Targeted Delivery of Nanoparticle-Conveyed Neutrophils to the Glioblastoma Site for Efficient Therapy.

Glioblastoma is a common brain tumor that poses considerable challenges in drug delivery. In this study, we investigated the potential of cell-based nanoparticles for targeted drug delivery to the glioblastoma sites. The anticancer drug of temozolomide (TMZ)-loaded T7-cholesterol nanoparticle micelles efficiently delivered nanoparticles to neutrophils and, subsequently, to the tumors. T7 is a cell-penetrating peptide that enhances the delivery of T7/TMZ to the target cells. T7 also serves as a transferrin target peptide, enabling targeted delivery to tumors. T7-conjugated cholesterol can self-assemble into micelles in aqueous solution and attach to the membrane of neutrophils. We confirmed that T7/TMZ nanoparticle micelles were efficiently located inside the neutrophils. Thereafter, T7/TMZ-conveyed neutrophils were administered to a glioblastoma mouse model, enabling neutrophils to penetrate the blood-brain barrier and deliver drugs directly to the tumor site. We evaluated the drug delivery efficiency and therapeutic effects of intravenous injection of T7/TMZ-conveyed neutrophils to a glioblastoma mouse model. These results demonstrate the promising role of neutrophil-based nanoparticle delivery systems in the targeted therapy of glioblastoma.

Pulmonary delivery of a recombinant RAGE antagonist peptide derived from high-mobility group box-1 in a bleomycin-induced pulmonary fibrosis animal model.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterised by irreversible fibrosis and destruction of the alveolar structure. Receptor for advanced glycation end products (RAGE) has been identified as one of the key molecules involved in IPF pathogenesis. A RAGE-antagonist peptide (RAP) was developed based on the RAGE-binding domain of high mobility group box-1 (HMGB-1). Anti-IPF effects of RAP were evaluated in a bleomycin-induced mouse model of IPF. Bleomycin was administered intratracheally, and then RAP was administrated twice by intratracheal instillation, 1 and 3 d after bleomycin challenge. Seven days after the bleomycin challenge, the mice were sacrificed and the lungs were harvested. The results showed that pulmonary hydroxyproline was reduced in mice administered RAP compared with the control group. Tumour growth factor-ß (TGF-ß), α-smooth muscle actin (α-SMA) and collagen were also reduced by RAP administration in a dose-dependent manner. Longer-term effects of RAP were investigated in mice challenged with bleomycin. RAP was administered intratracheally every 7 d for 28 d, after which lung samples were harvested and analysed. The results showed that hydroxyproline, TGF-ß, α-SMA and collagen were reduced by repeated RAP administration. Taken together, the results suggest that RAP is useful for treatment of IPF.

Dual-Functional Dendrimer Micelles with Glycyrrhizic Acid for Anti-Inflammatory Therapy of Acute Lung Injury.

Dendrimer micelles with glycyrrhizic acid (GA) were developed for anti-inflammatory therapy of acute lung injury (ALI). Cholesterol was conjugated to histidine- and arginine-grafted polyamidoamine (PamHR) for micelle formation. The cholesterol-conjugated PamHR (PamHRchol) was mixed with amphiphilic GA to produce PamHRchol/GA mixed micelles. The GA integrated into the micelles had two functions: it acted as an anti-inflammatory drug and facilitated intracellular gene delivery. The PamHRchol/GA micelles formed stable complexes with plasmid DNA. Integrating GA into the micelles increased their transfection efficiency. Confocal microscopy and flow-cytometry studies confirmed that the PamHRchol/GA micelles improved cellular uptake compared with PamHRchol. A competition assay with free GA suggested that the enhanced transfection efficiency of the micelles might be due to the interaction between GA and its receptor. In addition, GA has a membrane-destabilizing effect, and a chloroquine pretreatment assay confirmed that GA increased endosomal escape. Furthermore, the PamHRchol/GA micelles reduced tumor necrosis factor-α in lipopolysaccharide-activated Raw264.7 cells, suggesting a mechanism for its anti-inflammatory effects. To evaluate the therapeutic potential of the PamHRchol/GA micelles, the heme oxygenase-1 (HO-1) gene was delivered into the lungs of mice with ALI. The PamHRchol/GA micelles had higher gene delivery efficiency into the lungs than polyethylenimine (25 kDa, PEI25k) and the PamHRchol micelles. The combined effects of the HO-1 gene and GA produced effective anti-inflammation response in the lungs of the ALI animals. Therefore, the dual-function PamHRchol/GA micelles, which acted as an anti-inflammatory drug and a gene carrier, could be a useful therapy for inflammatory lung diseases.

Delivery of MiRNA-92a Inhibitor Using RP1-Linked Peptide Elicits Anti-Inflammatory Effects in an Acute Lung Injury Model.

Acute lung injury (ALI) is an inflammatory lung disease. miRNA-92a (miR92a) is induced in the lungs of ALI patients and mediates inflammatory reactions. In this study, a RP1-linked R3V6 (RP1R3V6) peptide was synthesized and evaluated as a carrier of anti-microRNA-92a oligonucleotide (AMO92a) into the lungs of an ALI animal model. In addition to the carrier function, the RP1-linked peptide can have anti-inflammatory effects in the lungs, since RP1 is an antagonist of the receptors for advanced glycation end-products (RAGEs). In a gel retardation assay, the RP1R3V6 peptide formed a spherical complex with AMO92a. In an in vitro delivery assay to L2 rat lung epithelial cells, RP1R3V6 had a lower AMO92a delivery efficiency than R3V6 and polyethyleneimine (PEI25k; 25 kDa). However, RP1R3V6 had an additional anti-inflammatory effect, reducing tumor necrosis factor-α (TNF-α) in lipopolysaccharide-activatedmacrophage cells. With the combined effects of AMO92a and RP1, the RP1R3V6/AMO92a complex reduced the miR92a level more efficiently than did the R3V6/AMO92a and PEI25k/AMO92a complexes. The RP1R3V6/AMO92a complex was administered into the lungs of ALI animals by intratracheal instillation. As a result, the expression of phosphatase and tensin homolog, a target of miR92a, was increased in the lungs. Furthermore, the RP1R3V6/AMO92a complex decreased the TNF-α and interleukin-1ß (IL-1ß) levels more efficiently than did the PEI25k/AMO92a and R3V6/AMO92a complexes, decreasing the damage in the lungs. These results suggest that RP1R3V6 is a useful carrier of AMO92a and has anti-inflammatory effects in an ALI animal model.

A RAGE-antagonist peptide potentiates polymeric micelle-mediated intracellular delivery of plasmid DNA for acute lung injury gene therapy.

Acute lung injury (ALI) is a severe inflammatory lung disease. A high mobility group box-1 (HMGB-1) derived RAGE-antagonist peptide (RAP) was previously developed for anti-inflammatory therapy for ALI. Due to its specific binding to RAGE on the surface of inflammatory cells, the RAP may facilitate polymer-mediated intracellular delivery of plasmid DNA (pDNA) into the inflammatory cells. To evaluate this hypothesis, a pDNA/polymer/RAP ternary-complex was produced and applied for ALI gene therapy. Dexamethasone-conjugated polyamidoamine G2 (PAM-D) was used as a gene carrier, and the adiponectin (APN) gene was employed as a therapeutic gene. First, the ratio of pDNA to PAM-D was optimized in terms of anti-inflammation and low toxicity. Then, the RAP was added to the pDNA/PAM-D complex, producing the pDNA/PAM-D/RAP complex. The transfection efficiency of the luciferase plasmid (pLuc)/PAM-D/RAP reached its maximum at a weight ratio of 1 : 2 : 9. At this weight ratio, pLuc/PAM-D/RAP had a higher transfection efficiency than pLuc/PAM-D or pLuc/RAP. The transfection efficiency of pLuc/PAM-D/RAP decreased due to competition with free RAPs, suggesting the RAGE-mediated endocytosis of the complex. In the LPS-activated ALI mouse models, intratracheal administration of APN plasmid (pAPN)/PAM-D/RAP induced higher APN expression and less pro-inflammatory cytokines in the lungs of ALI animal models than pAPN/PEI25k, pAPN/RAP, and pAPN/PAM-D. Hematoxylin and eosin staining confirmed the higher anti-inflammatory effect of pAPN/PAM-D/RAP than the other complexes in the ALI models. Therefore, RAP-mediated enhanced delivery of pAPN/PAM-D may be useful for the development of a treatment for ALI.

A self-assembled DNA-nanoparticle with a targeting peptide for hypoxia-inducible gene therapy of ischemic stroke.

A self-assembled nanoparticle composed of hypoxia-specific anti-RAGE peptide (HSAP), heme oxygenase-1 plasmid (pHO1), and deoxycholate-conjugated polyethylenimine-2k (DP2k) was developed for ischemic stroke therapy. RAGE is over-expressed and induces inflammation in the ischemic brain. To inhibit RAGE-mediated signal transduction, HSAP was produced by recombinant DNA technology, based on the RAGE-binding domain of high mobility group box-1. Because of the specific binding to RAGE, the nanoparticle with HSAP (HSAP-NP) may have dual roles as a cytoprotective reagent and a specific ligand to RAGE for receptor-mediated transfection. As a cytoprotective reagent, the HSAP-NP reduced RAGE expression on the surface of the brain cells by inhibiting the positive feedback of RAGE-mediated signal transduction. As a result, inflammation, apoptosis, and reactive oxygen species were decreased in hypoxic cells. As a gene carrier, HSAP-NP showed a higher transfection efficiency than polyethylenimine-25k, DP2k, and Lipofectamine. Particularly, HSAP-NP enhanced gene delivery to hypoxic cells. In the stroke animal models, HSAP-NP reduced the levels of RAGE, inducible nitric oxide synthase, and inflammation. Additionally, HSAP-NP with pHO1 (HSAP-NP/pHO1) increased HO1 expression in the ischemic brain. Gene expression was higher in hypoxia-inducible factor-1α (HIF-1α)-positive cells than in HIF-1α-negative cells, suggesting that HSAP-NP delivered the genes to ischemic tissues more efficiently. Cell death and infarct volume in the stroke models were significantly decreased by HSAP-NP/pHO1 compared with HSAP alone or the DP2k/pHO1 complex. Therefore, HSAP-NP may be a useful gene and peptide therapy system for stroke therapy with dual functions of hypoxia-specific gene delivery and cytoprotective effects.

Combined delivery of curcumin and the heme oxygenase-1 gene using cholesterol-conjugated polyamidoamine for anti-inflammatory therapy in acute lung injury.

BACKGROUND: Acute lung injury (ALI) is an inflammatory lung disease with a high mortality rate. In this study, combined delivery of the anti-inflammatory compound curcumin and the heme-oxygenase-1 (HO-1) gene using cholesterol-conjugated polyamidoamine was evaluated in a mouse model as a therapeutic option for ALI. METHODS: Curcumin was loaded into cholesterol-conjugated polyamidoamine (PamChol) micelles, and curcumin-loaded PamChol (PamChol-Cur) was then complexed with plasmid DNA (pDNA) through charge interactions. The pDNA/PamChol-Cur complex was physically characterized by dynamic light scattering, gel retardation, and heparin competition assay. Gene delivery efficiency was measured by luciferase assay. The HO-1 expression plasmid (pHO-1)/PamChol-Cur complex was administrated into the ALI model via intratracheal injection. The anti-inflammatory effect of the pDNA/PamChol-Cur complex was evaluated by ELISA, immunohistochemistry, and hematoxylin and eosin staining. RESULTS: The pDNA/PamChol-Cur complex had a size of approximately 120 nm with a positive surface charge. The in vitro plasmid DNA (pDNA) delivery efficiency of the pDNA/PamChol-Cur complex into L2 lung epithelial cells was higher than that of pDNA/PamChol. In addition, the curcumin in the pDNA/PamChol-Cur complex inhibited the nuclear translocation of NF-κB, suggesting an anti-inflammatory effect of curcumin. In the ALI animal model, the pHO-1/PamChol-Cur complex delivered the pHO-1 gene more efficiently than pHO-1/PamChol. In addition, the pHO-1/PamChol-Cur complex showed greater anti-inflammatory effects by reducing anti-inflammatory cytokine levels more than delivery of pHO-1/PamChol or PamChol-Cur only. CONCLUSION: The pHO-1/PamChol-Cur complex had a higher pHO-1 gene-delivery efficiency and greater anti-inflammatory effects than the pHO-1/PamChol complex or PamChol-Cur. Therefore, the combined delivery of curcumin and pHO-1 using PamChol-Cur may be useful for treatment of ALI.

Self-assembled polymeric micelles for combined delivery of anti-inflammatory gene and drug to the lungs by inhalation.

Acute lung injury (ALI) is a lung inflammatory disease for which pulmonary delivery of drug and gene could be a useful strategy. In this study, cholesterol-conjugated polyamidoamine (PAM-Chol) was synthesized and characterized as a carrier for combined delivery of anti-inflammatory gene and drug into the lungs by inhalation. The PAM-Chol formed self-assembled micelles in an aqueous solution with a critical micelle concentration of 0.22 mg ml-1. An in vitro transfection assay to L2 lung epithelial cells showed that the PAM-Chol micelle had higher transfection efficiency than lipofectamine and polyethylenimine (25 kDa, PEI25k). As the anti-inflammatory drug, resveratrol was loaded into the cores of the PAM-Chol micelles using the oil-in-water emulsion/solvent evaporation method. In lipopolysaccharide (LPS)-activated macrophage cells, resveratrol-loaded PAM-Chol (PAM-Chol/Res) reduced pro-inflammatory cytokines, confirming the anti-inflammatory effects of resveratrol. In in vitro transfection assays to L2 cells, the PAM-Chol/Res micelles had transfection efficiency similar to that of PAM-Chol. The delivery of resveratrol or the heme oxygenase-1 gene (pHO-1) by inhalation was evaluated in an ALI animal model. Resveratrol delivery using the PAM-Chol/Res micelles inhibited the nuclear translocation of nuclear factor-κB (NF-κB) and reduced pro-inflammatory cytokines in the lungs. pHO-1 delivery using PAM-Chol induced HO-1 expression and reduced pro-inflammatory cytokines. However, the highest anti-inflammatory effects were obtained with combined delivery of pHO-1 and resveratrol using the pHO-1/PAM-Chol/Res complex, as demonstrated in cytokine assays and immunohistochemical studies. Therefore, the PAM-Chol micelle is an efficient carrier of resveratrol and pHO-1 into the lungs and could be useful for the treatment of ALI by inhalation.

A ternary-complex of a suicide gene, a RAGE-binding peptide, and polyethylenimine as a gene delivery system with anti-tumor and anti-angiogenic dual effects in glioblastoma.

The receptor for advanced glycation end-products (RAGE) is involved in tumor angiogenesis. Inhibition of RAGE might be an effective anti-angiogenic therapy for cancer. In this study, a cationic RAGE-binding peptide (RBP) was produced as an antagonist of RAGE, and a ternary-complex consisting of RBP, polyethylenimine (2 kDa, PEI2k), and a suicide gene (pHSVtk) was developed as a gene delivery system with dual functions: the anti-tumor effect of pHSVtk and anti-angiogenic effect of RBP. As an antagonist of RAGE, RBP decreased the secretion of vascular-endothelial growth factor (VEGF) in activated macrophages and reduced the tube-formation of endothelial cells in vitro. In in vitro transfection assays, the RBP/PEI2k/plasmid DNA (pDNA) ternary-complex had higher transfection efficiency than the PEI2k/pDNA binary-complex. In an intracranial glioblastoma animal model, the RBP/PEI2k/pHSVtk ternary-complex reduced α-smooth muscle actin expression, suggesting that the complex has an anti-angiogenic effect. In addition, the ternary-complex had higher pHSVtk delivery efficiency than the PEI2k/pHSVtk and PEI25k/pHSVtk binary-complexes in an animal model. As a result, the ternary-complex induced apoptosis and reduced tumor volume more effectively than the PEI2k/pHSVtk and PEI25k/pHSVtk binary-complexes. In conclusion, due to its dual anti-tumor and anti-angiogenesis effects, the RBP/PEI2k/pHSVtk ternary-complex might be an efficient gene delivery system for the treatment of glioblastoma.

A curcumin-loaded polymeric micelle as a carrier of a microRNA-21 antisense-oligonucleotide for enhanced anti-tumor effects in a glioblastoma animal model.

A glioblastoma is a common primary brain tumor that expresses microRNA-21 (miR-21), which inhibits the expression of pro-apoptotic genes such as phosphatase and tensin homologue (PTEN) and programmed cell death 4 (PDCD4). Therefore, an antisense-oligonucleotide against miR-21 (miR21ASO) could have therapeutic effects for glioblastomas. In this study, curcumin was loaded into deoxycholic acid-conjugated polyethylenimine (DP) micelles. The curcumin-loaded DP micelle (DP-Cur) was evaluated as a carrier for the combined delivery of curcumin and miR21ASO. Gel retardation and heparin competition assays showed that DP-Cur formed stable complexes with miR21ASO. The anti-tumor effects of the combined delivery of curcumin and miR21ASO were evaluated in C6 glioblastoma cells. In vitro transfection showed that DP-Cur had an miR21ASO delivery efficiency similar to that of polyethylenimine (25 kDa, PEI25k) and DP. In the C6 cells, the delivery of miR21ASO using DP-Cur effectively reduced the miR21 level. The miR21ASO/DP-Cur complex induced apoptosis more effectively than the single delivery of curcumin or miR21ASO. The therapeutic effect of the miR21ASO/DP-Cur complex was also evaluated in an intracranial glioblastoma animal model. The miR21ASO/DP-Cur complex reduced the tumor volume more effectively than single therapy of curcumin or miR21ASO. Immunohistochemistry showed that PDCD4 and PTEN were induced in the miR21ASO/DP and miR21ASO/DP-Cur complex groups. Therefore, DP-Cur is an efficient carrier of miR21ASO and the combined delivery of miR21ASO and curcumin may be useful in the development of combination therapy for glioblastoma.

Production and application of HMGB1 derived recombinant RAGE-antagonist peptide for anti-inflammatory therapy in acute lung injury.

Acute lung injury (ALI) is an inflammatory lung disease caused by sepsis, infection, or ischemia-reperfusion. The receptor for advanced glycation end-products (RAGE) signaling pathway plays an important role in ALI. In this study, a novel RAGE-antagonist peptide (RAP) was produced as an inhibitor of the RAGE signaling pathway based on the RAGE-binding domain of high mobility group box-1 (HMGB1). Recombinant RAP was over-expressed and purified using nickel-affinity chromatography. In lipopolysaccharide- or HMGB1-activated RAW264.7 macrophage cells, RAP reduced the levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). RAP decreased the levels of cell surface RAGE and inhibited the nuclear translocation of nuclear factor-κB (NF-κB). These results imply that RAP decreases RAGE-mediated NF-κB activation and subsequent inflammatory reaction. For in vivo evaluation, RAP was delivered to the lungs of ALI-model animals via intratracheal administration. As a result, RAGE was down-regulated in the lung tissues by pulmonary delivery of RAP. Consequently, TNF-α, IL-6, and IL-1ß were also reduced in broncoalveolar lavage fluid and the lung tissues of RAP-treated animals. Hematoxylin and eosin staining indicated that inflammation was decreased in RAP-treated animals. Collectively, these results suggest that RAP may be a useful treatment for ALI.

Anti-Inflammatory Therapeutic Effect of Adiponectin Gene Delivery Using a Polymeric Carrier in an Acute Lung Injury Model.

PURPOSE: Adiponectin (APN) is an adipokine with anti-inflammatory and cytoprotective effects. In this study, the therapeutic effect of APN gene delivery using a polymeric carrier was evaluated in an acute lung injury (ALI) model. METHODS: Polyethylenimine (2 kDa, PEI2K), PEI25K (25 kDa), polyamidoamine (generation 2, PAMG2), dexamethasone-conjugated PEI2k (PEI2K-Dexa), and dexamethasone-conjugated PAMG2 (PAMG2-Dexa) were evaluated in vitro and in vivo as gene carriers. Formation of plasmid DNA (pDNA)/carrier complexes was confirmed by gel retardation and heparin competition assays. Delivery efficiency was measured by a luciferase assay and fluorescence microscopy. In an ALI animal model, pAPN/carrier complexes were delivered by intratracheal administration. Therapeutic effects were evaluated by cytokine assays and hematoxylin and eosin (H&E) staining. RESULTS: Gel retardation assays showed that PEI2K-Dexa and PAMG2-Dexa formed complexes with pDNA. In L2 lung epithelial cells, PAMG2-Dexa yielded higher transfection efficiency than PEI2K, PAMG2, PEI25K, lipofectamine, and PEI2K-Dexa. In vivo experiments showed that PAMG2-Dexa delivered DNA more efficiently to lung tissue than PEI2K-Dexa and PEI25K. Delivery of pAPN/PAMG2-Dexa complexes upregulated APN expression in the lungs of mice with ALI. As a result, the levels of pro-inflammatory cytokines such as TNF-α and IL-1ß were decreased. H&E staining showed that inflammation in the lungs of mice with ALI was reduced by delivery of the APN gene. CONCLUSION: Delivery of the APN gene using PAMG2-Dexa reduced inflammation in the lungs of mice with ALI. The APN gene could be a useful tool in the development of gene therapy for ALI.

Delivery of the high-mobility group box 1 box A peptide using heparin in the acute lung injury animal models.

In this study, the efficacy of the high-mobility group box-1 box A (HMGB1A)/heparin complex was evaluated for the treatment of acute lung injury (ALI). HMGB1A is an antagonist against wild-type high-mobility group box-1 (wtHMGB1), a pro-inflammatory cytokine that is involved in ALIs. HMGB1A has positive charges and can be captured in the mucus layer after intratracheal administration. To enhance the delivery and therapeutic efficiency of HMGB1A, the HMGB1A/heparin complex was produced using electrostatic interactions, with the expectation that the nano-sized complex with a negative surface charge could efficiently penetrate the mucus layer. Additionally, heparin itself had an anti-inflammatory effect. Complex formation with HMGB1A and heparin was confirmed by atomic force microscopy. The particle size and surface charge of the HMGB1A/heparin complex at a 1:1 weight ratio were 113nm and -25mV, respectively. Intratracheal administration of the complex was performed into an ALI animal model. The results showed that the HMGB1A/heparin complex reduced pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1ß, more effectively than HMGB1A or heparin alone. Hematoxylin and eosin staining confirmed the decreased inflammatory reaction in the lungs after delivery of the HMGB1A/heparin complex. In conclusion, the HMGB1A/heparin complex might be useful to treat ALI.