Multiscale Functional Metal Architectures by Antibody-Guided Metallization of Specific Protein Assemblies in Ex Vivo Multicellular Organisms.

Biological systems consist of hierarchical protein structures, each of which has unique 3D geometries optimized for specific functions. In the past decades, the growth of inorganic materials on specific proteins has attracted considerable attention. However, the use of specific proteins as templates has only been demonstrated in relatively simple organisms, such as viruses, limiting the range of structures that can be used as scaffolds. This study proposes a method for synthesizing metallic structures that resemble the 3D assemblies of specific proteins in mammalian cells and animal tissues. Using 1.4 nm nanogold-conjugated antibodies, specific proteins within cells and ex vivo tissues are labeled, and then the nanogold acts as nucleation sites for growth of metal particles. As proof of concept, various metal particles are grown using microtubules in cells as templates. The metal-containing cells are applied as catalysts and show catalytic stability in liquid-phase reactions due to the rigid support provided by the microtubules. Finally, this method is used to produce metal structures that replicate the specific protein assemblies of neurons in the mouse brain or the extracellular matrices in the mouse kidney and heart. This new biotemplating approach can facilitate the conversion of specific protein structures into metallic forms in ex vivo multicellular organisms.

Artificial Taste Buds: Bioorthogonally Ligated Gustatory-Neuronal Multicellular Hybrids Enabling Intercellular Taste Signal Transmission.

Heterogeneous tissue models require the assembly and co-culture of multiple types of cells. Our recent work demonstrated taste signal transmission from gustatory cells to neurons by grafting single-stranded DNA into the cell membrane to construct multicellular assemblies. However, the weak DNA linkage and low grafting density allowed the formation of large gustatory cell self-aggregates that cannot communicate with neurons efficiently. This article presents the construction of artificial taste buds exhibiting active intercellular taste signal transmission through the hybridization of gustatory-neuronal multicellular interfaces using bioorthogonal click chemistry. Hybrid cell clusters were formed by the self-assembly of neonatal gustatory cells displaying tetrazine with a precultured embryonic hippocampal neuronal network displaying trans-cyclooctene. A bitter taste signal transduction was provoked in gustatory cells using denatonium benzoate and transmitted to neurons as monitored by intracellular calcium ion sensing. In the multicellular hybrids, the average number of signal transmissions was five to six peaks per cell, and the signal transmission lasted for ∼5 min with a signal-to-signal gap time of 10-40 s. The frequent and extended intercellular signal transmission suggests that the cell surface modification by the bioorthogonal click chemistry is a promising approach to fabricating functional multicellular hybrid clusters potentially useful for cell-based biosensors, toxicity assays, and tissue regeneration.

Protein-induced metamorphosis of unilamellar lipid vesicles to multilamellar hybrid vesicles.

Protein encapsulation into nanocarriers has been extensively studied to improve the efficacy and stability of therapeutic proteins. However, the chemical modification of proteins or new synthetic carrier materials are essential to achieve a high encapsulation efficiency and structural stability of proteins, which hinders their clinical applications. New strategies to physically incorporate proteins into nanocarriers feasible for clinical uses are required to overcome the current limitation. Here we report the spontaneous protein-induced reorganization of 'pre-formed' unilamellar lipid vesicles to efficiently incorporate proteins within multilamellar protein-lipid hybrid vesicles without chemical modification. Epidermal growth factor (EGF) binds to the surface of cationic unilamellar lipid vesicles and induces layer-by-layer self-assembly of the vesicles. The protein is spontaneously entrapped in the interstitial layers of a multilamellar structure with extremely high loading efficiency, ~99%, through polyionic interactions as predicted by molecular dynamics simulation. The loaded protein exhibits much higher structural, chemical, and biological stability compared to free protein. The method is also successfully applied to several other proteins. This work provides a promising method for the highly efficient encapsulation of therapeutic proteins into multilamellar lipid vesicles without the use of specialized instruments, high energy, coupling agents, or organic solvents.

Development of microRNA-21 mimic nanocarriers for the treatment of cutaneous wounds.

Rationale: Of the regulatory microRNAs expressed in the wounded skin, microRNA-21 (miR21) plays a pivotal role in wound repair by stimulating re-epithelialization, an essential feature to facilitate healing and reduce scar formation. Despite their crucial roles in wound healing, synthetic exogenous microRNAs have limited applications owing to the lack of an appropriate delivery system. Herein, we designed an miR21 mimic nanocarrier system using facial amphipathic bile acid-conjugated polyethyleneimines (BA-PEI) for the intracellular and transdermal delivery of synthetic miR21 molecules to accelerate wound repair. Methods: To design miR21 mimic nanocarriers, BA-conjugated PEIs prepared from three different types of BA at molar feed ratios of 1 and 3 were synthesized. The intracellular uptake efficiency of synthetic miR21 mimics was studied using confocal laser scanning microscopy and flow cytometry analysis. The optimized miR21/BA nanocarrier system was used to evaluate the wound healing effects induced by miR21 mimics in human HaCaT keratinocytes in vitro and a murine excisional acute wound model in vivo. Results: The cell uptake efficiency of miR21 complexed with BA-conjugated PEI was dramatically higher than that of miR21 complexed with PEI alone. Deoxycholic acid (DA)-modified PEI at a molar feed ratio of 3:1 (DA3-PEI) showed the highest transfection efficiency for miR21 without any increase in toxicity. After effective transdermal and intracellular delivery of miR21/DA3 nanocarriers, miR21 mimics promoted cell migration and proliferation through the post-transcriptional regulation of programmed cell death protein 4 (PDCD4) and matrix metalloproteinases. Thus, miR21 mimic nanocarriers improved both the rate and quality of wound healing, as evident from enhanced collagen synthesis and accelerated wound re-epithelialization. Conclusion: Our miRNA nanocarrier systems developed using DA3-PEI conjugates may be potentially useful for the delivery of synthetic exogenous miRNAs in various fields.

Development of Biocompatible HA Hydrogels Embedded with a New Synthetic Peptide Promoting Cellular Migration for Advanced Wound Care Management.

In the past few years, there have been many efforts underway to develop effective wound healing treatments for traumatic injuries. In particular, wound-healing peptides (WHPs) and peptide-grafted dressings hold great promise for novel therapeutic strategies for wound management. This study reports a topical formulation of a new synthetic WHP (REGRT, REG) embedded in a hyaluronic acid (HA)-based hydrogel dressing for the enhancement of acute excisional wound repair. The copper-free click chemistry is utilized to form biocompatible HA hydrogels by cross-linking dibenzocyclooctyl-functionalized HA with 4-arm poly(ethylene glycol) (PEG) azide. The HA hydrogels are grafted with the REG peptide, a functional derivative of erythroid differentiation regulator1, displaying potent cell motility-stimulating ability, thus sustainably releasing physiologically active peptides for a prolonged period. Combined with the traditional wound healing benefits of HA, the HA hydrogel embedded REG (REG-HAgel) accelerates re-epithelialization in skin wound healing, particularly by promoting migration of fibroblasts, keratinocytes, and endothelial cells. REG-HAgels improve not only rate, but quality of wound healing with higher collagen deposition and more microvascular formation while being nontoxic. The peptide-grafted HA hydrogel system can be considered as a promising new wound dressing formulation strategy for the treatment of different types of wounds with combinations of various natural and synthetic WHPs.

Laser-Induced CO2 Generation from Gold Nanorod-Containing Poly(propylene carbonate)-Based Block Polymer Micelles for Ultrasound Contrast Enhancement.

Poly(propylene carbonate) (PPC) decomposes at high temperature to release CO2. This CO2-generation temperature of PPC can be reduced down to less than 80 °C with the aid of a photoacid generator (PAG). In the present work, we demonstrate that using an additional helper component, surface plasmonic gold nanorods (GNRs), the PPC degradation reaction can also be initiated by infrared (IR) irradiation. For this purpose, a PPC-containing nanoparticle formulation was developed in which PPC-based amphiphilic block copolymers (BCPs), poly(poly(ethylene glycol) methacrylate- b-propylene carbonate- b-poly(ethylene glycol) methacrylate) (PPEGMA-PPC-PPEGMA), were self-assembled with GNRs and PAG molecules via solvent exchange. Under IR irradiation, GNRs produce heat that can cause PPC to decompose into CO2, and PAG (after UV pretreatment) catalyzes this PPC degradation process. Two PPEGMA-PPC-PPEGMA materials were used for this study: PPEGMA7.3K-PPC5.6K-PPEGMA7.3K ("G7C6G7") and PPEGMA2.1K-PPC5.6K-PPEGMA2.1K ("G2C6G2"). Addition of CTAB-coated GNRs dispersed in water to a G2C6G2 solution in DMF produced individually G2C6G2-encapsulated GNRs, whereas the same solvent exchange procedure resulted in the formation of polymer-coated GNR clusters when G7C6G7 was used as the encapsulating material. GNR/G2C6G2 NPs exhibited a surface plasmon resonance peak at 697 nm. The clustered morphology of G7C6G7-encapsulated GNRs caused a blue shift of the absorbance maximum to 511 nm. As a consequence, GNR/G2C6G2 NPs showed a greater absorbance/heat generation rate under IR irradiation than did GNR/G7C6G7 NPs. The IR-induced CO2 generation rate was about 4.2 times higher with the GNR/G2C6G2+PAG sample than that with the GNR/G7C6G7+PAG sample. Both GNR/G7C6G7+PAG and GNR/G2C6G2+PAG systems produced ultrasound contrast enhancement effects under continuous exposure to IR light for >20 min; contrast enhancement was more spatially uniform for the GNR/G2C6G2+PAG sample. These results support the potential utility of PPC as a CO2-generating contrast agent in ultrasound imaging applications.

PEG-PLA-Coated and Uncoated Radio-Luminescent CaWO4 Micro- and Nanoparticles for Concomitant Radiation and UV-A/Radio-Enhancement Cancer Treatments.

Currently, there is great interest in the development of ways to achieve the benefits of radiation treatments with reduced negative effects. The present study demonstrates the utilization of radio-luminescent particles (RLPs) as a means to achieve radio-sensitization and enhancement and their ability to affect head- and neck-cancer-cell cultures (in vitro) and xenografts (in vivo). Our approach utilizes a naturally abundant radio-luminescent mineral, calcium tungstate (CaWO4), in its micro or nanoparticulate form for generating secondary UV-A light by γ ray or X-ray photons. In vitro tests demonstrate that unoptimized RLP materials (uncoated CaWO4 (CWO) microparticles (MPs) and PEG-PLA-coated CWO nanoparticles (NPs)) induce a significant enhancement of the tumor-suppressive effect of X-rays and γ rays in both radio-sensitive- and radio-resistant-cancer models; uncoated CWO MPs and PEG-PLA-coated CWO NPs demonstrate comparable radio-sensitization efficacies in vitro. Mechanistic studies reveal that concomitant CaWO4 causes increased mitotic death in radio-resistant cells treated with radiation, whereas CaWO4 sensitizes radio-sensitive cells to X-ray-induced apoptosis and necrosis. The radio-sensitization efficacy of intratumorally injected CaWO4 particles (uncoated CWO MPs and PEG-PLA-coated CWO NPs) is also evaluated in vivo in mouse head- and neck-cancer xenografts. Uncoated CWO MPs suppress tumor growth more effectively than PEG-PLA-coated CWO NPs. On the basis of theoretical considerations, an argument is proposed that uncoated CWO MPs release subtoxic levels of tungstate ions, which cause increased photoelectric-electron-emission effects. The effect of folic acid functionalization on the in vitro radio-sensitization behavior produced by PEG-PLA-coated CWO NPs is studied. Surface folic acid results in a significant improvement in the radio-sensitization efficiency of CaWO4.

Near-Infrared Plasmonic Assemblies of Gold Nanoparticles with Multimodal Function for Targeted Cancer Theragnosis.

Here we report a novel assembly structure of near-infrared plasmonic gold nanoparticles (AuNPs), possessing both photoacoustic (PA) and photothermal (PT) properties. The template for the plasmonic AuNP assembly is a bioconjugate between short double-strand DNA (sh-dsDNA) and human methyl binding domain protein 1 (MBD1). MBD1 binds to methylated cytosine-guanine dinucleotides (mCGs) within the sequence of sh-dsDNA. Hexahistidine peptides on the engineered MBD1 function as a nucleation site for AuNP synthesis, allowing the construction of hybrid conjugates, sh-dsDNA-MBD1-AuNPs (named DMAs). By varying the length of sh-dsDNA backbone and the spacer between two adjacent mCGs, we synthesized three different DMAs (DMA_5mCG, DMA_9mCG, and DMA_21mCG), among which DMA_21mCG exhibited a comparable photothermal and surprisingly a higher photoacoustic signals, compared to a plasmonic gold nanorod. Further, epidermal growth factor receptor I (EGFR)-binding peptides are genetically attached to the MBD1 of DMA_21mCG, enabling its efficient endocytosis into EGFR-overexpressing cancer cells. Notably, the denaturation of MBD1 disassembled the DMA and accordingly released the individual small AuNPs (<5 nm) that can be easily cleared from the body through renal excretion without causing accumulation/toxicity problems. This DMA-based novel approach offers a promising platform for targeted cancer theragnosis based on simultaneous PA imaging and PT therapy.

Synergistic antitumor effects of combination treatment with metronomic doxorubicin and VEGF-targeting RNAi nanoparticles.

Conventional cancer treatment strategies have been aimed at eradicating all cancer cells. To this end, standard chemotherapeutic approaches have relied on the maximum tolerated dose (MTD) of cytotoxic drugs with a long off-therapy interval, leading to heavy toxic side effects accompanied by drug resistance. To avoid the problems associated with the traditional MTD chemotherapy, metronomic chemotherapy with relatively low dose continuous treatments of cytotoxic drugs has been proposed as an alternative to the predominant paradigm of directly killing all cancer cells. Low-dose metronomic (LDM) chemotherapy is expected to have not only antitumor effects without toxicity and drug resistance, but also beneficial anti-angiogenic effects by causing selective apoptosis of tumor endothelial cells. In an attempt to keep the drug resistance under control and halt exponential tumor growth, herein, we combined LDM chemotherapy with a second anti-angiogenic strategy. The selective blockade of vascular endothelial growth factor (VEGF) in combination with metronomic doxorubicin (Dox) induced synergistic antitumor effects mainly through an antiangiogenic mechanism. For specific VEGF suppression, VEGF-targeting siRNA was delivered to tumor tissue using polymerized siRNA/thiolated glycol chitosan (poly-siVEGF/tGC) nanoparticles, leading to efficient VEGF gene knockdown in tumor tissue with a sequence-specific manner. Although the single treatment with metronomic Dox and poly-siVEGF/tGC nanoparticles alone showed some antitumor activity, notably, the combination of the two therapies resulted in superb tumor regression without causing systemic toxicity or drug resistance. Thus, these results suggest that the VEGF-targeted RNAi using poly-siRNA/tGC nanoparticles in combination with LDM chemotherapy could be a promising synergistic strategy for controlling tumor growth by enhancing the efficacy of anti-angiogenesis while minimizing toxicity and drug resistance.

Genetic Assembly of Double-Layered Fluorescent Protein Nanoparticles for Cancer Targeting and Imaging.

Hepatitis B virus capsid (HBVC), a self-assembled protein nanoparticle comprised of 180 or 240 subunit proteins, is used as a cage for genetic encapsulation of fluorescent proteins (FPs). The self-quenching of FPs is controlled by varying the spacing between FPs within the capsid structure. Double-layered FP nanoparticle possessing cancer cell-targeting capabilities is also produced by additionally attaching FPs and cancer cell receptor-binding peptides (affibodies) to the outer surface of the capsid. The generically modified HBVC with double layers of mCardinal FPs and affibodies (mC-DL-HBVC) exhibit a high fluorescence intensity and a strong photostability, and is efficiently internalized by cancer cells and significantly stable against intracellular degradation. The mC-DL-HBVC effectively detects tumor in live mice with enhanced tumor targeting and imaging efficiency with far less accumulation in the liver, compared to a conventional fluorescent dye, Cy5.5. This suggests the great potential of mC-DL-HBVC as a promising contrast agent for in vivo tumor fluorescence imaging.

Synergistic anti-tumor effects of bevacizumab and tumor targeted polymerized VEGF siRNA nanoparticles.

A variety of VEGF inhibitors have been reported to treat cancers by suppressing tumor angiogenesis. Bevacizumab, a monoclonal VEGF antibody, was the first FDA approved anti-angiogenic agent for cancer treatments. However, bevacizumab shows modest therapeutic efficiency and often cause resistant problem in significant populations of cancer patients. To solve these problem, we investigated the therapeutic efficacy of siRNA drugs targeting VEGF and combination of the RNAi drug with bevacizumab for cancer treatments. For efficient VEGF siRNA delivery, chemically polymerized siRNAs were complexed with thiolated-glycol chitosan (psi(VEGF)/tGC). The poly-VEGF siRNA and thiolated-glycol chitosan formed stable nanoparticles via electrostatic interaction and chemical crosslinking, and showed high accumulation in tumor tissues resulting in efficient gene silencing. Both VEGF siRNA nanoparticles and bevacizumab had efficient therapeutic effects in tumor xenograft mouse models. Interestingly, most pronounced therapeutic efficacy was observed when the two distinct VEGF inhibitors were treated in combination revealing synergistic effects. The results showed that the psi(VEGF)/tGC nanoparticle mediated knockdown of VEGF exerts anti-tumor effects and the combination treatments with bevacizumab can extend the treatments options to conventional bevacizumab treatments for cancer therapy.

Targeted Nanotheranostics for Future Personalized Medicine: Recent Progress in Cancer Therapy.

Recently, many theranostic nanomaterials have been developed by integrating therapeutic and diagnostic agents in a single regimen. Real-time visualization of nano drug carrier biodistributions, drug release processes and therapeutic responses can provide critical information needed for dynamically optimizing treatment operations in a personalized manner in real time. This review highlights recent progresses in the development of multifunctional nanoparticles possessing both therapeutic and imaging functionalities for cancer therapy. The advantages of using nanoparticle platforms are discussed. Examples demonstrating various combinations of imaging and therapeutic modalities are highlighted.

Chemical and structural modifications of RNAi therapeutics.

Small interfering RNA (siRNA), a 21-23nt double-stranded RNA responsible for post-transcriptional gene silencing, has attracted great interests as promising genomic drugs, due to its strong ability to silence target genes in a sequence-specific manner. Despite high silencing efficiency and on-target specificity, the clinical translation of siRNA has been hindered by its inherent features: poor intracellular delivery, limited blood stability, unpredictable immune responses and unwanted off-targeting effects. To overcome these hindrances, researchers have made various advances to modify siRNA itself and to improve its delivery. In this review paper, first we briefly discuss the innate properties and delivery barriers of siRNA. Then, we describe recent progress in (1) chemically and structurally modified siRNAs to solve their intrinsic problems and (2) siRNA delivery formulations including siRNA conjugates, polymerized siRNA, and nucleic acid-based nanoparticles to improve in vivo delivery.

Chitosan-g-hematin: enzyme-mimicking polymeric catalyst for adhesive hydrogels.

Phenol derivative-containing adhesive hydrogels has been widely recognized as having potential for biomedical applications, but their conventional production methods, utilizing a moderate/strong base, alkaline buffers, the addition of oxidizing agents or the use of enzymes, require alternative approaches to improve their biocompatibility. In this study, we report a polymeric, enzyme-mimetic biocatalyst, hematin-grafted chitosan (chitosan-g-hem), which results in effective gelation without the use of alkaline buffers or enzymes. Furthermore, gelation occurs under mild physiological conditions. Chitosan-g-hem biocatalyst (0.01%, w/v) has excellent catalytic properties, forming chitosan-catechol hydrogels rapidly (within 5 min). In vivo adhesive force measurement demonstrated that the hydrogel formed by the chitosan-g-hem activity showed an increase in adhesion force (33.6 ± 5.9 kPa) compared with the same hydrogel formed by pH-induced catechol oxidation (20.6 ± 5.5 kPa) in mouse subcutaneous tissue. Using the chitosan-g-hem biocatalyst, other catechol-functionalized polymers (hyaluronic acid-catechol and poly(vinyl alcohol)-catechol) also formed hydrogels, indicating that chitosan-g-hem can be used as a general polymeric catalyst for preparing catechol-containing hydrogels.

Small interfering RNA nunchucks with a hydrophobic linker for efficient intracellular delivery.

The high stiffness and low spatial charge density of siRNA limit the effectiveness of the electrostatic condensation of siRNA with cationic polyelectrolytes. Here, a facile method to stabilize nanoscale siRNA/polyelectrolyte complexes by introducing a reductively cleavable alkyl chain to siRNA as a hybrophobic linker of dimeric siRNA conjugates is reported. The increased length of the hydrophobic linker increases the intracellular translocation and gene silencing activity of the dimeric siRNA conjugates when they are complexed with linear polyethylenimine (LPEI). The results suggest that the introduction of a hydrophobic linker in the dimeric siRNA conjugates can facilitate the intracellular delivery of siRNA through effective condensation with cationic polyelectrolytes.

Cationic lipid-coated gold nanoparticles as efficient and non-cytotoxic intracellular siRNA delivery vehicles.

PURPOSE: Cationic lipid-coated gold nanoparticles were developed for efficient intracellular delivery of therapeutic siRNA. METHODS: Particle formation was characterized by UV-visible spectroscopy, atomic force microscopy, and dynamic light scattering analysis. Cellular uptake, gene silencing effect, and cytotoxicity were investigated in multiple human cancer cell lines. RESULTS: Nanoparticles had a spherical nanostructure with highly cationic surface charge and could form stable nanosized polyelectrolyte complexes with siRNA via electrostatic interactions; complexes exhibited efficient intracellular uptake and significant gene silencing effect with markedly low cytotoxicity compared to the widely used polycationic carrier, linear polyethyleneimine. CONCLUSIONS: We demonstrated that cationic lipid-coated gold nanoparticles could be widely utilized as efficient and safe siRNA nanocarriers for diverse therapeutic and diagnostic applications.

Reducible siRNA dimeric conjugates for efficient cellular uptake and gene silencing.

In this study, dimerized siRNAs linked by a cleavable disulfide bond were synthesized for efficient intracellular delivery and gene silencing. The reducible dimerized siRNAs showed far enhanced complexation behaviors with cationic polymers as compared to monomeric siRNA at the same N/P ratio, as demonstrated by microscopic techniques and gel characterization. Dimerized siRNAs targeting green fluorescent protein (GFP) or vascular endothelial growth factor (VEGF) were complexed with linear polyethylenimine (LPEI), and treated to various cell lines to examine gene transfection efficiencies. In comparison to monomer siRNA/LPEI complexes, dimeric siRNA/LPEI complexes showed greatly enhanced cellular uptake and gene silencing effects in vitro. These results were mainly due to the higher charge density and promoted chain flexibility of the dimerized siRNAs, providing more compact and stable siRNA complexes. In addition, the conjugation strategy of reducible siRNA dimers was further extended: poly(ethylene glycol) (PEG)-modified dimerized siRNAs and heterodimers of siRNAs targeting two different genes (e.g., GFP and VEGF) were synthesized, and their gene silencing efficiencies were characterized. The dimerized siRNA complex system holds great potential for in vivo systemic gene therapy.

L-leucine increases [3H]-thymidine incorporation in chicken hepatocytes: involvement of the PKC, PI3K/Akt, ERK1/2, and mTOR signaling pathways.

This study examined how L-leucine affected DNA synthesis and cell cycle regulatory protein expression in cultured primary chicken hepatocytes. L-Leucine promoted DNA synthesis in a dose- and time-dependent manner, with concomitant increases in cyclin D1 and cyclin E expression. Phospholipase C (PLC) and protein kinase C (PKC) mediated the L-leucine-induced increases in [3H]-thymidine incorporation and cyclin D1/CDK4 and cyclin E/CDK2 expression, as U73122 (a PLC inhibitor) or bisindolylmaleimide I (a PKC blocker) inhibited these effects. L-Leucine also increased PKC phosphorylation and intracellular Ca2+ levels. L-Leucine-mediated increases in [3H]-thymidine incorporation and cyclin/CDK expression were sensitive to LY 294002 (PI3K inhibitor), Akt inhibitor, PD 98059 (MEK inhibitor). It was also observed that L-leucine-induced increases of cyclin/CDK expression were inhibited by PI3K siRNA and ERK siRNA; L-leucine increased extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt phosphorylation levels. Bisindolylmaleimide I attenuated L-leucine-induced phosphorylation of ERK1/2 but did not influence Akt phosphorylation, and PI3K siRNA and LY 294002 inhibited L-leucine-induced ERK1/2 phosphorylation, suggesting some cross-talk between the PKC and ERK1/2 or PI3K/Akt and ERK1/2 pathways. L-Leucine also increased the levels of phosphorylated molecular target of rapamycin (mTOR) and two of its targets, ribosomal protein S6 kinase (p70S6K), and 4E binding protein 1 (4E-BP1); furthermore, rapamycin (an mTOR inhibitor) blocked all of the mitogenic effects of L-leucine. In addition, Akt inhibitor blocked L-leucine-induced mTOR phosphorylation. In conclusion, L-leucine stimulated DNA synthesis and promoted cell cycle progression in primary cultured chicken hepatocytes through PKC, ERK1/2, PI3K/Akt, and mTOR.