Dataset Information

Bone-Targeted Delivery of Cell-Penetrating-RUNX2 Fusion Protein in Osteoporosis Model.

ABSTRACT: The onset of osteoporosis leads to a gradual decrease in bone density due to an imbalance between bone formation and resorption. To achieve optimal drug efficacy with minimal side effects, targeted drug delivery to the bone is necessary. Previous studies have utilized peptides that bind to hydroxyapatite, a mineral component of bone, for bone-targeted drug delivery. In this study, a hydroxyapatite binding (HAB) tag is fused to 30Kc19α-Runt-related transcription factor 2 (RUNX2) for bone-targeting. This recombinant protein can penetrate the nucleus of human mesenchymal stem cells (hMSCs) and act as a master transcription factor for osteogenesis. The HAB tag increases the binding affinity of 30Kc19α-RUNX2 to mineral deposition in mature osteoblasts and bone tissue, without affecting its osteogenic induction capability. In the osteoporosis mouse model, intravenous injection of HAB-30Kc19α-RUNX2 results in preferential accumulation in the femur and promotes bone formation while reducing toxicity in the spleen. These findings suggest that HAB-30Kc19α-RUNX2 may be a promising candidate for bone-targeted therapy in osteoporosis.

SUBMITTER: Kim S

PROVIDER: S-EPMC10558633 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Bone-Targeted Delivery of Cell-Penetrating-RUNX2 Fusion Protein in Osteoporosis Model.

Kim Seoyeon S Lee Haein H Hong Jiyeon J Kim Seung Hyun L SHL Kwon Euntaek E Park Tai Hyun TH Hwang Nathaniel S NS

Advanced science (Weinheim, Baden-Wurttemberg, Germany) 20230813 28

The onset of osteoporosis leads to a gradual decrease in bone density due to an imbalance between bone formation and resorption. To achieve optimal drug efficacy with minimal side effects, targeted drug delivery to the bone is necessary. Previous studies have utilized peptides that bind to hydroxyapatite, a mineral component of bone, for bone-targeted drug delivery. In this study, a hydroxyapatite binding (HAB) tag is fused to 30Kc19α-Runt-related transcription factor 2 (RUNX2) for bone-targetin ...[more]

PMID: 37574255

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Project description:Study Design Randomized, controlled animal study. Objective Recombinant human bone morphogenetic protein-2 (rhBMP-2) is frequently utilized as a bone graft substitute in spinal fusions to overcome the difficult healing environment in patients with osteoporosis. However, the effects of estrogen deficiency and poor bone quality on rhBMP-2 efficacy are unknown. This study sought to determine whether rhBMP-2-induced healing is affected by estrogen deficiency and poor bone quality in a stringent osteoporotic posterolateral spinal fusion model. Methods Aged female Sprague-Dawley rats underwent an ovariectomy (OVX group) or a sham procedure, and the OVX animals were fed a low-calcium, low-phytoestrogen diet. After 12 weeks, the animals underwent a posterolateral spinal fusion with 1 μg rhBMP-2 on an absorbable collagen sponge. Representative animals were sacrificed at 1 week postoperative for alkaline phosphatase (ALP) and osteocalcin serum analyses. The remaining animals underwent radiographs 2 and 4 weeks after surgery and were subsequently euthanized for fusion analysis by manual palpation, micro-computed tomography (CT) imaging, and histologic analysis. Results The ALP and osteocalcin levels were similar between the control and OVX groups. Manual palpation revealed no significant differences in the fusion scores between the control (1.42 ± 0.50) and OVX groups (1.83 ± 0.36; p = 0.07). Fusion rates were 100% in both groups. Micro-CT imaging revealed no significant difference in the quantity of new bone formation, and histologic analysis demonstrated bridging bone across the transverse processes in fused animals from both groups. Conclusions This study demonstrates that estrogen deficiency and compromised bone quality do not negatively influence spinal fusion when utilizing rhBMP-2, and the osteoinductive capacity of the growth factor is not functionally reduced under osteoporotic conditions in the rat. Although osteoporosis is a risk factor for pseudarthrosis/nonunion, rhBMP-2-induced healing was not inhibited in osteoporotic rats.

Project description:Many neurodegenerative disorders (NDDs) are characterized by aggregation of aberrant proteins and extensive oxidative stress in brain cells. As a treatment option for NDDs, RNA interference (RNAi) is a promising approach to suppress the activation of abnormal genes and negative regulators of antioxidant genes. Efficient neuro-targeted siRNA delivery requires a delicate optimization of nucleic acid carriers, quite distinct from putative pDNA carriers in regard to stable condensation and serum protection of siRNA, blood-brain barrier (BBB) bypass, effective siRNA delivery to brain cells, and functional release of bioactive siRNA at therapeutic levels. Here, we propose that a myristic acid conjugated, cell-penetrating peptide (transportan; TP), equipped with a transferrin receptor-targeting peptide (myr-TP-Tf), will lead to stable encapsulation of siRNA and targeted delivery of siRNA to brain cells overcoming the BBB. Myr-TP-Tf was successfully prepared by solid-phase peptide synthesis with high purity. Myr-TP-Tf-siRNA complexes formulated at a 20:1 (peptide-siRNA) molar ratio provided prolonged siRNA stability against serum and ribonuclease treatment. Fluorescence images clearly indicated that siRNA uptake was successfully achieved by myr-TP-Tf complexes in both a murine brain endothelioma and a human glioma cell line. The luciferase assay and the human placental alkaline phosphatase (hPAP) reporter assay results demonstrated the functional gene silencing effect of myr-TP-Tf-siRNA complexes in a human glioma cell line as well as in primary murine neurons/astrocytes, supportive of successful release of bioactive siRNA into the cytosol. Finally, the transcytosis assay revealed that favorable siRNA transport via receptor-mediated transcytosis was mediated by myr-TP-Tf complexes. In summary, these data suggest that myr-TP-Tf peptides possess promising properties as a vehicle for neuro-targeted siRNA delivery. We will further study this peptide in vitro and in vivo for transport mechanism kinetics and to validate its capability to deliver siRNA to the brain, respectively.

Project description:The entry of proteins through the cell membrane is challenging, thus limiting their use as potential therapeutics. Seven cell-penetrating peptides, designed in our laboratory, were evaluated for the delivery of proteins. Fmoc solid-phase peptide synthesis was utilized for the synthesis of seven cyclic or hybrid cyclic-linear amphiphilic peptides composed of hydrophobic (tryptophan (W) or 3,3-diphenylalanine (Dip) and positively-charged arginine (R) residues, such as [WR]4, [WR]9, [WWRR]4, [WWRR]5, [(RW)5K](RW)5, [R5K]W7, and [DipR]5. Confocal microscopy was used to screen the peptides as a protein delivery system of model cargo proteins, green and red fluorescein proteins (GFP and RFP). Based on the confocal microscopy results, [WR]9 and [DipR]5 were found to be more efficient among all the peptides and were selected for further studies. [WR]9 (1-10 µM) + protein (GFP and RFP) physical mixture did not show high cytotoxicity (>90% viability) in triple-negative breast cancer cells (MDA-MB-231) after 24 h, while [DipR]5 (1-10 µM) physical mixture with GFP exhibited more than 81% cell viability. Confocal microscopy images revealed internalization of GFP and RFP in MDA-MB-231 cells using [WR]9 (2-10 μM) and [DipR]5 (1-10 µM). Fluorescence-activated cell sorting (FACS) analysis indicated that the cellular uptake of GFP was concentration-dependent in the presence of [WR]9 in MDA-MB-231 cells after 3 h of incubation at 37 °C. The concentration-dependent uptake of GFP and RFP was also observed in the presence of [DipR5] in SK-OV-3 and MDA-MB-231 cells after 3 h of incubation at 37 °C. FACS analysis indicated that the cellular uptake of GFP in the presence of [WR]9 was partially decreased by methyl-β-cyclodextrin and nystatin as endocytosis inhibitors after 3 h of incubation in MDA-MB-231 cells, whereas nystatin and chlorpromazine as endocytosis inhibitors slightly reduced the uptake of GFP in the presence of [DipR]5 after 3 h of incubation in MDA-MB-231. [WR]9 was able to deliver therapeutically relevant proteins (Histone H2A) at different concentrations. These results provide insight into the use of amphiphilic cyclic peptides in the delivery of protein-related therapeutics.

Dataset Information

Bone-Targeted Delivery of Cell-Penetrating-RUNX2 Fusion Protein in Osteoporosis Model.

Publications

Bone-Targeted Delivery of Cell-Penetrating-RUNX2 Fusion Protein in Osteoporosis Model.

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