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.     

Peptide-derivatized shell-cross-linked nanoparticles. 1. Synthesis and characterization

The conjugation of the protein transduction domain (PTD) from the HIV-1 Tat protein to shell-cross-linked (SCK) nanoparticles is reported as a method to facilitate cell surface binding and transduction of SCK nanoparticles. Attaching increasing numbers of peptide sequences to SCK nanoparticles in a global solution-state functionalization strategy has been devised as a method for increasing the efficiency of the cell-penetrating process. The numbers of peptides per SCK were controlled through stoichiometric balance and measured experimentally by two independent methods, UV-visible spectroscopy and phenylglyoxal analysis. PTD was conjugated in (0.005, 0.01, and 0.02) molar ratios, relative to the acrylic acid residues in the shell, to the SCK nanoparticles resulting in SCK populations possessing nominally 52, 104, and 210 (41, 83, and 202 as measured by phenylglyoxal analysis) PTD peptides per particle, respectively. The methodologies for the block copolymer and nanoparticle syntheses, peptide derivatization, and characterization of peptide-functionalized SCK nanoparticles are reported and the feasibility and efficiency of intracellular internalization of the respective SCKs were quantified.     

Suppression of transmitter release by Tat HPC-1/syntaxin 1A fusion protein.

It has been reported that the fusion protein with the protein transduction domain (PTD) peptide of HIV-1 Tat protein can be internalized through the cell membrane of intact cells, although the exact mechanism is unknown. In this report, we investigated whether this new method could be used for the molecular analysis of exocytosis via HPC-1/syntaxin 1A, which plays an important role in transmitter release. When applied to PC12 cells, Tat PTD fusion proteins were rapidly internalized into most cells. In order to show that the internalized protein remained biologically active, the H3 domain of HPC-1/syntaxin 1A was fused to Tat PTD (Tat-H3). Transmitter release in PC12 cells was suppressed by Tat-H3 treatment. These results indicate that the Tat fusion protein is a useful tool for analyzing the process of transmitter release.     

Characteristics of HIV-Tat protein transduction domain

The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD), which contains rich arginine and lysine residues, is responsible for the highly efficient transduction of protein through the plasma membrane. In addition, it can be secreted from infected cells and has the ability to enter neighboring cells. When the PTD of Tat is fused to proteins and exogenously added to cells, the fusion protein can cross plasma membranes. Recent reports indicate that the endogenously expressed Tat fusion protein can demonstrate biodistribution of several proteins. However, intercellular transport and protein transduction have not been observed in some studies. Therefore, this study examined the intercellular transport and protein transduction of the Tat protein. The results showed no evidence of intercellular transport (biodistribution) in a cell culture. Instead, the Tat fusion peptides were found to have a significant effect on the transduction and intercellular localization properties. This suggests that the HIV-1 PTD passes through the plasma membrane in one direction.     

Enhanced uptake of a heterologous protein with an HIV-1 Tat protein transduction domains (PTD) at both termini

Poor membrane permeability of proteins is a major limitation of protein therapy. In a previous study, we showed that the minimal sequence required for efficient transduction of Tat-GFP is the basic domain from 49-57 of HIV-1 Tat called the protein transduction domain (PTD. Here we have generated HIV-1 Tat PTD GFP fusion proteins in which HIV-1 Tat PTD is fused with the N- and/or C-termini of GFP. The various GFP fusion proteins were purified from Escherichia coli and characterized for their ability to enter mammalian cells using Western blot analysis, confocal microscopy and flow cytometry. The GFP fusion protein with Tat PTD at its C-terminus was taken up as efficiently as the GFP fusion protein with Tat PTD at its N-terminus. However, the same protein with PTDs at its both termini was taken up even more efficiently. All the GFP fusion proteins were present in both the nucleus and cytosol of the transduced cells. Uptake was lower at 4 degrees C than at 37 degrees C. The availability of the expression vectors developed in this study may help to devise novel strategies in the rational development of protein-based drugs.     

Enhanced transduction of Cu,Zn-superoxide dismutase with HIV-1 Tat protein transduction domains at both termini

The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD) is responsible for highly efficient protein transduction across plasma membranes. In a previous study, we showed that Tat-Cu,Zn-superoxide dismutase (Tat-SOD) can be directly transduced into mammalian cells across the lipid membrane barrier. In this study, we fused the human SOD gene with a Tat PTD transduction vector at its N- and/or C-terminus. The fusion proteins (Tat-SOD, SOD-Tat, Tat-SOD-Tat) were purified from Escherichia coli and their ability to enter cells in vitro and in vivo compared by Western blotting and immunohistochemistry. The transduction efficiencies and biological activities of the SOD fusion protein with the Tat PTD at either terminus were equivalent and lower than the fusion protein with the Tat PTD at both termini. The availability of a more efficient SOD fusion protein provides a powerful vehicle for therapy in human diseases related to this anti-oxidant enzyme and to reactive oxygen species.     

Resonant Broadband Field Enhancement in Cylindrical Plasmonic Structure Surrounded by Perovskite Environment

We demonstrate the optical response of metal nanoparticles and their interaction with organic-inorganic perovskite (methyl ammonia lead halide (CH₃NH₃PbI₃)) environment using discrete dipole approximation (DDA) simulation technique. Important optical properties like absorption, scattering, and electric field calculations for metal nanoparticle using different geometry have been analyzed. The metal nanoparticles embedded in the perovskite media strongly support surface plasmon resonances (SPRs). The plasmonic interaction of metal nanoparticles with perovskite matrix is a strong function of MNP’s shape, size, and surrounding environment that can manipulate the optical properties considerably. The cylindrical shape of MNPs embedded in perovskite environment supports the SPR which is highly tunable to subwavelength range of 400–800 nm. Wide range of particle sizes has been selected for Ag, Au, and Al spherical and cylindrical nanostructures surrounded by perovskite matrix for simulation. The chosen hybrid material and anisotropy of structure together make a complex function for resonance shape and width. Among all MNPs, 70-nm spherical silver nanoparticle (NP) and cylindrical Ag NP having diameter of 50 nm and length of 70 nm (aspect ratio 1.4) generate strong electric field intensity that facilitates increased photon absorption. The plasmonic perovskite interaction plays an important role to improve the absorption of photon inside the thin film perovskite environment that may be applicable to photovoltaics and photonics.

     

Uptake and metabolism of a dual fluorochrome Tat-nanoparticle in HeLa cells

The ability to use magnetic nanoparticles for cell tracking, or for the delivery of nanoparticle-based therapeutic agents, requires a detailed understanding of probe metabolism and transport. Here we report on the development and metabolism of a dual fluorochrome version of our tat-CLIO nanoparticle termed Tat(FITC)-Cy3.5-CLIO. The nanoparticle features an FITC label on the tat peptide and a Cy3.5 dye directly attached to the cross-linked coating of dextran. This nanoparticle was rapidly internalized by HeLa cells, labeling 100% of cells in 45 min, with the amount of label per cell increasing linearly with time up to 3 h. Cells loaded with nanoparticles for 1 h retained 40-60% of their FITC and Cy3.5 labels over a period of 72 h in label-free media. Over a period of 144 h, or approximately 3.5 cell divisions, the T2 spin-spin relaxation time of cells was not significantly changed, indicating retention of the iron oxide among the dividing cell population. Using confocal microscopy and unfixed cells, both dyes were nuclear and perinuclear (broadly cytoplasmic) after Tat(FITC)-Cy3.5-CLIO labeling. Implications of the rapid labeling and slow excretion of the Tat(FITC)-Cy3.5-CLIO nanoparticle are discussed for cell tracking and drug delivery applications.     

Genetically modified adenoviral vector with the protein transduction domain of Tat improves gene transfer to CAR-deficient cells

The transduction efficiency of Ad (adenovirus) depends, to some extent, on the expression level of CAR (coxsackievirus and Ad receptor) of a target cell. The low level of CAR on the cell surface is a potential barrier to efficient gene transfer. To overcome this problem, PTD.AdeGFP (where eGFP is enhanced green fluorescent protein) was constructed by modifying the HI loop of Ad5 (Ad type 5) fibre with the Tat (trans-activating) PTD (protein transduction domain) derived from HIV. The present study showed that PTD.AdeGFP significantly improved gene transfer to multiple cell types deficient in expression of CAR. The improvement in gene transfer was not the result of charge-directed binding between the virus and the cell surface. Although PTD.AdeGFP formed aggregates, it infected target cells in a manner different from AdeGFP aggregates precipitated by calcium phosphate. In addition, PTD.AdeGFP was able to transduce target cells in a dynamin-independent pathway. The results provide some new clues as to how PTD.AdeGFP infects target cells. This new vector would be valuable in gene-function analysis and for gene therapy in cancer.     

超临界流体强化溶液分散法制备Eudragit S100纳米颗粒

目的:制备Eudragit S100纳米颗粒。方法:采用超临界流体强化溶液分散(SEDS)法制备,考察了Eudragit S100浓度、超临界CO2流速、溶液流速、压力、温度对Eudragit S100纳米粒形貌和粒径的影响,并用场发射扫描式电子显微镜、激光粒度分析仪、差示扫描量热仪、傅里叶变换红外光谱仪对样品进行表征。结果:SEDS法可以制备球形的、粒径分布窄的Eudragit S100纳米粒,所得纳米粒的平均粒径在90~220 nm之间。降低Eudragit S100浓度和温度、升高压力有利于制备形貌好、粒径小的纳米粒;提高超临界CO2流速和降低溶液流速也有利于制备粒径小的纳米粒,但当超临界CO2流速升高至4 kg/h或溶液流速降低至0.5 ml/min时,纳米粒的产率较低。SEDS处理后Eudratit S100仍以无定形态存在,且SEDS过程没有对Eudratit S100的化学键造成破坏。结论:采用SEDS法可用于Eudragit S100纳米粒的制备,工艺简单可行。     

Transduction of anti-apoptotic proteins into chondrocytes in cartilage slice culture

Peptides of the protein transduction domain (PTD) mediate the introduction of passenger proteins into cells in vitro and in vivo, where the domains are positively charged. This unusual ability can be exploited for medical applications in protein therapeutics. Chondrocytes are embedded in a dense extracellular matrix, whose components are highly negatively charged. We examined whether PTD mediates the delivery of functional proteins into chondrocytes through the matrix using the super anti-apoptotic protein FNK fused with Tat/PTD peptide (PTD-FNK), the FNK protein being constructed from anti-apoptotic Bcl-xL to enhance its activity. The PTD-FNK protein labeled with a fluorescent dye was incorporated into chondrocytes through the matrix and immunostaining confirmed the transduction into the cells. The PTD-FNK protein protected chondrocytes from cell death induced by Fas antibody and nitrogen oxide (NO). Thus, the PTD peptide has the ability to deliver passenger proteins into chondrocytes by penetrating the extracellular matrix of cartilage.     

Self-assembled cationic nanogels for intracellular protein delivery

An effective intracellular protein delivery system with self-assembled cationic nanogels is reported. Interaction of proteins with self-assembled nanogels of cationic cholesteryl group-bearing pullulans (CHPNH 2) was investigated by dynamic light scattering (DLS), transmission electron micrograph (TEM), fluorescence resonance energy transfer (FRET), and fluorescence correlation spectroscopy (FCS). The cationic nanogels strongly interacted with bovine serum albumin (BSA) and formed monodispersed nanoparticels (<50 nm). The complex more effectively internalized into HeLa cells than cationic liposomes and a protein transduction domain (PTD) based carrier even in the presence of serum. The higher efficiency of the nanogel carrier is probably due to the formation of colloidally stable nanoparticles with the protein. The enzymatic activity of beta-galactosidase (beta-Gal) was retained after internalization into cells. The nanogel carrier promoted nuclear delivery of a GFP-conjugated nuclear localization signal and Tat as a PTD (Tat-NLS-GFP). A blocking experiment with chemical inhibitors revealed the possible involvement of macropinocytosis in the uptake of the nanogel complex. After cellular uptake, the complex of the nanogel-protein was dissociated and the protein was released inside the cell. Such a self-assembled cationic nanogel system should create opportunities for novel applications of protein delivery.     

Spatio-temporal modeling of nanoparticle delivery to multicellular tumor spheroids

The inefficiency of nanoparticle penetration in tissues limits the therapeutic efficacy of such formulations for cancer applications. Recent work has indicated that modulation of tissue architecture with enzymes such as collagenase significantly increases macromolecule delivery. In this study we developed a mathematical model of nanoparticle penetration into multicellular spheroids that accounts for radially dependent changes in tumor architecture, as represented by the volume fraction of tissue accessible to nanoparticle diffusion. Parameters such as nanoparticle binding, internalization rate constants, and accessible volume fraction were determined experimentally. Unknown parameters of nanoparticle binding sites per cell in the spheroid and pore shape factor were determined by fitting to experimental data. The model was correlated with experimental studies of the penetration of 40 nm nanoparticles in SiHa multicellular spheroids with and without collagenase treatment and was able to accurately predict concentration profiles of nanoparticles within spheroids. The model was also used to investigate the effects of nanoparticle size. This model contributes toward the understanding of the role of tumor architecture on nanoparticle delivery efficiency.     

Composite nanoparticles based on hyaluronic acid chemically cross-linked with alpha,beta-polyaspartylhydrazide

In this paper, new composite nanoparticles based on hyaluronic acid (HA) chemically cross-linked with alpha,beta-polyaspartylhydrazide (PAHy) were prepared by the use of a reversed-phase microemulsion technique. HA-PAHy nanoparticles were characterized by FT-IR spectroscopy, confirming the occurrence of the chemical cross-linking, dimensional analysis, and transmission electron micrography, showing a sub-micrometer size and spherical shape. Zeta potential measurements demonstrated the presence of HA on the nanoparticle surface. A remarkable affinity of the obtained nanoparticles toward aqueous media that simulate some biological fluids was found. Stability studies showed the absence of chemical degradation in various media, while in the presence of hyaluronidase, a partial degradation occurred. Cell compatibility was evaluated by performing in vitro assays on human chronic myelogenous leukaemia cells (K-562) chosen as a model cell line and a haemolytic test. HA-PAHy nanoparticles were also able to entrap 5-fluorouracil, chosen as a model drug, and release it in a simulated physiological fluid and in human plasma with a mechanism essentially controlled by a Fickian diffusion.     

Intracellular delivery of glutathione S-transferase into mammalian cells

Protein transduction domains (PTDs) derived from human immunodeficiency virus Tat protein and herpes simplex virus VP22 protein are useful for the delivery of non-membrane-permeating polar or large molecules into living cells. In the course of our study aiming at evaluating PTD, we unexpectedly found that the fluorescent-dye-labeled glutathione S-transferase (GST) from Schistosoma japonicum without known PTDs was delivered into COS7 cells. The intracellular transduction of GST was also observed in HeLa, NIH3T3, and PC12 cells, as well as in hippocampal primary neurons, indicating that a wide range of cell types is permissive for GST transduction. Furthermore, we showed that the immunosuppressive peptide VIVIT fused with GST successfully inhibits NFAT activation. These results suggest that GST is a novel PTD which may be useful in the intracellular delivery of biologically active molecules, such as small-molecule drugs, bioactive peptides, or proteins.     

The cationic cell-penetrating peptide CPP(TAT) derived from the HIV-1 protein TAT is rapidly transported into living fibroblasts: optical, biophysical, and metabolic evidence

Cell-penetrating peptides (CPPs) are cationic peptides which, when linked to genes, proteins, or nanoparticles, facilitate the transport of these entities across the cell membrane. Despite their potential use for gene transfer and drug delivery, the mode of action of CPPs is still mysterious. It has even been argued that the observed transport across the cell membrane is an artifact caused by chemical fixation of the cells, a common preparation method for microscopic observation. Here we have synthesized a fluorescent derivative of the HIV-1 TAT protein transduction domain [Fg-CPP(TAT(PTD))] and have observed its uptake into nonfixated living fibroblasts with time-lapse confocal microscopy, eliminating the need for fixation. We observe that Fg-CPP(TAT(PTD)) enters the cytoplasm and nucleus of nonfixated fibroblasts within seconds, arguing against the suggested artifact of cell fixation. Using differential interference contrast microscopy, dense aggregates are detected on the cell surface. Several observations suggest that these aggregates consist of Fg-CPP(TAT(PTD)) bound to membrane-associated heparan sulfate (HS). The aggregates grow in parallel with Fg-CPP(TAT(PTD)) uptake and are detected only on fibroblasts showing Fg-CPP(TAT(PTD)) uptake. These observations resemble earlier reports of "capping" of cell surface molecules combined with a polarized endocytotic flow. Enzymatic removal of extracellular HS reduced the rate of both Fg-CPP(TAT(PTD)) uptake and aggregate formation, demonstrating that HS is involved in the uptake mechanism. The functionality of the fibroblasts during the CPP uptake was investigated with a cytosensor microphysiometer measuring the extracellular acidification rate (ECAR). Short exposures (2.5 min) to the CPP reduced the ECAR which was, however, reversible upon reperfusion with buffer only. In contrast, no recovery to baseline values was observed after repeated exposures to the CPP, suggesting that the CPP is toxic in long-term applications.     

Peptide-derivatized shell-cross-linked nanoparticles. 2. Biocompatibility evaluation

The conjugation of the protein transduction domain (PTD) from the HIV-1 Tat protein to shell cross-linked (SCK) nanoparticles is a method to facilitate cell surface binding and transduction. In the previous report, the preparation, derivatization, and characterization of peptide-functionalized SCK nanoparticles were reported in detail. Following assembly, the constructs were evaluated in vitro and in vivo to obtain a preliminary biocompatibility assessment. The effects of SCK exposure on cell viability were evaluated using a metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and a fluorescent apoptosis assay. Furthermore, stages of apoptosis were quantified by flow cytometry. Although higher levels of peptide functionalization resulted in decreased metabolic function as measured by MTT assay, significant apoptosis was not observed below 500 mg/L for all the samples. To evaluate the potential immunogenic response of the peptide-derivatized constructs, a real-time polymerase chain reaction (RT-PCR) system that allows for the in vitro analysis and quantification of the cellular inflammatory responses tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL1-beta) was utilized. The inflammatory response to the peptide-functionalized SCK nanoparticles as measured by RT-PCR show statistically significant increases in the levels of both TNF-alpha and IL1-beta relative to tissue culture polystyrene (TCPS). However, the measured cytokine levels did not preclude the further testing of SCKs in an in vivo mouse immunization protocol. In this limited assay, measured increases in immunoglobulin G (IgG) concentration in the sera were minimal with no specific interactions being isolated, and more importantly, none of the mice (>50) subjected to the three 100 microg immunization protocol have died. Additionally, no gross morphological changes were observed in postmortem organ histology examinations.     

Allometric scaling and morphological variation in sinking rate of phytoplankton

Since the first decades of the last century, several hypotheses have been proposed on the role of phytoplankton morphology in maintaining a favorable position in the water column. Here, by an extensive review of literature on sinking rate and cell volume, we firstly attempted to explore the dependency of sinking rate on morphological traits using the allometric scaling approach. We found that sinking rate tends to increase with increasing cell volume showing the allometric scaling exponent of 0.43, which is significantly different than the Stokes’ law exponent of 0.66. The violation of the 2/3 power rule clearly indicates that cell shape changes as size increases. Both size and shape affect how phytoplankton sinking drives nutrient acquisition and losses to sinking. Interestingly, from an evolutionary perspective, simple and complex cylindrical shapes can get much larger than spherical and spheroidal shapes and sink at similar rates, but simple and complex cylindrical shapes cannot get small enough to sink slower than small spherical and spheroidal shapes. Cell shape complexity is a morphological attribute resulting from the combination of two or more simple geometric shapes. While the effect of size on sinking rate is well documented, this study deepens the knowledge on how cell shape or geometry affect sinking rates that still needs further consideration.     

Zonal necrosis prevented by transduction of the artificial anti-death FNK protein

Protection of cells from necrosis would be important for many medical applications. Here, we show protein transduction domain (PTD)-FNK therapeutics based on protein transduction to prevent necrosis and acute hepatic injury with zonal death induced by carbon tetrachloride (CCl4). PTD-FNK is a fusion protein comprising the HIV/Tat PTD and FNK, a gain-of-function mutant of anti-apoptotic Bcl-x(L). PTD-FNK protected hepatoma HepG2 from necrotic death induced by CCl4, and additionally, increased the apoptotic population among cells treated with CCl4. A concomitant treatment with a pan-caspase inhibitor Z-VAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), which alone could not prevent the necrosis, protected these cells from the apoptosis. When pre-injected intraperitoneally, PTD-FNK markedly reduced zonal liver necrosis caused by CCl4. Moreover, injection of PTD-FNK accompanied by Z-VAD-FMK suppressed necrotic injury even after CCl4 administration. These results suggest that PTD-FNK has great potential for clinical applications to prevent cell death, whether from apoptosis or necrosis, and organ failure.