular delivery property of amphiphilic cyclic peptides in manuscript II, we synthesized several amphiphilic cyclic CPPs and their analogs, and investigated antibacterial activities against multidrug resistant pathogens. [R4W4] exhibited a potent antibacterial activity, exhibiting MIC value of 2.67 μg/mL against methicillinresistant Staphylococcus aureus (MRSA). Cyclic [R4W4] and the linear counterpart R4W4 exhibited MIC values of 42.8 and 21.7 μg/mL, respectively, against Pseudomonas aeruginosa. [R4W4] in combination with tetracycline enhanced the potency, by decreasing the MIC 4 fold (0.12 μg/mL), suggesting partial synergistic effect of the combination between [R4W4] and tetracycline against MRSA. Twentyfour hour time-kill studies evaluating [R4W4] in combination with tetracycline demonstrated bactericidal activity against MRSA and E. coli. [R4W4] showed cellpenetrating properties as expected, and exhibited more than 84% cell viability at 15 μM (20.5 μg/mL) concentration against three different human cell lines. This study suggests that amphiphilic cyclic CPPs, when used in combination with antimicrobials could provide additional benefit to defeat multi-drug resistant pathogens. In summary, the studies in this dissertation provided insights and a deep understanding of applications of cyclic cell-penetrating peptides to enhance intracellular uptake of cargo drugs, and their antimicrobial activities as drug alone or combination with other antibiotics. Amphiphilic bicyclic peptides are the first reported bicyclic peptides as CPPs and molecular transporters. Acylated cyclic polyarginines showed that short polyarginines can be utilized as CPPs to have cell-penetrating properties by combining fatty acylation and cyclization. Moreover, this study provided a potential of amphiphilic cyclic CPPs as antimicrobial agents that their potency could be maximized by the combination with other antibiotics possibly through their drug delivery properties. Overall, these findings will be beneficial for the scientific community in academia and industry working in the area of designing molecular transporters of cell impermeable compounds, and cellular delivery.

Cell-penetrating peptides (CPPs) have been studied as molecular transporters because of their cellular translocation properties. [1] Covalent or non-covalent CPPdrug conjugates have been designed to deliver cell-impermeable drugs, such as negatively charged phosphopeptides, oligonucleotides, and siRNAs. [2] Various CPPs, such as TAT peptide, antennapedia, or polyarginines, have been used as molecular transporters for a wide range of molecular cargos. [3,4] Among CPPs, cyclic peptides take advantage of their higher serum stability compared to the linear counterparts. [5] In addition, the presence of positively charged arginine and hydrophobic tryptophan amino acids were found to be critical due to their characteristic interactions with phospholipid membranes. [6][7][8] We have previously reported the development of homochiral cyclic peptides containing arginine and tryptophan [WR] 4 and [WR] 5 as nuclear-targeting CPPs. [9] These monocyclic peptides were found to be effective tools as covalent and non-covalent molecular transporters. [10,11] Our findings showed that in addition to the cyclic nature, the sequence of the peptide contributes significantly to the molecular transporter property of the peptide by keeping a balance between both hydrophobic and positively charged properties.
To the best of our knowledge, bicyclic peptides have not been explored as CPPs and molecular transporters. We hypothesized that an amphiphilic bicyclic peptide containing two hydrophobic and positively charged cyclic peptides could act an alternative cellular delivery tool. Amphiphilic bicyclic peptides containing tryptophan and arginine amino acids and appropriate controls were synthesized and evaluated as phosphopeptide.
Two bicyclic peptides containing triazole and β-alanine linkers were synthesized to determine the effect of spacers in cellular delivery. First, a monocyclic peptide containing five arginine residues and one lysine azide [K(N 3 )R 5 ] and a cyclic peptide To determine the ability of these amphiphilic peptides as molecular transporters, we used a negatively charged fluorescein-labeled phosphopeptide, F-GpYEEI (F = fluorescein), as a model cell-impermeable compound, which is known as a substrate of Src kinase SH2 domain. [12,13] 5 ] was found to be 1.8-fold more in comparison to our previously reported monocyclic F-[W 5 R 4 K] peptide [9] at 5 M concentration. These data indicate that the bicyclic peptide has higher cellular uptake when compared to the corresponding monocyclic peptide. Thus, this new bicyclic peptide could be used as an alternative CPP with more efficiency compared to the monocyclic peptide.
To examine the cellular uptake mechanism of F-[KW 4 E]-(-Ala)-[KR 5 ], a temperature control assay was carried out at 4 °C to inhibit the energy-dependent endocytosis. The uptake of the bicyclic peptide was significantly reduced at 4 °C, indicating that the cellular uptake mechanism was dependent on the endocytosis ( Figure 3a). [14] To further confirm the energy-dependent cellular uptake of the bicyclic peptide, SK-OV-3 cells were incubated with sodium azide (10 mM) and 2-deoxy-Dglucose (50 mM) for 1 h before and 1 h after adding the bicyclic peptide to induce ATP depletion. The result showed that there was inhibition by ATP depletion ( Figure   3a), which is consistent with the result of the temperature control assay at 4 °C, suggesting that endocytosis is the major pathway for the cellular uptake of the bicyclic peptide as shown for other systems. [15] The cellular uptake studies were also conducted in the presence of several endocytosis inhibitors, such as chloroquine, chlorpromazine, methyl -cyclodextrin,  Figure 3b). Since chlorpromazine is a clathrin-dependent endocytosis inhibitor and nystatin blocks the lipid raft-caveolae endocytosis, [16,17] we suggest that the cellular uptake mechanism of F-[KW 4 E]-(-Ala)-[KR 5 ] is a clathrin-and caveolin-dependent endocytosis. These data indicate that the cellular uptake of the bicyclic peptide as a CPP follows a different pattern from that of cyclic peptide [WR] 5 , which showed endocytosis-independent cellular uptake. [9] Bicyclic  5 ] was found to be a more efficient molecular transporter for the negatively-charged phosphopeptide compared to commonly used CPPs and our previous reported cyclic CPP. [9] We have previously reported the binding affinity between monocyclic [WR] 5  In conclusion, we report synthesized bicyclic peptides,

Synthesis of monocyclic peptides.
The synthetic procedure is same as the linear peptide except the cleavage and additional cyclization step as described in the general procedure. As a representative example, the synthesis of [W 5 G(propargyl)] is described here (Scheme S1). The side-chain protected linear W 5 G(propargyl) was synthesized as described above. are depicted in Schemes S1-S4, respectively.

Synthesis of fluorescein-labeled bicyclic peptide (F-[KW 4 E]-(-Ala)-[KR 5 ]).
The  Then the cells were incubated with the premixed solution at 37 C with 5% CO 2 for 1 h. The sample preparation for FACS analysis was carried out with the same protocol described for cellular uptake for fluorescein-labeled bicyclic peptide. DMSO and F-GpYEEI were used as a negative control ( Figure S3).   (C 14 R 11 ) was found to be the most efficient cell-penetrating peptide. 7 However, the fatty acylated polyarginine peptides that contain 7-15 arginine residues can potentially cause toxicity, and they can be easily degraded by proteases.

Cellular uptake of doxorubicin (DOX
Moreover, linear peptides carrying L-form are not stable in serum and therefore have a limited application for in vivo studies. 9 Replacing L-form amino acids with Dform to improve the peptide stability leads to high cost production. On the other hand, cyclic peptides show more proteolytic stability than linear counterparts. Thus, the synthesis and development of cyclic CPPs containing short amino acid sequence with less toxicity is desired. Herein, we designed acylated cyclic polyarginine peptides (ACPPs) containing five arginine residues and investigated their ability as cell-penetrating peptides. We   showed higher fluorescence intensity compared to that of F′-dodecanoyl-(R 5 ) in SK-OV-3 cells. Therefore, ACPP F′-dodecanoyl- [R 5 ] was found to be more efficient cell-penetrating peptide compared to the linear counterpart. As shown in Figure 4, the fluorescence signal is extended through the whole cells, suggesting that F′dodecanoyl-[R 5 ] can get localized in the nucleus as well as cytoplasm.

Cellular Uptake Mechanistic Study of F′-Dodecanoyl-[R 5 ]
The mechanism of the cellular internalization of F′-dodecanoyl-[R 5 ] was investigated by a temperature control assay at 4 °C along with ATP depletion assay.
These two assays have been widely used to examine the energy-dependent endocytosis. 11 FACS results showed that the intracellular uptake of F′-dodecanoyl- [R 5 ] was significantly reduced at 4 °C, indicating that the mechanism of internalization was mainly dependent on the endocytosis pathways ( Figure 5). 12 Furthermore, ATP depletion assay was performed to investigate receptor-mediated endocytosis. 13  In this study, the intracellular uptake of F′-GpYEEI was monitored in the presence and absence of synthetic peptides after 1 h incubation by flow cytometry. As it is exhibited in Figure 6 arginine. 8 However, we discovered that both cyclization and acylation in a short pentaarginine can significantly improve the delivery of a cell-impermeable phosphopeptide in SK-OV-3 cells.
The major driving forces for the intracellular delivery are presumed to be structural rigidity through cyclization of the peptide and the interaction of the fatty acid with the cell membrane. It has been previously reported that the cellular uptake of the peptide can be increased due to the structural rigidity by cyclization of argininerich peptides. 3 They proposed that the maximal distance between guanidine groups of arginine residue can lead to an efficient transduction of arginine-rich peptides. Our investigations showed that dodecanoyl-[R 6 ] is able to deliver more efficiently by 1.6fold higher F′-GpYEEI uptake compared to that of dodecanoyl- [R 5 ]. Increasing the number of positively charged arginine residues can enhance the cellular uptake through ionic interactions with the negatively charged phosphopeptide and/or phospholipid in the cell membrane through ionic interactions. However, the higher number of arginine residue is not the only responsible element for the efficient cellular internalization. For example, it has been reported that polyarginine containing eleven amino acids (R 11 ) showed higher cellular uptake compared to the polyarginine containing thirteen amino acids (R 13 ). At the same time, R 11 was found to be more potent transporter compared to R 9 in prostate cancer cells. 19 These investigations showed that an optimal number of arginine residues are required for the highest degree of functionality. However, the more number of amino acid residues in cyclic peptides can decrease the structural rigidity, which lower the ability of the peptide to get into cells.
Dodecanoyl-[R 5 ] was also compared with several commonly CPPs, such as CR 7 and TAT (YGRKKRRQRRR) peptides. The ACPP improved the cellular uptake of the phosphopeptide by 1.4-and 1.8-fold higher than those of CR 7 and TAT, respectively ( Figure 6B). Thus, these results revealed that ACPP dodecanoyl-[R 5 ] can be used as an efficient molecular transporter even it has shorter peptide sequence than CR 7 and TAT.

The Effect of Fatty Acid Chain Length on the Cellular Uptake of ACPPs
To investigate the effect of the chain length on the cell penetration potency, we

Effect of Addition of Tryptophan Residues in Molecular Transporter Property
After ACPPs were found to act as CPP and molecular transporter, a systematic investigation was performed to modify the fatty acid to another hydrophobic moiety.
Two other conjugates were synthesized. In the first conjugate,            Moreover, MRSA rapidly evolves resistance against new commercial antibiotics.
Currently, vancomycin is employed commonly for the treatment of MRSA.
However, vancomycin-resistant Staphylococcus aureus was reported in 1996. 5 Daptomycin is a cyclic lipopeptide having a broad spectrum against Gram-positive bacteria, and it shows fast antibacterial responses. Its novel mechanism of action involves membrane depolarization resulting in efflux of potassium ions, followed by bacterial cell death. 6 Despite its novelty of the mechanism, daptomycin-resistance by MRSA was reported in 2005, only two years after FDA approval. The resistance mechanism against daptomycin remains to be determined. 7 Thus, new classes of antibiotics with different mechanisms of action are urgently needed.
Antimicrobial peptides (AMPs) have emerged as alternative therapeutics against antibiotic resistant pathogens because they can act as effectors and regulators of the immune system as well as inhibitors of bacterial cell growth. 8 Cationic AMPs target negatively charged bacterial membrane lipids, which may reduce the occurrence of bacterial resistance. 9 AMPs have been found as host defense peptides in various organisms, such as insects, amphibians, and mammalians. 10,11 AMPs such as magainin and omiganan are in clinical trials or in development. 12 Cell-penetrating peptides (CPPs) are short hydrophilic and/or amphiphilic peptides. Because of their ability to translocate across the eukaryotic cell membrane, they have been studied as molecular vehicles to deliver other drugs inracellularly. 13,14 Some AMPs and CPPs share similar physical properties, such as amphiphilicity and cationic properties. Thus, CPPs have potential application as AMPs with dual actions as both antibiotics and possible molecular transporter properties.
We have synthesized and evaluated several cyclic CPPs as molecular transporters of other cargo drugs. For example, we recently reported that synthetic cyclic peptides [WR] 4 and [WR] 5 enhanced the cellular uptake of phosphopeptides, doxorubicin, and anti-HIV drugs. 15 These peptides are expected to be more stable than linear peptides towards human serum because the cyclization decreases proteolytic degradation. 16 It has been previously reported that the rigidity in the peptides can enhance the cellpenetrating property. 17 According to our recent study, the acylation and cyclization of short polyarginine peptides enhance the intracellular delivery of cell-impermeable phosphopeptides.
In general, AMPs contain hydrophobic and hydrophilic portions that interact with the lipid part and hydrophilic negatively charged heads in bacteria membranes, respectively. Many linear AMPs adopt amphipathic α-helix conformations with the hydrophobic side chains arranged along one side of the helical structure and the hydrophilic side chains organized on the opposite side. This arrangement results in the ideal amphipathic helical structures. 18 Some AMPs form amphipathic -sheet conformation to interact with cell membranes. 18   where the limit of detection was 2.0 log 10 CFU/mL. 21 Bactericidal activity (99.9% kill) was defined as a ≥3 log 10 CFU/mL reduction at 24 h in colony count from the initial inoculum. Bacteriostatic activity was defined as a <3 log 10 CFU/mL reduction.

Chemistry
All fourteen peptides 1-14 (Table 1) were synthesized by Fmoc/tBu solid-phase peptide synthesis as described above. Peptides 1-6 were synthesized for this study and showed promising results as an antimicrobial peptide against MRSA. Thus, it was selected for further synergistic studies.

Combination effect of peptides and tetracycline against MRSA
Based on antibacterial activity screening, peptides 1, 9, 10, and 13 were selected to determine whether they could increase the antibacterial activity when co-incubated with tetracycline. Non-acylated cyclic peptide 7 was used as a control since it showed the lowest potency and was structurally different from other cyclic peptides. Peptide 1 in combination with tetracycline showed the most potent antibacterial activity with a MIC value of 0.12 µg/mL against MRSA (Table 3) with FIC > 4. 23,26,27 According to this definition, the combination of peptide 1 and tetracycline can be defined as partial synergy (Table 3).

Time-kill Studies against MRSA and E. coli
Time-kill studies were conducted to evaluate the antimicrobial activity of peptide 1 alone and in combination with tetracycline against MRSA and E. coli over 24 h. More studies are required to determine the mechanism of synergism. One assumption is that amphiphilic peptide 1 acts as cell-penetrating peptide and can deliver tetracycline at higher concentration. The fluorescence studies using peptide 1 and tetracycline were challenging because emission and excitation range for tetracycline were outside the range determined by the flow cytometer.

Cytotoxicity Assay of Peptide 1
The cytotoxicity of peptide 1 was evaluated by MTS proliferation assay against human ovarian adenocarcinoma SK-OV-3, human leukemia CCRF-CEM, and human embryonic kidney HEK 293T cell lines. In both cancer and normal cell lines, the compound showed more than 84% cell viability at 15 µM (20.5 µg/mL) concentration ( Figure 3). Peptide 1 alone showed 2.67 µg/mL MIC against MRSA that is significantly lower than the cytotoxic concentration of peptide 1. Furthermore, the therapeutic index peptide 1 was increased when was combined with tetracycline showing a MIC value of 0.12 µg/mL, exhibiting partial synergistic effect. Therefore, the combination of peptide 1 with tetracycline can be used to enhance the therapeutic index.

Cell-Penetrating Property of Peptide 1 ([R 4 W 4 ])
Peptide 1 was originally designed as a CPP to have cell-penetrating property. was added to SK-OV-3 cells and incubated for 1 h at 37 C. Confocal laser scanning microscope (CLSM) images showed that the fluorescein-labeled peptide was dispersed into the nucleus and cytosol (Figure 4), but no significant fluorescence was observed in the cells treated with fluorescein alone under the similar condition (data not shown).
The mechanism of cellular uptake was investigated by a temperature control assay at 4 °C and ATP depletion assay. The cellular uptake of peptide 1 was decreased about 59% at 4 °C, indicating that the cellular uptake occurred by both endocytotic and nonendocytotic pathways ( Figure 5). ATP depletion assay also supports this mixed pathways because there was 80% intracellular transportation even though all energy source was blocked by sodium azide and 2-deoxy-D-glucose. These results were consistent with the previous studies that indicated that intracellular transportation can be controlled by several mixed pathways. 28