Jin Sook Suh

Cell-penetrating chitosan/doxorubicin/TAT conjugates for efficient cancer therapy

In this study, a cell‐penetrating peptide, the transactivating transcriptional factor (TAT) domain from HIV, was linked to a chitosan/doxorubicin (chitosan/DOX) conjugate to form a chitosan/DOX/TAT hybrid. The synthesized chitosan/DOX/TAT conjugate showed a different intracellular distribution pattern from a conjugate without TAT. Unlike both free DOX and the conjugate without TAT, the chitosan/DOX/TAT conjugate was capable of efficient cell entry. The chitosan/DOX/TAT conjugate was found to be highly cytotoxic, with an IC50 value of approximately 480 nM, 2 times less than that of chitosan/DOX (980 nM). The chitosan/DOX/TAT provided decreases in tumor volume of 77.4 and 57.5% compared to free DOX and chitosan/DOX, respectively, in tumor‐bearing mice. Therefore, this study suggests that TAT‐mediated chitosan/DOX conjugate delivery is effective in slowing tumor growth.

The systemic delivery of siRNAs by a cell penetrating peptide, low molecular weight protamine

Small interfering RNAs (siRNAs), used for specific down-regulation of targeted genes, have garnered considerable interest as an attractive new class of drugs for broad clinical applications. The polyanionic charges carried by these siRNAs, however, restrain cellular uptake and consequently limit effects on gene regulation. Herein the authors describe a peptide/siRNA complex containing the cell penetrating peptide derived from natural protamine, termed low molecular weight protamine (LMWP), for the treatment of cancer. Fluorescently-tagged siRNAs were localized with the peptide in the cytoplasm shortly after incubation of LMWP/siRNA complex with carcinoma cells. The increased cell uptake of siRNA that was achieved using the LMWP resulted in significant down-regulation of model protein luciferase as well as therapeutic cancer target, vascular endothelial growth factor (VEGF) expression. In vivo studies with tumor-bearing mice further demonstrated that the peptide could carry and localize siRNA inside tumors and inhibit the expression of VEGF through systemic application of the peptide complex, thereby suppressing tumor growth. In addition, no detectable increase in the serum level of inflammatory cytokines including interferon (IFN)-alpha and interleukin (IL)-12 was observed under the LMWP/siRNA complex treatment, indicating systemic delivery of LMWP/siRNA did not exert measurable immunostimulatory effect. The LMWP-based systemic delivery method could be a reliable and safe approach to maximize effectiveness of therapeutic siRNA for treatment of cancer and other diseases.

Efficient labeling of mesenchymal stem cells using cell permeable magnetic nanoparticles.

For the purpose of successfully monitoring labeled cells, optimum labeling efficiency without any side effect is a prerequisite. Magnetic cellular imaging is a new and growing field that allows the visualization of implanted cells in vivo. Herein, superparamagnetic iron oxide (SPIO) nanoparticles were conjugated with a non-toxic protein transduction domain (PTD), identified by the authors and termed low molecular weight protamine (LMWP), to generate efficient and non-toxic cell labeling tools. The cells labeled with LMWP-SPIO presented the highest iron content compared to those labeled with naked SPIO and the complex of SPIO with poly-L-lysine, which is currently used as a transfection agent. In addition to the iron content assay, Prussian staining and confocal observation demonstrated the highest intracellular LMWP-SPIO presence, and the labeling procedure did not alter the cell differentiation capacity of mesenchymal stem cells. Taken together, cell permeable magnetic nanoparticles conjugated with LMWP can be suggested as labeling tools for efficient magnetic imaging of transplanted cells.

The identification of a heparin binding domain peptide from bone morphogenetic protein-4 and its role on osteogenesis

The presence of heparin binding has been become crucial in exerting growth factor related tissue formation. Receptor-mediated osteoblastic differentiation by bone morphogenetic protein (BMP)-4 and supportive function of its heparin binding has been proposed, direct role of the heparin binding site of BMP-4 on osteogenesis has not yet been fully investigated. If the binding site itself plays role on osteogenesis, the site domain can be useful in bone formation in combination with biomaterial. Herein, we synthesized a peptide sequence corresponding to residues 15-24 of BMP-4 (HBD, RKKNPNCRRH), as potential heparin binding sequence. The HBD peptide-induced ostoegenic differentiation by activating extracellular signal-regulated kinase (ERK1/2), one of the key regulators in hMSC. Also, treatment of cultured hMSCs with heparinase blocked both HBD peptide-induced osteogenic differentiation and GAG chain detection while abolishing the increased phospho-ERK level. These results suggest that the identified heparin binding domain peptide (HBD) stimulated osteoblastic differentiation via interaction with heparin and the ERK signaling. In vivo results further demonstrated that HBD, as a form of complex with alginate gel, was able to induce bone formation in the bone defect.

Cell Penetrating Peptides for Tumor Targeting

Anticancer drug delivery has been hindered due to cell membrane permeability and the lack of a selective marker for tumor cells. Cell permeability is related to the bioavailability of drugs and has therefore been considered to be an essential step for achieving therapeutic efficacy. While different types of transporters currently exist, cell penetrating peptides (CPPs) have become one of the most popular and effective tools for intracellular drug delivery. Most of the original CPPs are short peptides with basic residues. The mechanism of CPP cell entry remains to be established; however, the CPPs can deliver any type of molecular cargo including solid nanoparticles. Herein, this paper will discuss the classification of CPPs, the mechanism of cell entry, the application of CPPs in tumor therapy, and recent advances in targeted cell penetration that involve CPPs.

A Cell-permeable Fusion Protein for the Mineralization of Human Dental Pulp Stem Cells:

Human dental pulp stem cells (hDPSCs) are the only mesenchymal stem cells in pulp tissue that can differentiate into osteoblasts, odontoblasts, and adipose cells. The transcriptional co-activator with PDZ-binding motif (TAZ) protein has been reported to modulate osteogenic differentiation in mouse MSCs. Therefore, we examined whether the TAZ protein plays the same role in human pulp stem cells. In this study, TAZ was applied to cells directly with low-molecular-weight protamine (LMWP) as a cell-penetrating peptide (CPP). The LMWP-TAZ fusion proteins were expressed in an E. coli system with a pET-21b vector and efficiently transferred into hDPSCs without producing toxicity in the cells. The efficient uptake of TAZ was shown by Western blot with an anti-TAZ antibody, fluorescence-activated cell sorting, and confocal microscopy in live cells. The delivered TAZ protein increased osteogenic differentiation, as confirmed by alkaline phosphatase (ALP) staining, RT-PCR, and Western blotting. In addition, TAZ also inhibited adipogenic differentiation, regulating peroxisome proliferator-activated receptor-γ (PPAR-γ), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein (aP2) mRNA levels. These in vitro studies suggest that cell-permeable TAZ may be used as a specific regulator of hard-tissue differentiation.

Identification of a cell-penetrating peptide domain from human beta-defensin 3 and characterization of its anti-inflammatory activity

Human beta-defensins (hBDs) are crucial factors of intrinsic immunity that function in the immunologic response to a variety of invading enveloped viruses, bacteria, and fungi. hBDs can cause membrane depolarization and cell lysis due to their highly cationic nature. These molecules participate in antimicrobial defenses and the control of adaptive and innate immunity in every mammalian species and are produced by various cell types. The C-terminal 15-mer peptide within hBD3, designated as hBD3-3, was selected for study due to its cell- and skin-penetrating activity, which can induce anti-inflammatory activity in lipopolysaccharide-treated RAW 264.7 macrophages. hBD3-3 penetrated both the outer membrane of the cells and mouse skin within a short treatment period. Two other peptide fragments showed poorer penetration activity compared to hBD3-3. hBD3-3 inhibited the lipopolysaccharide-induced production of inducible nitric oxide synthase, nitric oxide, and secretory cytokines, such as interleukin-6 and tumor necrosis factor in a concentration-dependent manner. Moreover, hBD3-3 reduced the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. Further investigation also revealed that hBD3-3 downregulated nuclear factor kappa B-dependent inflammation by directly suppressing the degradation of phosphorylated-IκBα and by downregulating active nuclear factor kappa B p65. Our findings indicate that hBD3-3 may be conjugated with drugs of interest to ensure their proper translocation to sites, such as the cytoplasm or nucleus, as hBD3-3 has the ability to be used as a carrier, and suggest a potential approach to effectively treat inflammatory diseases.

Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis.

Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP), and a transcriptional coactivator with a PDZ-binding motif (TAZ) protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC) differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in alginate gel for the purpose of localization and controlled release. The LMWP-TAZ fusion protein-loaded alginate gel matrix significantly increased bone formation in rabbit calvarial defects compared with alginate gel matrix mixed with free TAZ protein. The protein transduction of TAZ fused with cell-penetrating LMWP peptide was able selectively to stimulate osteogenesis in vitro and in vivo. Taken together, this fusion protein-transduction technology for osteogenic protein can thus be applied in combination with biomaterials for tissue regeneration and controlled release for tissue-engineering purposes.

The mineralization inducing peptide derived from dentin sialophosphoprotein for bone regeneration.

Dentin sialophosphoprotein (DSPP) has been shown to play a primary role in the formation and growth of hydroxyapatite crystals in an extracellular matrix of hard tissue such as bone and teeth. We hypothesized that the mineralization ability of DSPP might depend on a specific domain within it. Three peptides, which have hydroxyapatite (HA) binding affinity, denoted as mineralization inducing peptide (MIP1, MIP2, and MIP3) were identified from DSPP. The both of MIP2 and MIP3 had HA nucleation activity demonstrated by XRD. Among three MIPs, MIP3 significantly supported the human bone marrow stromal cell differentiation into osteoblastic cells. An immunoblot with antibodies specific for the phosphorylated forms of ERK was conducted with cells treated by MIP3. MIP3 transduced intracellular signals via the ERK pathways and was able to induce osteoblastic differentiation, as seen by high expression of ALP, type 1 collagen, OC, OPN, and Runx2 in accordance with applied MIP3 concentration. The Asp, Glu, and Ser residues in MIP3 play important roles for the affinity of calcium in HA bone mineral. Further animal experiment with MIP3 in combination with hydroxyapatite mineral induced marked new bone formation for 4 weeks at rabbit calvarial defect model. The new bone area was much higher in test group, implying that the peptide modified group had excellent biocompatibility when compared with the unmodified group. Taken together, the MIP from DSPP has potential to enhance mineralization followed by to enhance osteoblastic differentiation and bone regeneration.

Selective osteogenesis by a synthetic mineral inducing peptide for the treatment of osteoporosis

Mineralization in mammalian cells is accomplished by concerted regulation of protein-based extracellular matrix (ECM) components, such as non-collagenous proteins and collagen fibrils. In this study, we investigated the ability of a collagen-binding motif (CBM) peptide derived from osteopontin to selectively affect osteogenic or adipogenic differentiation in vitro and in vivo. In particular, increased osteogenic differentiation and decreased adipogenic differentiation were observed in human mesenchymal stem cells (hMSCs). Osteocalcin (OCN) protein expression in MC3T3-E1 cells without osteogenic inducers was then investigated following treatment with the CBM peptide. In ovariectomized (OVX) mice, estrogen deficiency induced osteoporosis and increased fat tissue deposition. However, after the CBM peptide or estradiol was injected into the OVX mice for 2 months, the increased serum OCN concentration and alkaline phosphate (ALP) activity were decreased in the estradiol-treated group (OVX-E) and the high-concentration CBM peptide-treated group (OVX-HP). Significant bone loss was also observed in the ovariectomized mice (OVX-PBS). In particular, the bone volume per total volume (BV/TV) and bone mineral density (BMD) were significantly decreased in the OVX mice; however, both of these markers were restored in the OVX-HP group, which also had significantly well-developed bone structure and bone formation. In contrast to the bone structural change, adipose tissue was increased in the OVX-PBS. However, a significant decrease in total fat and subcutaneous fat was observed in the low-concentration CBM peptide-treated group (OVX-LP) and the estradiol-treated group (OVX-E). Taken together, these results suggest that the CBM peptide could be an effective therapeutic agent for osteoporosis due to its selective stimulation of osteogenic differentiation, rather than adipogenesis.